International Review of Cytology: A Survey of Cell Biology, Volume 155

VOLUME 155

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth Anthony P. Mahowald M. Melkonian Keith E. Mostov

Audrey Muggleton-Harris Andreas Oksche Muriel J. Ord Vladimir R. Pantic M. V. Parthasarathy Thomas D. Pollard Lionel I. Rebhun L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Alexander L. Yudin

Edited by Kwang W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 155

ACADEMIC PRESS San Diego New York

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This book is printed on acid-free paper.

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Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NWl 7DX International Standard Serial Number:

0074-7696

International Standard Book Number:

0-12-364558-1

PIUNTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 E B 9 8 7 6

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4

3 2

1

Contributors .......................................................................................

vii

Phylogeny and Ontogeny of Chemical Signaling: Origin and Development of Hormone Receptors G. Csaba I. 11. 111. IV.

Introduction ........................ Signal Molecules and Receivers in Unicellular Organisms Ontogeny of Hormone Receptors . Conclusions ....................... References ........................

.............

37

Growth Factor-Induced Cell Migration: Biology and Methods of Analysis Marianne Manske and Ernesto G. Bade I. II. 111. IV.

Introduction ... Analysis of Cell Migration ............. Growth Factors That Modu .................................. Conclusions and Outlook .................................................................. References ................................................................................

49 53 67 81 81

Physiological and Biochemical Aspects of Cytoplasmic Streaming Teruo Shimmen and Etsuo Yokota I. introduction ................................................................................ V

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CONTENTS

vi II. 111 . IV. V.

Mechanism of Motive Force Generation .................................................. DemembranatedCell Models of Characeae and Characteristicsof Cytoplasmic Streaming Extracellular Factors Affecting Cytoplasmic Streaming ................................... Concluding Remarks ...................................................................... References ................................................................................

98 110 120 129 131

Cell and Molecular Biology of Flagellar Dyneins David R . Mitchell I. II. 111. IV. V. VI . VII . VIII.

Introduction .................................................. Dyneins in Flagellar Motility .... .................... Outer Row Dyneins ............ ................ Inner Row Dyneins: 57 Varieties Distinct Roles for Outer and Inner Row Dyneins The Cross-Bridge Cycle ........ Dynein Regulation .............. ................. Conclusions .................... References ..........

.......................

.............

141 142 146 162 166 168 170 173 175

Morphological and Functional Reorganization of Human Carcinomas in Vitro Petra Kopf.Maier. Birgit Kolon. and Markus Bugenings I. II. 111 . IV.

Introduction ................................................... Cell Culture Systems for Growing Human Carcinomas ...... High-Density (Organoid) Culture .................... Concluding Remarks ........... References ..........

..................... .....................

Index .............................................................................................

181 182 187 245 252 259

Numbers in parentheses indicate the pages on which the authors' contributions begin

Ernest0 G. Bade (49),Arbeitsgruppe Zellbiologie-Tumorbiologie,Fakultat fur Biologie, Universitat Konstanz, 78434 Konstanz, Germany Markus Bugenings (181), InstitutfiirAnatomie,freie Universitat Berlin, D- 14195BerlinDahlem, Germany G. Csaba (1), Department of Biology, Semmelweis University of Medicine, 1445Budapest, Hungary

Birgit Kolon (181), lnstitut fur Anatomie, Freie Universitat Berlin, D- 14195 BerlinDahlem, Germany

Petra Kopf-Maier (181), lnsfifut fur Anatomie, Freie Universitat Berlin, D- 14195 BerlinDahlem, Germany Marianne Manske (49),Arbeiisgruppe Zellbiologie-Tumorbiologie,Fakultat fur Biologie, Universitat Konstanz, 78434 Konstanz, Germany David R. Mitchell (141), Department of Anatomy and Cell Biology, and Program in Cell and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210 Teruo Shimmen (97), Department of life Science, faculty of Science, Himeji lnstitute of Technology, Harima Science Park City, Hyogo 678-12, Japan Etsuo Yokota (97), Department of life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-12, Japan

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Phylogeny and Ontogeny of Chemical Signaling: Origin and Development of Hormone Receptors G.Csaba Department of Biology, Semrnelweis University of Medicine, 1445 Budapest, Hungary

1. Introduction A. Communication in the Lowest Level of Phylogeny Unicellular organisms live in aqueous environment and thus the molecules of the medium determine the environment itself. These molecules can be beneficial for the cell (e.g., nutrients) or harmful (e.g., toxins). It is essential that an encounter with these molecules result in a change in future behavior, a memory, because this will give the cell an advantage in its search for beneficial molecules and in its avoidance of harmful ones. The communication is unilateral in this case, the cell detecting its environment and orienting itself with the help of signals (reception). Since the environment of the unicellular organism is very variable, it is not possible to have previously defined-fixed-receptors on the plasma membrane. Only the continuous extension and withdrawal of structures can provide the sensitivity required in the plasma membrane. The encounter of receptor structures and signal molecules fixes the reception. Everything points to the fact that only the subunits of the plasma membrane are genetically coded in unicellular organisms (Koch er al., 1979), and the way in which they are assembled is accidental. Following an encounter with signaling molecules, the number of structures that receive the signals increases in the plasma membrane (or the structures are produced as a complex of subunits); with the help of this “memory,” the ability to respond promptly is assured (Csaba, 1986a). The lifespan of the unicellular organism is brief and can be measured in hours or minutes. This means that receptor memory has value for the species as a whole if it is transferred to following generations. Protozoa Inlernalionol Review of C.vIolog.v, Vol. 155

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Copyright 0 1994 by Academic Press,lnc. All rights of reproduction in any form reserved.

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are able to transfer information acquired by imprinting to their progeny (Csaba e? al., 1982~); the presence of this memory was observed over a long period (in a thousand generations at least). The unicellular organism does not have the ability to transfer cultural information as do the higher ranks of animals or human beings. Nevertheless, the transmission of receptors (memory) that develop in an encounter performs the same role as transmission of cultural information. It is probable that the formation of receptors in the membrane is preceded by other activities because isolated intracellular compartments have to find each other and this requires the ability to recognize each other. When a lysosome containing enzymes approaches and fuses with the phagosome which enters the cell, there is a process of recognition. For this process to take place, glycoprotein structures are required in the intracellular membranes. These structures build signals and signal-receiver units (marker and receptor-like structures), which are considered to be the ancestors of the plasma membrane receptors. The unicellular organism can detect signals and synthesize signal molecules to be secreted into the environment (Le Roith e? al., 1980, 1981, 1982; Roth et al., 1982). Interestingly, ancestors of signal molecules that have a hormonal function in higher animals have been demonstrated in unicellular eukaryotes, though their exact function is still unclear. Insulin or an adrenocorticotropic hormone produced by a protozoan might be a side product of the protein synthesis of a similar hormone which has the capacity to influence either the cell itself in an autocrine way or to affect other unicellular organisms. Whether or not an autocrine function exists, the protozoan has a complete endocrine system because it is able to produce a hormone that interacts with the membrane receptors. Since the unicellular organism has its own second messenger system (Kuno et al., 1979; Schultz e? al., 1983; Nakaoka and Ooi, 1985; Nagao er al., 1981; Kovhcs and Csaba, 1987b, 1990c; Kovacs e? al., 1989a), the hormone is also able to induce a response. However, it is possible that the material secreted to the environment can influence other related cells, as happens in the case of Dictyostelium discoideum (Ray and Lerner, 1987). Here secreted cAMP is the signal which induces grouping of amebas because the amebas have cAMP receptors. Similarly, special hormone-like materials (pheromones) are secreted during the conjugation of protozoa (Nobili e? al., 1987). Probably the receptors are developed earlier than the signal molecules (hormones) at this stage of phylogeny, because the ability for receptors to recognize signals and molecules is the critical element in the cell-environment relationship. In the first phase of development, the communication is such that the cell “understands but does not speak”; in the second phase, hormone-like materials appear and the cell “both speaks and under-

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

3

stands.” At the same time, if we agree with Blalock’s hypothesis (Bost et ul., 1985a,b), this suggests that the DNA strand coding for the receptor also contains a code for the hormone (Schwabe, 1990). 6 . Fundamentals of Communication in Multicellular Organisms

At the unicellular level, the receptor becomes stronger in the presence of signal molecules because it is not preprogrammed. However, it is usual for the receptors of multicellular organisms to be preprogrammed (Csaba, 1986a,b). This means that the types of receptors appearing on the surface (or intracellularly) are determined at the gene level in each cell. Similarly, the determination of which cell will synthesize a certain kind of hormone is also programmed. These traits are derived from the multicellular character of the organism, which is structured so that the signal and the receiver are built on the information of the same genome. However, if there is a community of cells, certain regulator molecules are able to influence only special groups of cells. Although the signal molecule (hormone) may be present in the whole organism, only the receptor possessing the proper cells will respond to it. In this way the gene program permits inquiry by receptors. In this case, the interaction between the signal molecule and the receiver is completed in a closed system. This does not exclude the subsequent emission of some signal molecules, as in the case of the emission and reception of pheromones. Thus the older system of signaling persists.

II. Signal Molecules and Receivers in Unicellular Organisms A. Receptors in Protozoa and Microbes

When substances which function like hormones in the higher ranks of animals are added to the environment of protozoa and suitable indexes are applied, the reaction of the unicellular organism can be described. Phagocytosis in Tetruhymena was successfully induced by histamine, which acts as a phagocytosis hormone in higher ranks of phylogeny, and also by serotonin (Csaba and Lantos, 1973). The sequence of the phagotrophic effect is similar to the order described in higher animals. Hormones influencing the carbohydrate metabolism of higher cells, insulin (Csaba and Lantos, 1975a) and epinephrine (Csaba and Lantos, 1976) are

4

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able to induce sugar metabolism in Tetrahymena while they are ineffective in several other reactions (Csaba and Lantos, 1975b). The morphogenetic hormone of higher animals, thyroxine and its precursors, can influence the division of Tetrahymena. Other hormones and hormone-like substances are able to induce positive or negative chemotaxis in Tetrahymena (Nobili et al., 1987; Csaba and Kovacs, 1994a). Hormones that have no target reaction in Tetrahymena cause alterations in protein synthesis, and so they are not ineffective (Csaba and Ubornyak, 1981). From these facts and other works (Castrodad et al., 1988; Renaud et al., 1991; De Jesus and Renaud, 1989; Quinones-Maldonado and Renaud, 1987; Wyroba, 1989; Zagon and McLaughlin, 1992), we can conclude that Tetrahymena is affected by the hormones of higher animals. At the same time, the biological view requires us to pose the question inversely, since it seems that it is not the hormones of higher animals but those substances that became hormones and signal molecules during evolution that have the capacity to evoke some specific reaction in Tetrahymena. Tetrahymena has no endocrine system and the hormone is only one substance of many which the cell is able to recognize; the presence of this material in its environment or its interaction with the cell membrane elicits some reaction by the cell. In this way perhaps some materials were selected as hormones from the materials surrounding the protozoa if they did not already have another evolutionary purpose, and if they were able to bind to receptors to transmit information into an intracellular system. It is probable that a material suitable for becoming a hormone has to fulfill many additional conditions. The information carried by a signal molecule becomes valuable and usable only when a cell has a receptor for the molecule. Such receptors are present in the plasma membrane of unicellular organisms (Kovacs and Csaba, 1990a). For a protozoan it is essential to recognize the environment, to select the useful molecules as nutrients, and to avoid the harmful ones (Leick and Hellung-Larsen, 1992). The existence of nutrient receptors (Lenhoff, 1968, 1974)for amino acids or polypeptides makes this possible. Tetrahymena recognizes an amino acid and the hormone derived from the amino acid as similar molecules. This cell also has the ability to differentiate the hormone, the analog of the hormone, and its antagonist (Csaba and Lantos, 1975c; Csaba and Darvas, 1986) (Fig. 1). The nutrient receptor detects the binding of similar molecules by an altered response. Tetrahymena possesses not only nutrient receptors but mating receptors (Rosati and Verni, 1991) and it is postulated that the latter have a role in the development of hormone receptors (pheromone receptors). The development of membrane receptors that are rigidly determined at the gene level is impossible because of the lifestyle of the protozoa. This organism lives in a changing environment that includes a wide variety

5

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS P.C.1

sc C=l .o

0.6

FIG. 1 Effect of histamine and histamine antagonists on the phagocytosis of Tetrahymena related to the control as 1 .O (broken line). P.C., Phagocytic coefficient; C, control (untreated); C-H, histamine-treated cells; CICim, cimetidine; C/s, chloropyramine; C/D, tripelennamine; and SC, significance to control. The scale represents the range of scattering. In the case of cells that were not treated with histamine earlier, only the histamine treatment elevated the phagocytotic capacity. (Reproduced from Csaba and Darvas, 1991, with permission. Copyright The Faculty Press, Cambridge, England.)

of surrounding molecules. This makes the presence of plastic structures in the plasma membrane important because these structures are able to form the most diverse configurations and thus are able to recognize the molecules of the surroundings. It seems to be essential to keep the receptor-type structures in the membrane to facilitate recognition. The hormone receptors have to be able to recognize everything; this capacity supports the formation of permanent structures (receptors) more than of other molecules. The location of recognition structures in the plasma membrane does not seem to be accidental at this level of evolution because this component of the cell maintains the cell’s connection with the environment. Though Tetrahymena is a first-class model cell for studying receptor evolution, the effects of hormones have been demonstrated not only in Tetrahymena but in Amoeba (Csaba et al., 1985c; Mayers and Couillard, 1990; Couillard et al., 1989) and even in fungi and bacteria (Le Roith et al., 1987; Lummis, 1992). Since the protozoa are representative of a lower level of evolution, we have to take into account the fact that the molecules which are hormones of the higher animals appear in some form in a lower rank of evolution. That is why it is assumed that in Tetrahymena, the precursors of hormones have greater importance than the higher rank hormone itself (Csaba and Nemeth, 1980). It has been shown that the less complex molecules of the thyroxine series-triiodothyronine, diiodothyronine, and monoiodothyrosine-are able to induce the multiplication of cells better than the thyrox-

6

G. CSABA

ine itself. These studies also support the theory of the origin of nutrient receptors. The low concentration of the base molecule of the thyroxine series, tyrosine, is as effective as diiodothyrosine. The difference is that diiodothyrosine works like a hormone, which means that the effect decreases in low doses and there is a fall following the peak because of toxicity. Tyrosine works as a nutrient; it induces a more intensive response (actually the division of cells) in the highest concentration applied. It is conceivable of course that this phenomenon could be observed where the complexity of the hormone is greater, (consider the jump from tyrosine to thyronine in the example of the thyroxine series, while in the case of the serotonin series, the hormone serotonin itself is most effective).

6. Hormones in Protozoa and Microbes The recognition of hormones by protozoa led to the study of endogenous hormones in unicellular organisms and microbes. In Tetrahymena pyriformis, the following substances were demonstrated: insulin, somatostatin, adrenocorticotropin (ACTH), @-endorphin,relaxin, vasotocin, and calcitonin (Le Roith et al., 1987);from the steroid hormones, dehydroepiandrosterone (DHEA) and estradiol (Csaba et al., 1985a); and from the amino acid-type hormones, serotonin and adrenalin (Blum, 1967; Brizzi and Blum, 1970; Janakidevi et al., 1966; Csaba and Kovacs, 1994a). In microbes thyrotropin (TSH) was detected in Clostridium perfringens, and chorionic gonadotropic hormone (HCG) was detected in many bacteria. Neurotensin, somatostatin, calcitonin, and insulin were detected in Escherichia coli (Lenard, 1992). The presence of neurotensin, insulin, and somatostatin was proved in other bacteria the same way. Neurotensin is present in Caulobucter crescentus and in Rodopseodomonas palustrus; insulin is present in Aspergillus fumigatus, Halobacterium solinarium, and in Bordatella pertussis; while somatostatin was demonstrated in Bacillus subtilis. Among fungi, calcitonin, arginine, and vasotocin have been detected in Neurospora crassa; cholecystokinin in Candida albicans; and farnesol-type mating hormones were detected in Saccharomyces cerevisiae (Le Roith et al., 1987). There are references to the presence of prostaglandin in Tetrahymena but this is uncertain (Csaba and Nagy, 1987). The presence of thyroxine and triiodothyronine could not be proved though it was checked (Csaba and Nagy, 1987). The presence of hormones does not mean that they are secreted by the cell. This is shown clearly by DHEA, which is not present in the medium of Terrahymena though the cells can be induced to secrete DHEA by dexamethasone (Csaba et al., 1985a). Tetrahymena also secretes insulin and serotonin into its medium (Csaba and Kovacs, 1994a).

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

7

As in the case of the hormone receptors discussed earlier, the question arises whether the hormones of higher animals also have hormone-like functions in Tetrahymena or whether they are simply the side products of amino acid, polypeptide, or steroid synthesis. It is probable that nature made several trials in the highly developed ciliates and those molecules that have not been detected have not been sought. The absence of triiodothyronine and thyroxine contradicts this, but the presence of such a nonhormone but steroid-type molecule as digoxin supports this premise (Csaba et al., 1984b; Darvas et al., 1985b). Considering that Tetrahymena has receptors, the presence of autocrine regulation is also imaginable, as is a paracrine-like intercellular regulation. In this case the Tetrahymena hormones have value in communication, and this ability, combined with receptors and hormones, may serve as a basis for the evolution of hormone and receptor alike. According to Roth and his co-workers, who played a pioneer role in the detection of hormones, there are well-conserved regions in the hormones of Tetrahymena and higher animals, and for this conservation the presence of a function is required (Le Roith er al., 1987). On the basis of this, it is conceivable that the molecules fulfill a communicative role. However, the excessively high number of molecules with this role in unicellular organisms raises doubts about their function. C. Specificity of Hormone and Receptor

Tetrahymena receptors have a good ability to discriminate. Receptors of the wall-less mutants of Neurospora crassu bind insulin in a specific way (Fawell et ul., 1988; Fawell and Lenard, 1988; McKenzie et al., 1988; Kole et al., 1991).Amebas possess specific opioid receptors and naloxone inhibits them ( Josefsson and Johansson, 1979). The biological mirrorversion of naloxone has no capacity to inhibit these receptors. Pseudomonus maltojilia has human chorionic gonadotropin (hCG) binding sites, and their affinity and specificity are similar to the HCG receptors of the human ovary. There are TSH binding sites in Yersiniu enterocolica and in Escherichia coli, and one can replace the labeled hormone with the unlabeled hormone. Paracoccidiodes brasiliensis has high-affinity binding sites for estrogen (Le Roith et af., 1987). Also, Saccharomyces cerevisiae is able to bind estrogens, and the biochemistry of the binding protein is similar to the steroid receptors of higher animals. Candida albicans binds corticosterone and its affinity lies between the affinities of cortisol binding protein (CBG) and the glucocorticoid receptor (Loose and Feldman, 1982). Trypanosoma cruzi has beta adrenergic receptors with radioligand binding criteria characteristic of mammalian cells (de Castro and Oliveira, 1987).More-

8

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over, the opioid receptor of Tetrahymena has a sequence homology with the identical receptors for leeches and rats (O'Neil et al., 1988; Zipser er al., 1988). All of this is meant to show that there are binding sites in protozoa which are similar to the binding sites of higher animals in several respects, although we have to consider the fact that the binding sites for steroids were found in cells that live in host organisms and thus the cells are not isolated from the influence of the host. Among the hormones of unicellular organisms, the alpha-type yeast mating factor has similarities to the gonadotropin-releasing hormone of mammals. Administration of this substance induces the production of hormones in the pituitary of mammals (Loumaye et al., 1983). Terrahymenu insulin is able to influence the level of blood glucose in vertebrates (Le Roith er al., 1983). On the basis of all these facts, the specificity of Tetrahymena hormones is acceptable and demonstrates that these molecules, which are present at a low level of phylogeny, can influence, at least in some part, the regulation of higher animals. D. Second Messenger Systems

The hormone itself and the presence of the membrane structures which bind it do not ensure the responsiveness of the cell. The system will only work properly when one terminal of the receptor performs its mediator function in a way similar to the epidermal growth factor (EGF) or insulin receptors, or when second messenger systems are present. It has been well known for a long time that cAMP serves as a starvation signal in bacteria and that the presence of these molecules is able to induce the assembly of Dicryosrelium discoideum (Roy and Lerner, 1987). Dictyostelium has cAMP receptors and there are different types of Gproteins which are able to generate and induce the liberation of different second messengers such as CAMP, cyclic guanosine monophosphate (cGMP), inositol trisphosphate, and calcium (Snaar-Jagalska et al., 1988a,b; Kesbeke and Van-Haastert, 1988; Kesbeke er al., 1988). It is probable that the structure of the G-protein is also similar to the identical molecule in higher animals. It has been shown that the isolated a-subunit is similar to the a-subunits of G-proteins of vertebrates in its function and physical parameters. In Trypanosoma cruzi, where the effect of isoproterenol on cell division was observed, a synchronous cAMP increase was also reported (de Castro et al., 1987). The presence of adenylate cyclase, the effect of CAMP, and the participation of the guanosine triphosphate (GTP) regulatory protein were demonstrated in Saccharomyces cereuisiae (Levitzki, 1988; Becker er al., 1988; Engelberg et al., 1989). Protein kinases and tyrosine kinase, which has a role in the function

9

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

of receptors, were also found. The GTP binding protein was also detected in Neurospora crassu (Hasunuma and Funadera, 1987; Hasunuma et al., 1987a,b; Furukawa et ul., 1987). There were mutants of Neurospora in which the concentrations of both the cyclic phosphodiesterase and the CAMPwere decreased and the 24-hr oscillation of CAMP was also characteristic. In this mutant, white light also decreased the level of cGMP. It is clear that second messengers are present at the lowest levels of phylogeny, at the unicellular level of organization. In the protozoan Tetrahymenu, the work and cooperation of several second messenger systems was observed. They are the adenylate cyclase-CAMP system (Nagao et al., 1981; Csaba and Sudar, 1978; Kovacs and Csaba, 1986a; Kovacs, 1986; Csaba et al., 1976, 1978, 1987a), the guanylate cyclase-cGMP system (Kovacs et ul., 1989a), and the calcium-calmodulin system (Kovacs and Csaba, 1987a,b) (Figs. 2 and 3). Hormonal effects modify the level of second messengers working in the systems. Inositol phospholipids were also detected (Kovacs and Csaba, 1990~). Considering that (1) all three members of a typical endocrine system are present in unicellular organisms because the hormones are there and they are secreted, (2) the identical receptors are there, and (3) the second

I--

-A

0

C

FIG. 2 Guanylate cyclase activity after insulin imprinting in Tetrahymena. There is a highly significant difference ( p <0.01) between the experimental and control groups. A. control; B. control + IO-'M insulin; C, insulin imprinting; D, insulin imprinting 10-6M insulin. (Reproduced from Kovacs et al., 1989a. with permission. Copyright The Faculty Press, Cambridge. England. )

+

10

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0

FITC-insulin

Intensibof fluorescence

control

~FITC-TSH

insulin

TSH

TMBB

TMB8

Intensity of fluorescence

TMB 8

+

insulin

+

TSH

FIG. 3 Intact calcium mediation is an essential prerequisite for normal hormonal imprinting. Top: insulin imprinting and TSH imprinting of Tetruhymena cells; label is fluorescein isothiocyanate (FITC). Bottom: TMBS, a calcium release inhibitor of the sarcoplasmic reticulum, does not allow insulin or TSH imprinting of Tetrahyrnenu. (Reproduced from KovAcs and Csaba, 1987b, with permission.)

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

11

messenger system and a change in its level following the binding of the hormone are also observed, it is obvious that unicellular organisms have a true endocrine system. This endocrine system may work in an autocrine way; it may serve intercellular communication and may also respond to exogenous hormones or hormone-like materials. In this way the system equally serves the survival of unicellular organisms and establishes the total specificity and the fixing at the gene level of signal molecules and receivers through the process of evolution. E. Role of Signal Molecules in Receptor Development

It is clear from the previously mentioned facts that there are structures in the plasma membrane of the unicellular organisms which react to the molecules in the environment. Recognition by these receptors is more or less specific and is able to induce the cell to respond. However, these structures are no more than binding sites and are not true receptors. Thus they have the characteristics of membrane receptors of higher cells only in part. Following an encounter with a hormone (signal molecule), these circumstances change. The offspring generations of the protozoa will bind the formerly recognized signal molecule (Fig. 4) in an altered, mostly heightened, way (Csaba, 1981, 1984, 1986a). The binding represents the specificity characteristic of the receptor and has kinetics similar to those of the receptors of higher animals, which are dependent on temperature and pH (Kovacs and Csaba, 1990a). The binding capacity of a hormone increases in a concentrationdependent manner. Although this is hard to study using the classic methods of receptor kinetics because of the uniqueness of the unicellular membrane, studies applying fluorescent and confocal microscopic techniques support the alteration of binding capacity following an encounter with a hormone (Christopher and Sunderman, 1992). Similar results were demonstrated by immunological investigations. When rabbits are immunized with untreated or insulin-treated Tetrahymena, the binding of the developed antibodies on the insulin receptors of the liver demonstrates the immunological relationship (Kovacs et a/., 1985). The binding of the antibody developed against Tetrahymena pretreated with insulin is much higher than the binding of the antibodies developed against untreated Tetrahymena cells. Conversely, the treatment of Tefrahymenri with antibody developed against the insulin receptor of liver is able to induce the binding of insulin like the insulin itself, while the targets of this antibody are the insulin receptors (Csaba et al., 1984d). Thus the specificity seems to be acceptable. This supports the hypothesis that an imprinting type of mechanism

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G. CSABA S

T. pyriformis

T. thermophila

FIG. 4 Imprinting can cause positive (increasing) or negative (decreasing) effects on later hormone binding, depending on the reaction of the imprinted cell. Binding of FITC-labeled insulin to insulin-pretreated (imprinted) T, pyriformis and T . rhermophila (related to the control as 100). s = p <0.01. Insulin provoked a positive imprinting in T . pyriformis and a negative imprinting in T . thermophila. (Reproduced from Kovhcs and Csaba, 1987a. with permission.)

is developed in the presence of a hormone and the result is prolonged and alters the binding capacity of cells in the offspring generations. The similarity to the receptors of higher animals is shown also by the phenomenon of downregulation (Csaba and KBhidai, 1986, 1989). It has been demonstrated in the unicellular Tetrahymena and in the higher animals that downregulation is concentration and time dependent. The mechanism of receptor formation is also similar to that of the higher organisms because the participation of the cytoplasmic membrane pool in the development of receptors (in their recirculation) was also demonstrated, as was the internalization of receptors in coated vesicles (Csaba et a f . , 1984~). Similar to the higher animals (Horvat, 1978; Horvat and Katsoyannis, 1975; Burven and Jones, 1987), in protozoa the nuclear envelope also has hormone receptors (Csaba and Hegyesi, 1992; Hegyesi and Csaba, 1992 a,b) (Figs. 5 , 6, and 7). These receptors could be translocated plasma membrane receptors (as in mammals: Podlecki et a f . , 1987),justifying the internalization and recirculation of the receptor-containing membrane and the unity of the membrane pool in protozoa; or they could be members

13

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

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8 3 Y

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60 40-

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FIG. 5 FITC-insulin binding of the nuclear (.) and plasma (+) membranes of Tetrahymena treated (imprinted) with different concentrations of insulin 4 hr earlier. Control was 100% M and fluorescence intensity. All points are significant ( p
of a receptor-transmitter system working in the nuclear envelope (Rubins et al., 1990). The imprintability and the change in the specificity of these nuclear membrane receptors has been observed (Figs. 8 and 9). Upon an encounter with a hormone, probably the whole system of membranes bursts into activity and the structures ready for binding become detectable and altered both inside the cell and on its surface. 140

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FIG. 6 FITC-insulin binding of the nuclear (.) and plasma ( + ) membranes of Tetrahymena treated (imprinted) with different concentrations of insulin 24 hr earlier. Control was 100% fluorescence intensity. All points are significant ( p 10.01) except IO-'OM and M for the nucleus. In each concentration tested, imprinting resulted in increased FITC-insulin binding. (Reproduced from Hegyesi and Csaba, 1992b. with permission. Copyright The Faculty Press, Cambridge, England.)

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cc insulin(M)

FIG. 7 FITC-insulin binding of the nuclear (.) and plasma ( + ) membranes of Tetrahyrnena

treated (imprinted) with different concentrations of insulin 48 hr earlier. Control was 100% fluorescence intensity. All points are significant ( p <0.01) except lo-" M and M for the nucleus. In each concentration tested, the imprinting resulted in increased FITC-insulin binding. In the case of the plasma membrane, the power of binding was directly proportional to the increase in the concentration of hormone participating in imprinting. For the nuclear envelope, the optimal concentration was lo-' M. At very low concentrations (!O-''M), the binding resulted in imprinting that was insignificant in the plasma membrane, but was still present in the nucleus. (Reproduced from Hegyesi and Csaba. 1992b. with permission. Copyright The Faculty Press, Cambridge, England.)

0 C 0 v)

0

L

2I) 6 0 c 0

c

0

x c : ..C 40 v)

-

0

c

C .-

20 -

10-9

10-8

10-7

10-6

10-5

cc insulin (M) FIG. 8 Binding of FITC-insulin after treatment with different concentrations of "cold" insulin by the Tetrahyrnena plasma membrane, = control, * = 24 hr, x = 168 hr. With the passage of time, the effect ofimprinting becomes more apparent. (Reproduced from Csaba and Hegyesi, 1992. with permission. Copyright The Faculty Press, Cambridge, England.)

15

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

120

-s

100

Y

8 5 80 v)

!??

2

60

0

.$ 40 rn c a, c

c

.- 20 0 10-9

10-8

10.7

10-5

10-4

cc insulin (M) FIG. 9 Binding of FITC-insulinafter treatment of Tetrahymena nuclei with different concentrations of “cold” insulin. +, control; 0, 24 hr; 0 , 168 hr. In the case of the nucleus, the

specificity increases parallel with time (with more generations). (Reproduced from Csaba and Hegyesi, 1992, with permission. Copyright The Faculty Press. Cambridge, England.)

An intact plasma membrane is required for the development of imprinting. Any disturbance of the membrane will make the development of imprinting impossible. Changes in fluidity, perturbation by anesthetics, alteration of membrane potential, influencing the saccharide components of the membrane, treatment with alcohol, all are able to inhibit the development of imprinting (Kovacs et al, 1984, 1987; Nozawa et af., 1985a,b,c, Kovacs and Csaba, 1986b; KBhidai ef al., 1986a,b,c). However, once developed, imprinting is practically impossible to destroy; only very strong disturbances have the capacity to do this if they are applied just after imprinting (Csaba ef al., 1982a, 1983a; Csaba and Kovacs, 1986). The pathway from the receptor to the effector system also seems to be essential during the development of imprinting and any perturbation hinders imprinting (Kovacs and Csaba, 1992a,b). It seems that an intact cell is required by the sensitive mechanism of imprinting but whenever imprinting is developed, its fixation is very strong. Receptors are developed in protozoa by recognition of molecules in the environment and by the fixing of the obtained information. This makes it possible to imprint every substance, not only amino acid-type hormones but also amino acids (Csaba and Darvas, 1987a); not only polypeptidetype hormones but indifferent polypeptides, for example, bovine serum albumin (Csaba and Darvas, 1987b; Csaba et al., 1985~1,or some molecules of drugs (Fig. 10) acting at the receptor level (Csaba et al., 1982b,

16

G. CSABA

grains/ cell

20

10

n

n

FIG. 10 Digoxin and ouabain can imprint Tetrahymena. Autoradiographic detection of the incorporation (binding) of labeled glucoside by Tetrahymena cells not treated with digoxin not treated with ouabain, ( O K ) , and treated with (D/C), pretreated with digoxin (D/D), ouabain (O/O). The small boxes above the columns indicate the level of significance ( p <0.01). (Reproduced from Csaba et a/.. 1986d. with permission.)

1984b; Swydan et al., 1986; Darvas et al., 1985a). However, the strength and duration of imprinting is not the same for every substance. Molecules which become signal molecules during evolution have a significant advantage. Tetrahymena has no steroid receptor, as mentioned earlier; at least it is not detectable by classic techniques (Csaba et al., 1985b). At the same time, prolonged treatment of the protozan with a highly potent fluorinated steroid hormone such as triamcinolone, or dexamethasone will induce the development of receptors. The labeled hormone is displaced by unlabeled hormone, though the kinetics of receptor binding are not equivalent to those of higher animals. The specificity of receptors which were developed in this way is incomplete because there are effects that overlap with the steroid receptors of higher animals. The steroid receptor induced by dexamethasone does not bind testosterone, not to mention the nonhormone-type steroids like digoxin or ouabain (Csaba and InczefiGonda, 1989a). The inducibility of the steroid receptor raises the question of whether other receptors such as polypeptide receptors are also developed in the presence of a hormone. In this case the binding site would be detectable only after an encounter with the hormone. This seems to be an actual situation in the case of many hormones, as experiments with labeled antibodies have shown (Kovacs et al., 1989~).

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

17

The individual lifespan of a protozoan is short and the fixing of “harmful or useful” recognition (discrimination) has its true value when this knowledge is transferred to following generations. This happens in the case of imprinting and its receptor-level effect, which is embodied both in the binding and in the responsiveness of the cell after several hundred generations (Csaba er al., 1982~).While there is some reduction in responsiveness (but not in the binding; see Fig. l l ) , the difference from the control is significant both mathematically and biologically. An intermediate encounter with the hormone strengthens imprinting and reduces its loss. Extending the life of individual protozoans (by keeping them in anaerobic conditions) does not result in a loss of imprinting (Csaba ef af., 1984a). When the conditions of division are reduced, it requires some time to rebuild imprinting. This means that the imprinting is located in structures of the cell that are not used as energy sources during starvation. In this way imprinting lasts until the end of the life of an individual cell as well as in the offspring generations, where it is unrestrictedly transferred. In the case of imprinting, the effective groups of the molecule and not just the individual molecule are bound to receptors. For example, in insulin the P-chain has a greater ability to induce imprinting, than the a-chain; however both the a- and the P-chains are required for complete imprinting (Csaba and Kovacs, 1990a). On the other hand, the dimers of insulin, which have no biological activity, do not imprint. The absence of the terminal phenylalanine or of the five C-terminal amino acids does not

”-

riI 0

100

‘a

90

____....._. ................................................................................ ~

-

3

-

9

64

129

193

258

323

516

664

generation changes

FIG. 11 FITC-insulin binding in the offspring generations of Tefrahymena cells treated with the hormone on a single occasion (control = 100%). After 664 generations, the effect of imprinting was expressed more strongly than it was 1 day after treatment. (Reproduced from KBhidai et al., 1990, with permission.)

18

G. CSABA

affect imprinting regardless of the biological activity they induce in mammals (Fig. 12). This refers to the fact that binding to the receptors is more important during imprinting than the biological activity of the hormone, in spite of the fact that biological activity can be detected for protozoan insulin. It is worth mentioning that liver cells of higher animals (in cell culture) and Tetrahymena cells were very similar from the point of view of imprinting, which may mean that a mechanism developed at the unicellular level is present in the higher animals (Kovacs et a/., 1989b). Imprinting alters the activity of second messengers. Probably this is the reason for different directions of responses to imprinting in various taxa as well as different quantitative values (Kovacs and Csaba, 1987a, 1989, 1990b). Imprinting is able to influence both the adenylate cyclase-CAMP and the calcium-calmodulin systems. It affects the function of the guanylate cyclase system in the same way it affects the phosphoinositol system. The messenger system changes as a result of these alterations, and in a repeated encounter with a hormone, the second messenger system will not change its levels as it did at the first encounter; in a manner of speaking, it switches to an energy-saving mode. However, the paralysis of second messenger systems inhibits the development of imprinting in a way similar to the inhibition of receptor-dependent kinases, and imprinting will not develop under these circumstances (KovBcs and Csaba, 1992a).

-I $- 150 Q)

0 C 0 Q)

2 0

E 0

100

._ 0 In

al

c

c -

0

Insulin

BZ9dimer

B,dimer

Des-Phe-B, insulin

Des[(B,,.,,)pentapeptide] insulin

FIG. 12 Biological activity and imprinting capacity of insulin and insulin derivatives related to the control as 100%. y = p
19

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

F. Mechanism of Imprinting As imprinting is fixed and transferred to offspring generations, the possibil-

ity of different factors being involved in the storage of the transmitted information must be assumed. One factor is the membrane itself; the second is the nucleus; there would also be some intracellular material or compartment located between the membrane receptor and the nucleus. The genetic dogma contradicts the possibility of nuclear fixation o r the transmission of imprinting by the nucleus because this dogma does not accept the possibility of transcription from protein to DNA. Though in protozoa there are some signs that this does not work like it does in bacteria or multicellular organisms (Sonneborn, 1965; Nelsen et al., 1989), it seems to be accepted that there is a nonnuclear fixation. However, we should not exclude the DNA itself while the DNA of the plasma membrane (Gabor and Bennett, 1984; Moyer, 1979; Sudar et af., 1986; Csaba et al., 1986c) is required for imprinting (SudBr et a / . , 1986). Experiments (Fig. 13) have demonstrated that DNase treatment of Tefrahymenaprevents the development of imprinting though the treatment does not affect imprinting once it is developed (Csaba et a/., 1989a). However, it is hard to discard the hypothesis that the total integrity of the cell membrane is essential for the development of imprinting, and this is altered following digestion by enzymes. A further possibility is that the imprinting information is located in the membrane because the receptors are there or underneath in the membrane

DNase treatment (5min)

DNase treatment (10 min)

FIG. 13 DNase treatment reduces the FITC-insulin binding capacity of Tetrahymena and prevents imprinting when it is applied before or after insulin treatment. Open column, control (DNase/C); checked column, insulin-treated (DNase/I); A . immediately; B . 24 hr; C. 48 hr after treatment. = p <0.05; * = p <0.01 related to the control as 100. (Reproduced from Csaba cf a/., 1989a. with permission. Copyright The Faculty Press, Cambridge, England.)

20

G. CSABA

pool. Division of the cell or the plasma membrane pool may be followed by the separation of the imprinted receptors also, and this would present the possibility of a self-assembly driven to completion in the offspring generations. This possibility is also not precluded. In cultured cells of higher animals, it was observed that only 30% of the cells with membrane receptors bind the hormone at the encounter with the hormone, but protein kinase activity is altered in the entire cell population (Murray and Fletcher, 1984; Fletcher and Greenan, 1985). This is because the intercellular metabolic communication, with the help of gap junctions, is able to transmit the information that was originally transmitted by the hormone and thus direct contact with the hormone is not necessary. The same phenomenon can be observed in Tetrahymena (Figs. 14 and 15) when hormone-treated, imprinted cells transmit the information to nontreated (virgin) cells (Csaba and Kovacs, 1987), while direct contact is less imaginable. Studies point to a substance in the medium that has the ability to transmit the imprinted information. Imprinted cells may secrete some material which reaches or enters other cells and imprints them.

S

control

Pyr.

Pyr.

Pyr.

Ther.

Ther.

Ther.

FIG. 14 FITC-insulin binding of (virgin) T . pyriformis in the media of different T. pyriformis and T. rhermophila (pretreated) groups, related to the control and medium-treated groups (C-Pyr; C-Ther) as 100. The media OF the insulin pretreated and mixed (C + insulin) groups increased hormone binding of virgin cells irrespective of the donor taxon. z = p c0.05; s = p CO.01. (Reproduced from Csaba and Kovacs. 1987, with permission.)

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

C Ther.

Insulin Ther.

C+lnsulin Ther.

C Pyr.

21

Insulin Pyr.

C+lnsulin Pyr.

FIG. 15 FITC-insulin binding of (virgin) T . therrnophilu in the media of different T . pyriformis and T . rhermophila (pretreated) groups, related to the control and medium-treated groups (C-Pyr.; C-Ther) as 100. The media of the insulin pretreated and mixed (C + insulin) groups decreased the hormone binding of virgin cells, irrespective of the donor taxon. s = p 10.01; z = p<0.005. (Reproduced from Csaba and Kovacs, 1987, with permission.)

The character of this substance is at the moment unknown. The possibility arises that the hormone itself, secreted from the imprinted cell, can influence the nonimprinted cells of the same population. This would mean that a protozoan, which takes up the hormone through the processes of downregulation and internalization, does not digest it but secretes it, and that this hormone would have its proper effect. The surprising thing is that a cell which encounters a certain hormone a single time is able to transmit the imprinted information after months and hundreds of generations (Csaba et al., 1990). This could mean either that the exogenously added hormone is still present in the cells after such a long time, which is the less probable option, o r that treatment with hormones (imprinting) has a role not only in the development of receptors but also in hormone production. Tetrahymena itself synthesizes hormones, for example, insulin, but the amount is not enough to transmit imprinted information because we compared cultures containing imprinted and virgin cells with controls. We should remember Blalock’s theory mentioned earlier. Experiments show that the hormone that was applied at imprinting is still present at an elevated level a t least six generations after the treatment (Kovacs et al., 1989~).

22

G. CSABA

G. Phylogenetic Conclusions

Previously only some hormone-producing cell (organ), the hormone itself, and the specific receptor fit into the concept of the endocrine system of multicellular organisms. This concept has been widened greatly in the past 10 years. In addition to the idea of endocrines with long-distance effects, paracrines, autocrines, and intracrines appeared, with their direct effects. Then it was demonstrated that in addition to the hormoneproducing cells, which were believed to do this exclusively, hormone production is more or less general and there are cells characterized as hormone producers which were not included in the endocrine system before; their actual classification is an unsolved problem (Norman and Litwack, 1987). Insulin has not been detected only in those cells where it has not been investigated, and this is true for several other hormones. At the same time, it appears that hormone effects previously thought to be specific are wide ranging. There are cells possessing receptors which either do not require hormonal induction or we do not know why would they require this type of induction. New substances were added to the hormone group, such as neurosecretory materials, neurotransmitters, and opioids; these are close to the endocrine system and the neuronal system in their function and in their classification. However, widening our knowledge about endocrine regulation is not sufficient if it is not done profoundly, if we do not study the presence of hormones and the development of hormone-receptor relations at different levels of evolution (Pertseva, 1991). There have been many comparative endocrinological investigations related to this (Gorbman et al., 1983) but only in the past two decades have we been encouraged to take the concept of the endocrine system-which was reserved for communication in multicellular organisms-down to the single cell-level of protozoa (Csaba, 1980, 1981, 1985, 1987). These investigations show that the unicellular eukaryotes not only have the basic units of an endocrine system but a mechanism similar to such a system has also been recognized. In protozoa we probably cannot sharply isolate hormone-receptor relations from general recognition of the environment; special circumstances are required to convert this interaction into a true hormone-receptor relation. At the same time, the capacity to recognize environment in general serves the basic needs of the evolution of an endocrine system, which means an increased specialization on one hand and the fixing of the recognitive capacity of the hormone at the gene level on the other. Hormonal imprinting has an essential role in this process. The structures in the protozoan plasma membrane are ready to recognize every nearby molecule in their environment-this is their job-but they do not memorize them in the same way. There is an advantage

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

23

in memorizing molecules suitable for signaling-hormones-and this is embodied in the transition to a true receptor from a binding site, and in the development of a hormone from a signal molecule when these alterations are beneficial for the cell. The last concept should include the possibility of transmission to the second messenger system, and with this, response coupling and the possibility of transmission to following generations. Here imprinting is activated like receptor memory-as the result of the first encounter with the hormone; furthermore, it cannot be destroyed. From this it clearly follows that the development of a hormone-receptor connection is a part of the general chemoreception. This is shown by the similar structures of cilia in the Ciliata and each chemoreceptor. This suitability is probably more quantitative than qualitative. It is conceivable that the evolutionary development of neuronal learning might be also ascribable to the learning process induced by imprinting, considering that there is a retroactive interference, tolerance, and dependence (e.g., in the case of opioids) and that an amplifier effect of repetitions is also observed (Csaba er al., 1984e). From these facts and hypotheses it seems to be obvious that the presence of a hormone is essential for the development of receptors. Hormones-at least the amino acid- and polypeptide-type hormones-develop by using amino acid-type nutrient receptors, primarily the configuration, imprintability and unused being reserved for other functions to determine its suitability. Discrimination of L and D forms of amino acids shows the specificity of the unicellular eukaryotic binding site for amino acids (Darvas et al., 1987).There is a greater capacity in these organisms to differentiate amino acids and oligopeptides. In the case of imprinting of oligopeptides, protozoa and isolated cells of higher animals show similar discriminatory capacity (Kovacs er a]., 1989b),which points to conservation of conditions of affinity once they are developed. Among amino acids, there are some types which are more hormonal than others (Slominsky and Paus, 1990; Csaba er al., 1986b; Csaba and Kovacs, 1994b; Ishii, 1988). The hormonal character is demonstrated in the capacity for imprinting amino acids and in the molecules built by them. Receptors appear to be more conservative than hormones (Muggeo er ul., 1979a,b; Ginsberg er al., 1977).In some hormones there are significant changes during evolution but they do not prevent the hormone from binding to a receptor at a lower evolutionary level. At the same time, the preference that receptors of lower organisms (Csaba et al., 1980a) show for molecules at a lower level of hormone development seems to verify the conservatism of receptors with respect to the dynamic alterations of hormones. While in unicellular eukaryotes the relation of hormones and receptors is present as a part of chemoreception (but as far as we know, without

24

G. CSABA

any special advantage) from the standpoint of hormone-receptor relations, the change of binding sites is significant. At the moment when a multicellular life is developed, receptors have to be determined at the gene level because the normal regulation process is imaginable only in this way. We do not know the mechanism that is able to transfer this information-because of the advantage of receptor-hormone connection-to the gene level. However, in a specific environment containing signal molecules (Csaba et al., 1982d), it is conceivable that there is a process that selects those cells which have the power to form more and higher affinity receptors. It is also possible that a redundant mass of genes is present for all receptors and only a rearrangement is required with methylation to increase the number of receptors. Though the results of experiments applying azacytidine (Csaba and Kovacs, 1990b) refer to this, the results still have only a hypothetical value.

111. Ontogeny of Hormone Receptors

A. Maturation of Receptors and Perinatal Hormonal Imprinting

In multicellular organisms, hormone receptors are determined at the gene level independently of whether they are localized in the cell membrane in the cytosol or in the nucleus. However, the development (maturation) of receptors is parallel with the differentiation of the cells (Hubbert and Miller, 1974)and this means that the embryonic or fetal hormone receptors are not identical to the receptors of adult animals in either their binding capacity, their number, or possibly also their structure. All of these are represented by the binding capacity of the receptors. For example, insulin receptors are present on the 15th day of the intrauterine life of rats but their binding capacity is only a portion of the values measured in adults; this binding capacity increases until the 2 1st day of pregnancy (Margolis et al., 1990). Following this, there is a small decrease which is succeeded by another increase. The circumstances are not different in the case of the glucagon receptor, which reaches the binding capacity characteristic of adults also by the 21st day of pregnancy (Blazquez et al., 1976, 1987; Fujii et al., 1989). In the case of other receptors, this tendency in development is not as well expressed, but the difference between the maturing and adult receptors has been demonstrated extensively (Warren et al., 1984; Maes et al., 1983; Csaba et al., 1977; Csaba and Sudar, 1978; Menon et al., 1991).

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

25

In multicellular organisms, the receptor and its hormone develop on the basis of information from the same genome but the two members of the regulatory system are manifested in different cells. This means that the cell producing the hormone has no knowledge about the capacities of the receptor, and the cell possessing the receptor has no information about the quantity of the hormone produced by the cell which regulates it, even when the structure of receptor is shaped to the quality of the hormone. Receptors and hormones have to experience each other, and the synchronism developed in this way will ensure the normal function of an organism. This kind of experience takes place just after birth in mammals, possibly because the presence of maternal hormones would disturb the normal setting. The first encounter of a hormone with the receptor develops hormonal imprinting (Csaba, 1980, 1981, 1984, 1986b). As a result of this, the receptor terminates its maturation and reaches the binding level which is characteristic of adults. The effect of imprinting is rarely embodied in a change of affinity of receptors but it is always involved in determining the maximum number of inducible receptors. The binding capacity of the imprinted cell alters and this is demonstrated by a change in responsiveness. Imprinting takes place at an appropriate time and its effect is present for life. This means that, as in the case of unicellular organisms, the imprinting is transmitted to the daughter cells because it is evident that the majority of the cells of an adult are not identical to the imprinted cell. Imprinting is not an accidental but an inevitable phenomenon. The maturation of a receptor requires an encounter with a hormone; without this encounter, maturation does not occur. In this way, not only the structure of a receptor but the necessity of an encounter with its hormone is written into the genetic program. When hormone production is suppressed in the crucial perinatal period, for example by administration of T3 or T4 to suppress TSH production, imprinting is not developed and the reaction of the thyroid gland to TSH is pathological; it remains unmatured in adults, with characteristics of the just maturing receptors (Csaba and Nagy, 1985). At the same time, the excess of the physiologic hormone may either strengthen the normal imprinting or weaken it. In adults these effects are measurable at the receptor level or functionally. Weakening occurs, for example, when there is an excess amount of TSH in a crucial period of receptor development (Csaba and Nagy, 1976). Insulin treatment of newborns could induce strengthening or weakening of hormone binding (Fig. 16), depending on the sex of the animal. Only one dose of the hormone treatment is needed to provoke these effects when it is given at a crucial time.

26

G. CSABA

I

0

t

102

5.102 103 5.103 104 nglml insulin

FIG. 16 A single neonatal insulin treatment decreased the insulin binding capacity of erythrocytes in adult male rats in the same way that hepatic insulin receptors respond to insulin exposure during the perinatal period or during liver regeneration in adulthood. When the erythrocytes of adult rats were exposed to insulin in ,uirro, after 48 hr the erythrocyte’s insulin binding capacity was depressed to the same degree whether or not the individuals had been treated with insulin when newborn. (Reproduced from Csaba and Inczefi-Gonda, 1989b. with permission.)

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

27

B. Hormone Specificity and Overlap of Hormones in the Critical Perinatal Period

Hormones are generally members of hormone families and originate from a common ancestor, most likely by duplication and differentiation of the gene (Ohta, 1989). Within the family, they are similar to each other; minimal differences ensure specificity. The a-subunits of the gonadotropic and thyrotropic hormones of the pituitary gland are the same, and there are great similarities also in the @subunits, with differences providing the specificity (Amir et al., 1978; Azukizawa et al., 1977). The receptors of these hormones are also homologous. In the amino acid sequence of extracellular parts of follicule-stimulating hormone (FSH) and luteinizing hormone (LH) receptors, there is a 46% homology; FSH is 39% and LH is 45% analogous with the appropriate part of the TSH receptor (Nagayama et al., 1991; Vassart et ul., 1991). The transmembrane regions of FSH and L H are similar in 72% of their amino acid sequence while the similarity of the transmembrane region of TSH to FSH is 78%, and to L H is 70%. This demonstrates not only that the receptors of gonadotropic hormones resemble each other but also that the TSH receptors are only minimally different. A similar likeness has been observed among ACTH, melanocyte-stimulating hormone (MSH) and endorphins, and among oxytocin and vasopressin (Norman and Litwack, 1987), for example. Despite the relationship between hormones and receptors of the same family, the differences are large enough to result in the specific binding of the hormone by the matching receptor in the adult animal. During the critical period of receptor development this is not so. A single high dose of gonadotropic hormone administered to newborn rats (Fig. 17) is followed by a reduced reaction to TSH in the thyroid gland in adults (Csaba and Nagy, 1976). The administration of a high dose of TSH to newborns is also able to decrease the reactivity of the thyroid gland (Csaba and Nagy, 1978) and it will inhibit the response of gonads to gonadotropic hormone (Dobozy et al., I98 I , 1985). There is an overlap between vasopressin and oxytocin receptors (Csaba et ul., 1980b); they have the capacity to permanently influence each other. The effects of overlapping in imprinting are also present in binding (Fig. 18) and function (Csaba et al., 1979, 1981). That is why it is hard to explain why hormones that are actually present do not induce overlapping imprinting during the physiological maturation of receptors. Possibly a higher dose of the hormone is required for imprinting, which is embodied in both binding and function. However, the peaks of individual hormones appear at different time points and possibly this ensures the development of imprinting

G. CSABA

28

/'

:150

/

600

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(5 0

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I

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i

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N

DUU SALINE

TSH 13.5

f25

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142%

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-

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i

I

t

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TS H

50 IU

100 IU

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3 IU

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- - - / III GTH

3.39

4.1

-

TSH

4.08

f0.6

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4.63

fop

-

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I

FIG. 17 The effect of hormonal imprinting by gonadotropin (GTH) or thyrotropin of newborn rats on the blood thyroxine (T4) level after hormonal treatment in adults compared with controls (1-3). A high dose of gonadotropin given to newborn rats suppresses the effect of TSH in adults (6). Similar, but more moderate effects are produced by neonatal treatment with TSH (4). (Reproduced from Csaba and Nagy, 1976, with permission.)

with the adequate hormone at a suitable time (Klett and Schonberg, 1986; Bidlingmayer et al., 1986). During the critical developmental period, a related (nonself) hormone that is ready to bind to a receptor and in this way disturb the binding of the appropriate hormone, is not able to modify the receptor. In these instances the receptor does not react more to the wrong hormone than it does to the correct one. When false imprinting develops in parallel with the perturbation of the membrane during the perinatal period, this results in an advantage for the foreign imprintor (Csaba and Nagy, 1986). This

29

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

ClTSH

TSH/FSH

FSHlTSH

FIG. 18 FSH and TSH similarly imprint the Chinese hamster ovary cell line for FSH. Labeled FSH binding of untreated (C) TSH or FSH-treated cells 48 hr after the first treatment ( p <0.01). (Reproduced from Csaba et al., 19842 with permission.)

means that though the structure of a receptor is fixed at the gene level, some epigenetic alterations (Campbell and Perkins, 1988) can take place. The plasticity or absence of discrimination for receptors in the critical perinatal period is possibly an ontogenetic projection of the nonspecificity of lower levels of phylogeny (Ishii, 1988; Salesse et al., 1991). For example, adult male frogs can be induced to ejaculate not only by gonadotropin but also by thyrotropin, and TSH has a stronger effect than FSH (Csaba et al., 1982e, 1983b). C. Optimal Time of Hormonal Imprinting

Hormonal imprinting takes place perinatally but over a relatively wide period. The imprintability possibly depends on the development of the receptor-hormone axis on the basis of a built-in program. Rats were successfully imprinted with insulin only in the first 24 hr after birth. This

30

G. CSABA

is the time when the binding capacity of insulin receptor decreases after the peak of the 21st day. In chicken embryos, imprinting was not developed on the 8th day but it was present on the 12th day (Nagy and Csaba, 1980). It is known that the hypothalamus-hypophysis-thyroid axis starts to develop between these two time points (Mess and Straznicky, 1970). Before this, the receptor is resistant; following the appearance of the axis, it turns to become imprintable. It even seems to be possible that imprinting might not take place on the receptors of different organs at the same time. The maturation of receptors differs according to the organ. Receptors of thyroid hormone appear first in the brain and then in the liver (in both cases still in the embryonic life) while the receptors for thyroid hormone in other tissues mature only in the perinatal period (Fisher and Polk, 1989). Probably their appearance depends on the presence of a functioning receptor: the brain requires a working thyroxine receptor in the embryonic stage but other organs require this activity later. The quantity of the imprinting inducer hormone and the maturation of its receptor probably are coordinated; for example, it has been shown that immunoreactive ACTH and corticosterone and the number of ACTH receptors correlate highly around the time of birth (Chatelain ef al., 1989). In this way physiological imprinting takes place with a defined amount of hormone and in a defined time (Fig. 19), ensuring the normal maturation of receptors. D. Pathological Imprinting

It became clear after a description of the harmful effects of gonadotropins on TSH receptors that normal imprinting developed only when the appropriate hormone was present at the proper time and the amount of the hormone was the one required. A hormone that is appropriate and that can be bound also to another receptor, whatever its physiological structure, is able to change the maturation of the receptor, activating a nonphysiological phenomenon. This may occur when hormones overlap in the perinatal period (Csaba, 1984, 1986b). It is not impossible that other materials, such as analogs with hormonal functions or molecules with similar structures but different functions, all having the capacity of binding to the receptor, occur in the environment of the developing receptors (Fig. 19). The chemical industry produces many kinds of hormone analogs that have agonist or antagonist effects after they bind to the receptor. It is typical for each that they have a structure more or less divergent from the physiological structure of the hormone. This results in abnormal imprinting in the crucial period of receptor development; the binding capacity, primarily the number of receptors, is changed. A characteristic exam-

31

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

Normal

Appropriate

-hormone \time

J

quantity

Normal development of the receptors and response

Abnormal

Appropriate hormone

1

/

Inappropriate time, quantity Inappropriate molecules

Abnormal receptor and response

FIG. 19 Possibilities of hormonal imprinting.

ple is treatment with the fluorinated glucocorticoid, dexamethasone, in newborns (Fig. 20). The result is a significant decrease in the binding capacity of glucocorticoid receptors in the thymus of adults (Inczefi-Gonda and Csaba, 1985). The steroid receptors belong to a big superfamily with receptors for thyroxine, vitamin D, retinoic acid, and aromatic hydrocarbons. In adults the relationship of receptors does not cause any problems but it does during a given critical period of receptor development. The binding of the appropriate hormone is inhibited by other molecules numerically superior to it and these molecules can also be bound to the receptor. On the other hand, these molecules themselves induce the development of receptors in a pathological direction. The basis of the pathological shift is the overlapping of receptors belonging to this superfamily. Following the treatment of newborn rats with a single dose of allylestrenol, there is a large-scale decrease in estrogen receptors in the adult uterus (Csaba et al., 1986a). There is also a decrease in the number of glycocorticoid receptors (Inczefi-Gonda et al., 1986). Perinatal treatment with the synthetic estrogen, diethylstilbestrol (DES), results in a decrease in the number of androgenic and estrogenic receptors (Turner et al., 1989), and neonatal estrogen treatment (imprinting) renders the uterine epithelium independent of hormonal influences for life (Gibson et al., 1991). Prenatal androgenization completely abolishes estrogen-induced hypertrophy of uterine epithelium (Arriaza et al., 1989). These experiments demonstrate that the synthetic hormone analog damages its own receptor by defective imprinting and develops defective imprinting by overlapping the glycocorticoid and androgen receptors with different structures. Nonphysiological molecules executing the imprinting have the ability to induce different receptors at the same time; this induction depends on the actual stage of receptor development.

G. CSABA

32

k,

0.3

treated

control

-00- 4

-++-

j-g

-0-6

0.2

0.1

1000

2000

3000

Dexarnethasone Bound (fmoVmg protein)

FIG. 20 Scatchard plots of the data from control and neonatal rats treated with dexamethasone. The data points were calculated from displacement of [3H]dexamethasone. KD and B,,, values were obtained by linear regression analysis. Control female groups, KD = 8.8 x lO-'M; B,,, = 2278 fmol/mg protein (corr. coeff. 0.9942). Treated female groups, K O = 6.25 X lo-' M; B,,, = 2107 fmolhg protein (corr. coeff. 0.9888). Control male groups, KD = 10 x 10-'M; B,,, = 3044 fmollmg protein (corr. coeff. 0.9912). Treated male groups, KO = 11.8 X 10-'M; B,,, = 985 fmol/mg protein (corr. coeff. 0.9848). The results unequivocally demonstrate a decrease in glycocorticoid receptor numbers after neonatal dexamethasone treatment. (Reproduced from Csaba and Inczefi-Gonda, 1990, with permission.)

Imprinting of Steroid Receptors Induced by Aromatic Hydrocarbons The aromatic hydrocarbons, with their steroid-like structure, have their own receptors, named Ah receptors. These resemble the receptors for glycocorticoid hormones (Mason et al., 1988).Probably this is why during the critical period of receptor maturation the aromatic hydrocarbons can overlap on the steroid receptors and are able to evoke abnormal imprinting. A single dose of benzpyrene given to newborn rats decreased the number of glycocorticoid receptors in the adult thymus (Csaba and Inczefi-Gonda, 1984). This overlap is wider, as the number of estrogen receptors also decreases (Csaba and Inczefi-Gonda, 1993). This means that benzpyrene is able to effect and wrongly induce the maturation not only of receptors similar to Ah receptors but also of steroid receptors which are slightly different. The other aromatic hydrocarbon, dioxin (TCDD), has similar effects (Csaba et al., 1991b).

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

33

The period critical for benzpyrene is very long lasting. Treatment of 15- (Csaba and Inczefi-Gonda, 1992) and 19- (Csaba et al., 1991a) dayold rat embryos induced a durable pathologic form of imprinting, as did treatment at 3 and 6 weeks after birth (Csaba and Inczefi-Gonda, 1984; Csaba et al., 1991a). In the latter case, there were differences between males and females; the sensitivity of males was lower. These results verify that it is not the biological effect that is essential for imprinting but the structure of the molecule. Molecules with physiologically totally different effects, even when they are nonphysiological molecules, are able to develop imprinting in a critical period of receptor maturation if they can link to the receptor. This means that this kind of openness of the system leads to the normal development of receptors when the circumstances are also normal. However, this openness also presents the possibility of foreign intervention, which may distort the receptor. This directs our attention to those frequently observed cases in which a wellregulated biological mechanism might become dangerous in an abiological environment and activate a pathological phenomenon instead of the normal one. E. Hormonal Imprinting in the Developing Cells of Adult Organisms

By the end of the ontogenetic processes, the development of the organism is completed but there are continuously developing cells in the mature organism. These developing cells might be subjected (or are subjected) to the effects of imprinting like the cells of the developing organism. The wide period observe for the effects of benzpyrene has already shown that imprinting takes place in cells of cytogenic organs (thymus) or in the continuously renewing cells of the noncytogenic organs (uterus) of adults. Continous imprinting was demonstrated in experiments in which hormone analogs were administered in puberty (Csaba et al., 1991a; Csaba and Inczefi-Gonda, 1990). Imprinting is not an age-dependent but a stagedependent phenomenon and the characteristic phase of development may be present at every age (Fig. 21). Imprinting occurs in all organs capable of regeneration (Fig. 22), as shown by successful imprinting of cells in regenerating liver (Csaba et af., 1989b). It is clear then that around the perinatal period, imprinting sets the cells and their daughter cells to a certain level of receptor production and capacity. In the adult organism, there is the possibility of changing these settings when the differentiation of new cells takes place. At this time, the imprinting of the newly developed cells can be changed and the receptors in their offspring will reflect the new settings.

G. CSABA

34

':h Bound (fmollmg protein)

FIG. 21 Scatchard plots of the data from control and dexamethasone-treated (at 4 weeks) rats. The data points were calculated from displacement of ['H]dexamethasone by dexamethasone. K D and B,, values were calculated by linear regression analysis. Control female groups, KD = 5.2 x M ; B,,, = 1017 fmol/mg (corr. coeff. 0.9800). Treated female M ; B,,, = 754 f m o l h g protein (corr. coeff. 0.9525). Control groups, KD = 3.88 x male groups, K D = 7.3 x W 8 M ;B,,, = 2785 fmol/mg protein (corr. coeff. 0.9947). Treated male groups, K D = 5.5 x IO-'M; B,,, = 2022 fmol/mg protein (corr. coeff. 0.9929). The results demonstrate the effect of late dexamethasone treatment on the number of glycocorticoid receptors in adults (a decrease in both sexes). (Reproduced from Csaba and InczefiGonda, 1990, with permission.)

F. Cell-to-Cell Transmission of Hormonal Imprinting

On the basis of recent genetic knowledge, it is hard to imagine that alterations at a protein-receptor level could be reversely coded into the gene level. However, this would be required for the transmission of perinatal hormonal imprinting to daughter cells, if we suppose a gene-level fixation. This would be the situation also when imprinting develops in a stem cell of a cytogenic organ of a mature organism, and many generations of offspring show the effect of the imprinting. Following the description of gap junctions, we could understand how information could flow from cell to cell with the help of direct contact. This was verified also in the case of receptor-transmitted information, as referred to earlier (Lawrence ef al., 1978; Murray and Fletcher, 1984;

35

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

=-r.

' ,-('

Insulin Q Insulin d

Control Q - Control d

.---A

0-

Insulin ng/ml FIG. 22 Insulin treatment following partial hepatectomy caused a long-lasting change in the binding capacity of hepatic insulin receptors. Insulin binding increased in females and decreased in males in the insulin-treated animals 14 days after the operation (during regeneration). The tendency of changes is very similar to those caused by neonatal insulin treatment. The figure shows the binding '*'[I]insulin in the presence of different concentrations of nonlabeled insulin. (Reproduced from Csaba el a / ., 1989b, with permission.)

Fletcher and Greenan, 1985). Information passes from cell to cell and is transmitted by the hormone. In the case of hormonal imprinting, this was also observed when the information received by imprinting was transmitted into daughter cells (Csaba et al., 1987b). This would explain how the umpteenth generation of cells is able to produce a proper response to imprinting without the necessity of alterations in genetic material. Of course, this does not exclude the development of similar genetic alterations, for example, for emerging immune clones, but these have not been verified so far. G. Ontogenetic Conclusions

Not everything is determined with the same rigor by the developmental program. The seriousness of different physiological processes is not the same from the viewpoint of the survival of organisms. That is why one

36

G. CSABA

can assume that the housekeeping genes work according to more stringent criteria than those genes that are used to build connections with the environment or help adaptation. This is why there are totally closed, more or less closed, and open systems during ontogenesis. The accidental influence of the environment on these systems is different. Under physiological conditions, a totally closed system makes the intervention of the environment impossible, serving a survival function, and only drastic effects can disturb the program and cause misdevelopment. However, the program for an open system definitely requires the influence of the environment and the genetic program can be completed only this way (Csaba, 1991). This shows that genetic adjustment (imprinting) is a part of the program. Of course the program is not always inducible by the environment, but it is affected in the critical periods and the determination of these periods is also a part of the program. The best example of an open system is immunobiological adjustment, when the differentiation of self and nonself has to be done in the framework of this process. This means that an aggressive response is programmed in the cellular network of the immune system, but it develops only when the self and nonself are distinguished and all responses which would be aggressive against the self are inhibited. Otherwise, the immunobiological program would be a suicidal program. The situation is very much the same in the case of the adjustment of sexual behavior. The hypothalamus has the capability of adjusting for either sex, but male and female sexual behavior are determined by the quality and quantity of hormone which is actually present in a critical period. If this adjustment of the hypothalamus is not completed, the sex becomes uncertain; if the adjustment is defective, then the sexual behavior is the opposite of the chromosomal and gonadal sex. The endocrine system is a similar open system. Though the receptor and hormone are determined by the same genome, the receptor has to be adjusted to the quality and quantity of hormone during a critical period of development. This is a fine tuning of the genetically programmed system and the fitting of the transmitter and the receiver to each other. The method of adjustment is hormonal imprinting. The receptors of higher rank organisms are probably the result and a “copy” of the phylogenetically ancestral type of imprinting observed in unicellular eukaryotes. The more or less restricted survival of the totally open system is not simple atavism but exists because of a demand. It is needed to balance the hormone-receptor system during the whole life of the organism, but the sensitivity of the critical periods presents the possibility of defective (pathological) shifts. Imprintability is not age-dependent but depends on the phase of development, and in some types of cells this means imprintability during the entire

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

37

life of the cell. In contrast with imprinting during the critical period of perinatal life, imprintability in adults raises difficult medical problems in our age, which is characterized by intensive use of chemicals (Csaba, 1991; Brindak et ul., 1992; Sergeyev and Shimanovsky, 1990).

IV. Conclusions Though receptors, hormones, and second messenger systems have been demonstrated in protozoa, we cannot speak about an endocrine system in the sense in which it exists in higher organisms because the protozoan system does not involve regulation among the cells composing the same organism. At the same time, the protozoan system provides a basis for development of an endocrine system at a higher level, and the basis of the endocrine systems of higher organisms can be attributed to the unicellular organisms. Studies of evolution show that there are no unique signal molecules, only those which are selected to be hormones and which are more suitable than others to perform the hormone function. It is also probable that there are no unique hormone receptors, only those structures which are able to recognize the environment. These structures develop into receptors following an encounter with a molecule suitable for signal transmission. Hormonal imprinting occurs at the first encounter of the prospective hormone and the prospective receptor. This results in more specific receptors and more numerous receptors being produced by the cell. The receptors are transmitted to the offspring generations. The hormonal imprinting that occurs at the beginning of the evolutionary sequence retains its role in the higher organisms and influences the maturation of receptors around the period of birth. The imprinting of higher organisms (mammals) is also transmitted to the daughter cells and this ensures the life-long effect of imprinting. Imprinting is necessary to complete the program of receptor formation. While an open program makes it possible for molecules that are similar but not identical with the hormone to affect the receptor, this program deforms the latter’s development. This is how defective imprinting occurs and its effect is as life-long as physiological imprinting. Imprinting is not age-dependent but depends on the stage of development; it can be developed also in immature, differentiating cells in adults. The recognition system of cells is universal but there are several ways it can be expressed. In each method there are receptors and ligands. Examples of this kind of expression are cell-cell recognition, self-nonself recognition, cell and environment recognition, receptor-hormone recogni-

38

G. CSABA

tion, and so on. Each member of the system works in coordination with each other, and the more developed systems use the components and mechanisms of the less developed ones. Acknowledgments The author is grateful for the permission to use figures and tables from Faculty Press, Oxford, and the Publishing House of the Hungarian Academy of Sciences. The experiments done by the author and his co-workers were supported by National Research Fund (OTKA) grant No. 1067 and by the Scientific Research Council, Ministry of Welfare (grant T-2611, Hungary.

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the hormone binding capacity of thymocytic receptors in adulthood. Acra Physiol. Hung. 75, 195-199.

Csaba, G., and Inczefi-Gonda, A. (1992). Benzpyrene exposure at 15 days of prenatal life reduces the binding capacity of thymic glucocorticoid receptors in adulthood. Gen. Pharmacol. 23, 123-124. Csaba, G., and Inczefi-Gonda. A. (1993). Uterus estrogen receptors' binding capacity is reduced in rat if exposed by benzpyrene neonatally. J . Deuelopm. Physiol. 19, 217-219. Csaba. G., and KBhidai, L. (1986). Modelling the insulin receptor in the Tetrahymena. Timedependence of receptor formation, down-regulation and imprinting. Acra Protozool. 25, 33 1-338. Csaba, G . , and KBhidai, L. (1989). Interrelationship of hormone concentration, hormonal imprinting and receptor down-regulation in Tetrahymena. Acta Protozool. 28, 183- 186. Csaba, G . .and KovLs, P. (1986). Studies into disturbing receptor "memory" in a unicellular (Tetrahymena) model system: Changes in the imprinting potential on exposure to combinations of related and unrelated hormones. Exp. Cell Biol. 54, 333-337. Csaba, G., and Koviics, P. (1987). Transmission of hormonal imprinting in Tetrahyrnena cultures by intercellular communication. Z. Naturforsch. C: Biosci. 42C, 932-934. Csaba, G., and KovBcs, P. (1990a). Influence of imprinting with A and B chains of insulin on binding and functional changes in Tetrahymena. Biosci. Rep. 10, 431-436. Csaba, G., and Kovacs, P. (1990b). Impact of 5-azacytidine on insulin binding and insulininduced receptor formation in Tetrahymena. Biochem. Biophys. Res. Commun. 168, 709-713. Csaba, G., and KovBcs, P. (1994a). Effect of hormones and hormone induced imprinting on the serotonin level of Tetrahymena. lmmunocytochemical studies. Microbios (in press). Csaba, G., and KovBcs, P. (1994b). Role of proline in the imprinting developed by dipeptides-in Terrahymena. Possible role in hormone evolution. Experientia 50, 107-109. Csaba, G., and Lantos, T. (1973). Effect of hormones on protozoa. Studies on the phagocytotic effect of histamine, 5-hydroxytryptamine and indoleacetic acid in Tetrahymena p.vriformis. Cytobiologie I , 361-365. Csaba, G., and Lantos, T. (1975a). Effect of insulin on the glucose uptake of Protozoa. Experientia 31, 1097-1098. Csaba, G . . and Lantos, T. (1975b). Effect of amino acid and polypeptide hormones on the phygocytosis of Tetrahymena pyriformis. Acta Protozool. 37, 409-413. Csaba, G., and Lantos, T. (1975~).Specificity of hormone receptors in Tetrahymena. Experiments with serotonin and histamine antagonists. Cytobiologie 11, 44-49. Csaba, G., and Lantos, T. (1976). Effect of epinephrine on glucose metabolism in Tetrahymena. Endokrinologie 68, 239-240. Csaba, G., and Nagy, S. U. (1976). Plasticity of the hormone receptors and possibility of their deformation in neonatal age. Experientia 32, 651. Csaba, G., and Nagy, S. U . (1978). The binding of '"I-TSH to thyroid cell receptors previously deformed (in neonatal age) by gonadotropin treatment. Biol. Neonate 34, 275271. Csaba, G., and Nagy, S. U. (1985). Influence of the neonatal suppression of TSH production (neonatal hyperthyroidism) on response to TSH in adulthood. J . Endocrinol. Inuest. 8, 557. Csaba, G., and Nagy, S. U. (1986). Can neonatal treatment with a related hormone adapt the receptor for itself? Acta Physiol. Hung. 67, 65-69. Csaba, G., and Nagy, S. U. (1987). Presence (HPL. prostaglandin) and absence (triiodothyronine, thyroxine) of hormones in Tetrahymena: Experimental facts and open questions. Acta Physiol. Hung. 70, 105-1 10. Csaba, G., and NCmeth, G. (1980). Effect of hormones and their precursors on protozoa-the selective responsiveness of Tetrahymena. Comp. Biochem. Physiol. B 65B, 387-390.

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Csaba. G., and Sudar, F. (1978). Differentiation dependent alterations in lymphocytic triiodothyronine reception. Horm. Metab. Res. 10, 455-456. Csaba, G., and Ubornyak. L. (1981). Effects of hormones on the RNA-synthesis of Tetrahvmenu pyriformis. C o m p . Biochem. Physiol. C . 68C, 251-253. Csaba. G.. Nagy, S. U., and Lantos, T. (1976). Are biogenic amines acting on Tetrahymena through a cyclic AMP mechanism? Acta Biol. M e d . G e r . 35, 259-261. Csaba, G . . Sudar. F.. and Dobozy, 0. (1977). Triiodothyronine receptors in lymphocytes of newborn and adult rats. Horrn. Metab. R e s . 9, 499-501. Csaba, G.. Nagy, S . U., and Lantos, T. (1978). Cyclic AMP and its functional relationships in Tetrahvrnena: A comparison between phagocytosis and glucose uptake. Acta Biol. M a d . G e r . 37, 505-507. and Kaizer. G. (1979). Study of FSH-TSH functional overlap by Csaba, G.. Dobozy. 0.. cockerel testicle test. Horrn. Metah. R e s . 11, 689-692. Csaba, G.. Bierbauer. J., and FehCr. S. (1980a). Influence of melatonin and its precursors on the pigment cells of Planaria (Dugesia lrrgubris). Comp. Biochem. Physiol. C 67C, 207-209. Csaba. G.. Ronai, A., Laszlo, V., Darvas, Zs.. and Berzetei, I. (1980b). Amplification of hormone receptors by neonatal oxytocin and vasopressin treatment. Horm. M e t a b . R e s . U ,28-3 I . Csaba, G.. Dobozy. 0.. and Kaizer. G. (1981). FSH-TSH functional overlap in cockerel testicle. Durable amplification of the hormone receptors by treatment at hatching. Horm. M e t a b . R e s . 13, 177-179. Csaba, G . , Nemeth, G.. and Vargha, P. (1982a). Receptor memory in the unicellular Tetrahymenu. Impact of treatment with analogous hormones. Acfa Biol. Acad. Sci. Hung. 33, 425-427. Csaba, G.. Nemeth, G.. and Vargha P. (1982b). “Memory” of first interaction with physiological or biologically active foreign molecules (benzpyrene. gibberelline) in a unicellular (Tetrahymena) model system. Z . Naturforsch. C : Biosci. 37C, 1042-1044. Csaba. G., Nemeth, G., and Vargha, P. (1982~).Development and persistence of receptor “memory” in a unicellular model system. Exp. Cell B i d . 50, 291-294. Csaba, G., Nemeth, G., Juvancz, I . . and Vargha P. (1982d). Involvement of selection and amplification mechanisms in hormone receptor development in a unicellular model system. BioSysterns 15, 59-63. Csaba. G., Dobozy. O., and Deak. B. M.(l982e). HCG-TSH overlap and induction of GalliMannini reaction with TSH in adult male frogs. Horm. Metab. R e s . 14, 614-618. Csaba, G., Nemeth, G., and Vargha, P. (1983a). Attempt to disturb receptor memory in a unicellular (Tetrahymena) model system. Acta Physiol. Hung. 61, 131-136. and Deak, B. M. (1983b). Interaction of thyrotropin (TSH) and Csaba, G., Dobozy, 0.. gonadotropins in the function of genital organs. I. Investigations in the frog. Acta Physiol. Hung. 61, 137-140. Csaba, G., Darvas. Zs., Laszlo. V., and Vargha, P. (1984a). Influence of prolonged life span on receptor memory in a unicellular organism, Tetrahymena. Exp. Cell B i d . 52,211-216. Csaba, G., Darvas. Zs., Swydan, R., and Nagy. S. U. (1984b). Presence ofdigoxin detectable by radioimmunoassay in Tefrahyrnenu.Experienfia 41, 392-393. Csaba, G., Gruszczynska, M., Madarasz, B., and Sudar. F. (1984~).Coated pits and coated vesicles in unicellulars (Tetrahymena. Crithidia) and in Hydra. Actu Morphol. Acad. Sci. Hung. 32, 181-186. Csaba, G.. Kovacs, P.. and Inczefi-Gonda. A. (1984d). Insulin binding sites induced in the Tetrahymena by rat liver receptor antibody. Z. Naturforsch. C: Biosci. 39C, 183-185. Csaba, G., NCmeth, G., and Vargha, P. (1984e). Receptor memory in Tetrahymena: Does it satisfy the general criteria of memory? Exp. Cell B i d . 52, 320-325. Csaba, G.. Torok. O., and Kovacs. P. (1984f). Hormonal imprinting in cell culture 11.

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Growth Factor-Induced Cell Migration: Biology and Methods of Analysis Marianne Manske and Ernest0 G.Bade Arbeitsgruppe Zellbiologie-Tumorbiologie, Fakultat fur Biologie, Universitat Konstanz, 78434 Konstanz, Germany

I. Introduction Cell migrations are at the basis of metazoan life. Starting with the migration of the sperm and ending with the invasive behavior of malignant tumor cells that finally kill their host, cell translocations over considerable distances are an essential part of the dynamics of higher eukaryotes. The implantation and development of the embryo, the development of the nervous system and the gonads as well as metamorphosis and, in the adult organism, the renewal of surface epithelia, all require a precise control of cell migration at multiple stages (Trinkaus, 1984; Bade and Nitzgen, 1985). These precise and cell-specific controls are altered or lost when tumor cells become invasive and metastatic. Much of our current information on cell migration has been obtained from in uitro studies with embryonic and tumor cells. Malignant tumors have provided “motile” and “immortalized” cells, and have also been the source of proteins that were initially isolated as “growth” (i.e., proliferation-stimulating) factors. Surprisingly, however, their role as migration modulators was recognized only very gradually. The first growth factor known, nerve growth factor (NGF), discovered as a secreted product of a malignant tumor (Levi-Montalcini and Hamburger, 19511, and the transforming growth factors (TGFs), first detected in the conditioned media of cultured malignant tumor cells (De Larco and Todaro, 1978) are now known to have multiple functions as modulators of cell proliferation and migration. The concept of angiogenesis as a crucial step in tumor development (for review, see Folkman and Klagsburn, 1987) stimulated the search for and analysis of factors that modulate the proliferation and migration of endothelial cells. Furthermore, in addition to the polypeptides currently considered as bona j d e or classic growth factors, the lymphoInrernariunal Reuiew of Cyrology. Vol. 155

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kines (interleukins, cytokines), which are essential modulators of the immune system, also modulate cell migration. In this chapter however, both lymphokines/interleukins and the hematopoietic growth factors (colonystimulating factors CSFs) are discussed only in connection with their role in the migration control of nonhematopoietic cells. The action of growth factors was early found to be paracrine (Sporn and Todaro, 1980) in nature. In some cases, however, growth factors act autocrinely [e.g., platelet-derived growth factor (PDGF) and TGF-a; yuxtacrinely (TGF-a; Anklesaria et al., 1990) or intracrinely (PDGF)]. Furthermore, since some factors [insulinlike growth factor-] (IGF-1) and the TGF-P family; for review, see Massague, 1990al can reach their target cells also via the blood circulation, a clear differentiation from polypeptide hormones is difficult. In a generalized way, growth factors might thus be defined as polypeptide modulators of cell proliferation, differentiation, and migration, produced “diffusely” by various cell types, as opposed to hormones, which are produced by highly specialized endocrine glands. The induction of cell proliferation by growth factors was early recognized to proceed with a defined sequence of changes in gene expression (Greenberg and Ziff, 1984; Bravo et al., 1985). In contrast, the functions required for migration are only starting to become unraveled. It may be assumed, however (Bade and Feindler, 1988), that the induction of migratory behavior by growth factors requires the expression or modulation of ECM components and of the corresponding cell surface receptors, as well as of proteases and their specific inhibitors, and that these changes must occur in a specific sequence. Further complexity is likely, as in some cases the induction of a migratory phenotype was found to depend on the interaction of two growth factors (McKinnon et ul., 1991; Sato et al., 1991). Whether a similarly sequential action of factors is a generalized phenomenon is not clear at this time. A few definitions follow. Cell migration has often been also referred to as “cell motility,” but in this chapter “cell migration” will be used (Bade and Nitzgen, 1985; Bade and Feindler, 1988) to specifically indicate a cell translocation over a clearly measurable distance. In contrast, cell motility will be used to refer to cellular movements that do not result in translocation of the affected cell. With these restrictions, extensions and retractions of cell segments, or the movement of highly specialized cell appendices, for example, cilia, will thus be referred to as cell motility. Cell migration might then, by analogy, be envisioned as “jogging,” while motility might find its everyday equivalent in “body-building.” Random migratory reactions are generally described as chemokinesis, to be differentiated from migrations toward increasing concentrations of a soluble factor (chemotaxis), or along a concentration gradient of a substratum component (haptotaxis; Carter, 1967).

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For a systematic analysis, cell migration processes may be conveniently divided into three phases (Bade and Nitzgen, 1985):initiation or induction, the translocation process itself, and termination or inhibition. The most efficient controls may be expected to be exerted mainly at the initiation and termination phases (Bade and Nitzgen, 1985), with events during the actual translocation determining mostly the speed and direction of migration. Initiation requires sensitive cells, one or more factors that induce the migratory behavior, and permissive conditions in the immediate cellular environment. The results discussed in this review clearly emphasize growth factors as migration inducers. The actual translocation process has been studied mostly in connection with the dynamics of the actin cytoskeleton and by using spontaneously migrating cells. The composition of the extracellular matrix (ECM) encountered by neural crest cells guides and may even help determine the end of the process (Rovasio et a / . , 1983; Le Douarin, 1984). However, the specific mechanisms that inhibit cell migations are still largely unknown, even though some growth factors have been found that are able to prevent migration (Section 111, C and H). As indicated, ECM composition, but especially cell-cell interactions, are expected to play an essential role in this step through contact inhibition. This concept, developed by Michael Abercrombie (Abercrombie and Harkness, 1951; Abercrombie et al., 1970) in the course of his studies with migratory mesenchymal cells, has become a basic principle of vertebrate eukaryotic cell dynamics (Abercrombie, 1982). Cell surface proteins such as E-cadherin/uvomorulin (Nagafuchi et al., 1987; Takeichi, 1990, 1991; Chen and Obrink, 1991), initially shown to confer epithelial characteristics on fibroblastoid (migratory; see later discussion) cells (Nagafuchi et al., 1987), and later (Behrens et al., 1991; Chen and Obrink, 1991) found to decrease the invasive capacity of some tumor cells, may be assumed to play an essential role in this crucial step. More recently, cell surface proteins with presumably similar functions have been directly characterized in connection with the search for tumor supressors (Hedrick et al., 1992). Migratory reactions are cell- and tissue-specific. Epithelial cells may migrate either as sheets or as individual cells, while most other cells tend to migrate as single entities. Both the cell type and the substratum (ECM) determine if migration will be superficial (two-dimensional) as in conventional cell culture systems, or if it will be invasive. With the exception of the renewal or repair of surface epithelia, in metazoan organisms cell migrations are usually invasive (Bade and Nitzgen, 1985).The gene expression pattern of migratory cells may be expected to reflect these differences. Individually migrating cells usually adopt a fibroblastoid (=migratory or mesenchymal) morphology, with a relatively broad front (leading edge) and a thin cytoplasmic extension at the trailing end (Figs. lB, 3, and 6A,C).

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This morphological change (epithelial-mesenchymal transition; Hay, 1991) becomes accentuated when cells migrate invasively, for example, suspended in a collagen matrix (Hay, 1991). The migration of single cells requires an early interruption of cell-cell and cell-ECM junctions, which must be followed by the establishment of new cell-ECM interactions. At the end of the translocation of normal cells, stable cell-cell and cell-ECM interactions are (re)established and consolidate the tissue or organ structure. This sequence of events may be common to most singly migrating cells. In contrast, the migration of epithelial sheets requires in addition the maintenance of specific cell-cell junctions and mechanisms that transmit both the initiation and termination signals among all cells involved. These mechanisms remain to be elucidated, but cell-cell (E-cadherin/uvomorulin) and cell-ECM (integrins) receptors and various growth factors may be expected to be involved in these complex intercellular signaling processes. Throughout a migratory reaction, modification and degradation of ECM components and of surface receptors presumably alternates with their resynthesis. The required enzymes and their specific inhibitors must be synthesized, localized, activated, and inhibited in a highly coordinated fashion. Growth factors with the capacity of inducing migratory behavior have been shown to induce-although not always in context with a migratory behavior-the expression of ECM components (Hay, 1991; Massague, 1990b), as well as of a variety of proteolytic enzymes and their specific inhibitors (Dan@et al., 1985; Mignatti et ul., 1986; Matrisian, 1990). The (proteo)lytic activity required for a migratory and invasive behavior must be localized to the cell surface (Saksela and Rifkin, 1988; Laiho and Keski-Oja, 1989), and presumably close to the leading edge of the cell during the early invasive phase. Cell surface localization of secreted proteins may be achieved by polarized secretion and by binding to specific cell surface receptors. Several proteases have been found to become functionally localized to the cell surface through specific receptors (Saksela and Rifkin, 1988; Laiho and Keski-Oja, 1989; Kane and Gottesman, 1990). The receptor for the serine protease, urokinase-type plasminogen activator (uPA) localizes the activator to the cell surface, but even a complex devoid of proteolytic activity can stimulate migation (Odekon et ul., 1992). Our current knowledge of cell migration and its regulation has been obtained mainly through the analysis of cell culture systems. Accordingly, a brief review of a number of commonly used systems is included (Section 11). Since some systems might have been involuntarily overlooked, the authors would appreciate comments on these omissions. Most of the methods described allow some form of quantitative evaluation, but when comparing the effects of different agents or different

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systems, in addition to determination of the migration velocity, two parameters have proven useful (Bade and Feindler, 1988): ( 1 ) migration frequency (% of migratory cells in the population), and (2) migration efficiency (maximal/total distance covered by the cells). In particular, migration frequency is a good indicator of the biochemical capability of a cell system.

II. Analysis of Cell Migration A. Direct Observation and Time-Lapse Recording

Most of the current experimental work on cell migration is based on a conceptional framework developed on the basis of simple microscopy (Abercrombie et ul., 1970), photographic documentation (Fig. 1) or by time-lapse analysis (Abercrombie, 1982). Direct observation resulted not only in the discovery of the phenomenon of contact inhibition of movement (Abercrombie, 1982), a term inappropriately extended to the inhibition of cell proliferation observed in confluent cell cultures (Trinkaus, 1984), but also in essential information on the epidermal growth factor (EGF)induced motility of glia cells (Brunk et uf., 1976) and the collagenstimulated migration of NBT-I1 bladder carcinoma cells (Tchao, 1982). It should not be overlooked, however, that continuous microscopic controls of cells over extended periods requires specialized equipment to guarantee constant culture conditions. Time-lapse analysis, on the other hand, also suffers from the limitation of the small number of cells that can be analyzed simultaneously and might, therefore, be involuntarily selective. In addition, because large numbers of determinations are necessary to obtain reliable information about the behavior of whole cell populations, alternative methods are required for a correlation with biochemical data. The method is also not ideally suited for the quantitative analysis of tissue cell chemotaxis. Thus, a number of methods have been developed to overcome some of these limitations, but microscopy will remain the primary approach and essential control for the more complex methods of analysis. B. Boyden (Chemotaxisl Chamber Assays

The extensively used filter membrane assays, as originally introduced by Boyden for the analysis of leukocyte chemotaxis (Boyden, 19621, are based on a chamber of two medium-filled compartments separated by a microporous membrane (Figs. 2 and 3). A number of different Boyden chamber devices are available commercially (Neuro Probe Inc., Cabin

FIG. 1 Epithelial-mesenchymal transition of rat liver epithelial cells, induced by EGF. Morphology of stationary (epithelial) cells (A) and fibroblastoid morphology of migratory (B) cells cultured in serum-free medium supplemented with insulin (A) or with insulin

and EGF (B). Phase contrast. Bar

=

60 Nm.

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n

rr

55

n

I T

control medium

chemoattractant

n

i

CHEMOTAXIS

rT n

i

rr

CHEMOKINESIS

FIG. 2 Principle of the Boyden chamber assay. A detailed description of the method is given in the text.

John, MD). Generally cells are placed in the upper compartment and are allowed to migrate through the pores into the lower chamber, which typically contains the chemotactic agent. After an appropriate incubation time, the filters are fixed and stained, and the number of cells that have migrated to the lower side of the membrane is determined microscopically (Cates et ul., 1978). Chemotactic reactions can be easily distinguished from chemokinesis by addition of equimolar concentrations of the agent on both sides of the filter (checkerboard analysis). An interesting modification of the assay is obtained by coating the microporous membrane substratum with ECM components. Collagen IV (Thompson et al., 1991) or EHS-tumor extracts (Matrigel) (Hendrix et ul., 1987; Thompson et al., 1991) have been used for such approaches. The membrane filter assay can thus also be used to study aspects of invasion. However, when using EHS tumor extracts as coating, it should be taken into consideration that this material is prepared from transplanted tumors and will, as such, contain not only ECM and other materials produced by the EHS tumor cells, but also cell components and products from the host in which the tumor had developed. Therefore, any conclusion concerning specific (e.g. antibody-inhibitable) effects can at best be

FIG. 3 Boyden chamber analysis of Ha ras oncogene-transformed liver epithelial cells. Scanning electron microscopy shows the cells migrating over the filter surface ( 1 ) and entering the 12-pm pores of the filter (2). Trailing (Ir) and leading (*) edges of a migrating cell are indicated. Bar = 10 p m .

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taken as an indication that the given component is essential. The extent to which the component is sufficient to achieve the desired effect has to be determined by studies involving the pure substance. This restriction is not meant to discourage the use of complex ECM preparations of this type, but instead to help against undiscriminated generalizations that do not take into account the complexity of tumor extracts.

C. “Under Agarose“ Assay

This assay, originally described for explanted tissue fragments (R. R. Carpenter, 1963), is based upon the migration of cells under an agarose gel. In its simplest version, the cells are placed in the central one of three wells punched in a layer of agarose solidified on a culture dish (Fig. 4). The external wells are filled with solutions of the chemotactic factors and an appropriate nonchemotactic control medium, respectively. The distance the cells move toward the factor, minus the distance toward the control well (“spontanous migration”) is taken as a measure of the chemotactic activity. Although the method is primarily used to study leukocyte migration (Nelson et al., 1978), it has been recently applied successfully to the determination of chemotactic and chemokinetic effects of acidic fibroblast growth factor (FGF, FGF-1) on endothelial cells (Stokes et al., 1990).

/

(a

\

cells

control medium

chemoattractant

h chemokinesis

chemotaxis

FIG. 4 Principle of the under agarose assay. A detailed description of the method is given in the text.

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D. Migration Track Assays Two different approaches to migration track assays are currently used: the phagokinetic track assay (Albrecht-Buehler, 1977) and the ECM-track assay (Bade and Nitzgen, 1985). The phagokinetic track assay is a negative assay based on phagocytotic clearing of the culture substatum from protein-coated colloidal gold particles by migrating cells (Fig. 3,while the positive ECM track assay relies on immunohistological demonstration of the ECM proteins deposited by the migrating cells onto the culture substratum (Fig. 6). The phagokinetic track assay was initially used to study the migration of 3T3 cells (Albrecht-Buehler, 1977), and to demonstrate the different responses of capillary and large vessel endothelial cells to a tumor-derived angiogenic factor (Zetter, 1980). More recently the method was used to study the migration of single NIH 3T3 cells transfected with FGF-2 cDNA (Mignatti et al., 1991). The assay has the advantage that the migratory behavior can be observed directly throughout the course of the experiment. However, the large number of protein-coated gold particles internalized by many cells can influence their behavior and vital-

FIG. 5 Phagokinetic track assay. The liver epithelial cells have cleared their migration path of albumin-coatedgold particles from the glass coverslip, and the incorporated gold particles appear as dark aggregates within the cells. Phase contrast. Bar = 100 pm.

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FIG.6 ECM-track assay. Liver epithelial cells cultured in serum-free medium supplemented with insulin (A) or with insulin plus EGF (B). immunostained for laminin. Cells with a typical epithelial morphology (A) are surrounded by a delicate network of laminin fibrils. Elongated (fibroblastoid) migratory cells appear connected with a laminin-containing ECM track. The site of attachment and spreading of two migrated cells is clearly outlined by the ECM deposits from focal adhesion remnants. Migratory cells appear to be able to reuse the migration track of other (leader?) cells (C). Bar = 30 pm.

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ity, and these additional variables must be considered when interpreting the results. These problems are obviated in the ECM-track assay, which is based on the immunohistological demonstration of ECM components, e.g., laminin (Bade and Nitzgen, 1985), fibronectin (Bade and Feindler, 1988; Seebacher et al., 1988), tenascin (E. G. Bade and M. Strehle, unpublished results), or plasminogen activator inhibitor (PAI-1) (Seebacher et al., 1992) deposited by the cells on the substratum during their migration. This method requires serum-free or serum-reduced culture conditions to eliminate interference by serum components with the staining of the cell-derived proteins. Using the ECM track assay, the migration of liver epithelial cells has been analyzed extensively (Bade and Nitzgen, 1985; Bade and Feindler, 1988; Manske et al., 1990; Geimer and Bade, 1991; Seebacher et al., 1992). The high migration efficiencies (up to 70-90% of the cells in the culture, see also Fig. 1) observed with this system have allowed a correlation with biochemical data. A combination of different labeling procedures has allowed the simultaneous determination of migration and DNA synthesis at the single-cell level (Geimer and Bade, 1991). Using this method, it has also been observed that migratory cells may reuse the ECM tracks deposited by other, “leader,” cells (Fig. 6). Even though the method does not allow a continuous analysis of the progression of a migratory reaction as the phagokinetic track assay does, the change in morphology (epithelial to fibroblastic) and the scattering effect are good indicators of the migratory reaction. A further advantage of the method is the possibility of determining the composition of the migration tracks. Their morphology and that of the attachment and spreading sites yield retrospective information on the cell’s interaction with the culture substratum (Bade and Nitzgen, 1985; Bade and Feindler, 1988).

E. Coating Assays

These assays are essentially an extension of the methods used to study the requirement of ECM components for attachment and spreading. Typically, either the entire culture substratum (Bade and Nitzgen, 1985) or several small segments of it are coated with the ECM components of interest. The behavior of the plated cells on the coated substratum or at the interface to the blank dish (Rovasio et al., 1983) or to other ECM components (Goodman et a / . , 1989) allows conclusions on stimulating or inhibiting effects.

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F. In Vitro Wound Assay

The in uitro wound assay (Fig. 7) is based on the ability,of cells to migrate into a denuded space produced by scraping off a small portion of a cell monolayer (wounding) with a “rubber policeman”, a razor blade, or a plastic micropipette tip. This procedure has been successfully used to analyze the migration of 3T3 cells (Lipton et al., 1971; Burk, 1973), endothelial cells (Morimoto ef al., 1991; Pepper ef al., 1992), liver epithelial cells (Manske, 1991) and bladder carcinoma cells (Boyer et al., 1989; Feindler et al., 1993; Muller et al., 1993a). Since multiple wounded cultures

FIG. 7 Wound closure assay. Closure of a discontinuity (wound)introduced into a monolayer of NBT I1 (ATCC CRL 1655) rat bladder carcinoma cells with a micropipette tip. Migration was induced with Ultroser G (B), EGF (C), or TGF-a (D). The control without added growth factors is in panel (A). Bar = 100 Fm.

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are necessary to obtain a significant portion of migrating cells (Pepper et al., 1992), a correlation with biochemical data is difficult. However, the method can be ideally combined with immunohistologicaland in situ hybridization procedures (Pepper et al., 1992), thus allowing a direct comparison of the gene expression pattern of the migrating cells with that of the stationary ones. When using an in vitro wound assay, it must be taken into account that incompletely removed extracellular matrix components might still influence migratory behavior, and that not every cell type capable of forming a monolayer in culture must necessarily be capable of closing an in vitro wound by migration.

G. Spheroid and Microcarrier Assays Spheroids are tissue cell aggregates that result from the reassociation of single cells kept in suspension by slow rotation (Mareel et al., 1979) or on agar-coated culture dishes (Freshney, 1987). The use of spheroids has a long tradition in developmental biology for the study of specific cell aggregation (Holtfreter, 1939; Moscona, 1952). To analyze cell migration, spheroids are placed into a tissue culture dish and the outmigration of the cells onto the bottom of the dish is evaluated microscopically (Fig. 8, and Lund-Johansen et al., 1990). The assay, which, similarly to other systems, yields information about both the efficiency and the velocity of migration, has been recently used to analyze the EGF-induced migration of glioma

FIG. 8 Spheroid migration assay. Spheroids of RT 18 human colon carcinoma cells showing (9) the out-migration of numerous adherent carcinoma cells. Nonmigrated control (A). Bar = 100pm.

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cells (Lund-Johansen et al., 1990). A similar principle is utilized in the microcarrier migration assay, first described by Marucha et al. (1987; Jozaki et al., 1990). In this method, the downmigration of cells from microcarrier beads is assessed similarly to the outmigration from spheroids. This alternative has been used to analyze the migratory activity of endothelial, epithelial, and mesenchymal cells (Jozaki et al., 1990; Rosen et al., 1990).

H. Scatter Assay

The scatter assay is probably the easiest method for detecting factors able to induce migratory behavior. It was originally described for the detection of scattering activity for Madin-Darby canine kidney (MDCK) cells present in a culture medium of MRC-5 human embryo fibroblasts (Stoker and Perryman, 1985) and which was later identified as being identical to hepatocyte growth factor (see Section 111, G). The compact colonies of the epithelial cells used in this system are dispersed by hepatocyte growth factor/scatter factor (HGF/SF) and this scattering activity is easily evaluated microscopically after fixation and staining (Fig. 9). Since the compactness of the colonies formed is essential for a sensitive assay, use of a recloned subline of MDCK cells (Bomsel et al., 1989) is strongly recommended because the cells currently available from the American Type Culture Collection, Rockville, MD show a high background of single, migratory cells. The scatter assay, while ideally suited for HGF/SF, is not applicable to every growth factor discussed in this review (B. Eckstein, S. Feindler, and E. G. Bade, unpublished results). Similarly to the ECM track assay (Section 11, D), the scatter assay depends on the disruption of epithelial cell colonies. Both assays, therefore, represent a useful tool for analyzing the role of cell-cell adhesions in the regulation of cell translocation.

I. Invasion Assays Invasive processes depend considerably on the migratory behavior of the involved cells, and may thus also yield essential information about the regulation of cell migration. Various cell culture systems, tissues, and organs have been used for invasion assays, including cultured cell monolayers (Kramer and Nicolson, 1979; Verschueren et al., 1986), spheroids of embryonic chick heart cells (Mareel et al., 1979), rat brain aggregates (Lund-Johansen et al., 1990), chick chorioallantoic membrane (Ossowski

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FIG. 9 Scatter assay. Dissociation of MDCK cell colonies exposed to partially purified scatter factor (b). Control without addition of HGF/SF (a). Reprinted with permission from Nature (Stoker et al., 1987). Copyright (1987) Macmillan Magazines Limited.

and Reich, 1980; Poste et al., 1980), and human amnion membrane (Liotta et al., 1980; Mignatti et al., 1986). While the more complex approaches using cells or even organ fragments have the advantage of a resemblance to the in vivo situation, one main drawback is the difficulty of quantification. This problem can be obviated by using the more simple protein gel invasion assays [e.g., collagen gels (Fig. 10) and Schor et al. (1982)l or modified Boyden chamber assays with ECM-coated filters (Hendrix et al., 1987; Erkell and Schirrmacher,

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FIG. 10 Collagen invasion assay. Invasion of ras-transformed liver epithelial cells into a gel of rat tail collagen. The photographs taken at three different focusing steps reveal cells on the surface of the gel (A) and at two different depths within it. Bar = 16 pm.

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FIG. 11 Principle of the chorioallantoic membrane invasion assay. The membrane is held in position by an agar gel and Teflon rings. (Adapted with modification from Poste et al., 1980.)

1988; Thompson et al., 1991). In a slightly more complex approach (Erkell and Schirrmacher, 1988), the invasion of lymphoma cells was evaluated by the number of cells on the lower of two filters separated by a thin ECM layer. Invasion assays using biological membranes, for example, chicken corioallantois (Poste et al., 1980) or human amnion (Liotta et al., 1980; Mignatti et al., 1986), combine the advantages of a relatively complex biological system with the possibility of a quantitative evaluation. In these approaches the membranes are stretched in a device resembling a Boyden chamber (Fig. 1 1 ) and the number of invading cells is determined after staining or as membrane-bound radioactivity. J. Conclusion

This summary of methods for studying cell migration is probably incomplete. However, it is hoped that it provides critical introductory information and conveys the message that when studying cell migration processes, the answer will partially depend on the method used, and that generalizations should be made with extreme caution. Caution is especially indicated concerning extrapolations to in uiuo situations. For the study and identification of soluble factors that induce or stimulate migratory behavior, the available methods seem to be sufficient. However, the analysis of the inhibition of migration, which is more complex, may require additional methods or systems. Furthermore, for every study, more than one assay should be used if possible.

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111. Growth Factors That Modulate Cell Migration A. Epidermal Growth Factor and Transforming Growth Factor-Alpha

Epidermal growth factor was discovered in 1960as an activity that accelerated incisor eruption and eyelid opening in newborn animals (Cohen, 19621, and that could be purified in large amounts from male mouse submaxillary glands (Cohen, 1962). The protein was completely sequenced soon afterward (Savage et al., 1972), thus becoming the first growth factor to have a completely solved structure. Of the 53 amino acids of the factor, the six cysteine residues are essential for conferring the typically folded structure required for biological activity (Carpenter and Wahl, 1990). Interestingly, this characteristic structure has since been found as “EGF-like domains” in an ever-increasing number of biologically essential proteins. The cDNA of the mouse factor codes for a 1291-aminoacid transmembrane protein that contains nine EGF-homologous sequences in its extracellular domain, of which only those closest to the transmembrane segment correspond to the sequence of the mature growth factor (Carpenter and Wahl, 1990). The processing mechanisms, and the reasons for the remarkably restricted tissue and cell specificity of EGF expression are still unclear. However, recent data showing that during development EGF responsiveness is expressed by restricted groups of cells, for example, in the central nervous system (Ferrari et al., 1991), that were not previously detected when studying homogenized organs, suggest a need for reevaluation of the current interpretation of the factor’s biological roles. This analysis may confirm a biological role for EGF well beyond its capacity to stimulate DNA synthesis. While the proliferation-stimulating action of EGF still remains the most frequently analyzed effect, a number of nonproliferative actions have also been described. These include stimulation of cell migration (Bade and Nitzgen, 1985), induction of gene expression which may (Bade and Feindler, 1988; Manske et al., 1990) or may not be related (Carpenter and Wahl, 1990)to a migratory phenotype, induction of differentiated functions (Carpenter, 1979), and even an inhibition of cell proliferation (Bravo et al., 1985) and DNA synthesis (Geimer and Bade, 1991; Miiller et al., 1993a). Similarly to the factor itself and its sequence, the identification of the EGF receptor and its characterization as a transmembrane protein with intrinsic tyrosine kinase activity were “firsts” in the field of growth factor biology and biochemistry (Cohen, 1987; Carpenter and Wahl, 1990). The complete sequence of the EGF receptor molecule (Ullrich et al., 1984)

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also initiated a new phase in the study of transmembrane receptors and of (proto)-oncogenes. The human EGF receptor is a transmembrane protein of 1186 amino acids, with an extracellular N-glycosylated domain, which is folded by disulfide bridges into the biologically active structure. Ligand binding causes receptor aggregation and activates the intracellular tyrosine kinase domain that initiates the signal transduction process, which frequently culminates in DNA synthesis and cell division as late effects. The EGF receptor also binds, with seemingly equal efficiency and biological consequences, the structurally related, but genetically different transforming growth factor-alpha. TGF-a was initially discovered (De Larco and Todaro, 1978) as one of two factors (TGF-a and TGF-P) present in culture media conditioned by tumor cells and which, acting together, induced the morphologic transformation of cultured mesenchymal cells (“fibroblasts”). TGF-a is generally considered biologically equivalent to EGF, although functional differences have been suggested (Schreiber et al., 1986). However, a comparative analysis of the biological effectiveness of the two factors will require extensive standardization and controls. Similarly to EGF, TGF-a is also synthesized as a transmembrane protein (Kriegler et al., 1988), but this membrane-bound precursor has a signaling capacity (Anklesaria et al., 1990; Massague, 1990a) denominated as yuxtacrine (as opposed to endo-, para- and autocrine). Among the early effects of EGF, Chinkers et al. (1979) described a membrane ruffling of A 431 cells that accompanied changes in cell morphology. A specific migratory response was not reported at that time. At present, however, EGF and TGF-a are well established migrationinducing factors (Bade and Nitzgen, 1985; Bade and Feindler, 1988), a fact that seems to have been overlooked by some recent authors. Both factors have been shown to induce chemotaxis and chemokinesis of numerous normal (nontumorigenic) epithelial cells, including those from the oral cavity (Terranova and Lyall, 1986; Royce and Baum, 1991), intestinal lining (Blay and Brown, 1985; Saxena et al., 1992), and rat liver (Bade and Nitzgen, 1985; Bade and Feindler, 1988; Manske et al., 1990; Geimer and Bade, 1991 ; Seebacher et al., 1992),as well as keratinocytes (Nickoloff et al., 1988; Turksen et al., 1991) and thyrocytes (Westermark et al., 1991). Also, the migration of endothelial cells (McAuslan et al., 1985; Mawatari et al., 1991; Matsuda et ul., 19921, and of fibroblasts (Adelmann et al., 1990) is stimulated by these factors. EGF and TGF-a have also been found to induce the migration of several malignant cells, such as glioma cells (Westermark et al., 1982; Lund-Johansen et al., 1990), melanocytes (Morelli et al., 1992), epidermoid carcinoma cells (Koyasu et al., 1988), and embryonal carcinoma cells (Engstrom, 1986), and both EGF (Feindler et al., 1993) and TGF-a (Tucker et al., 1991; Feindler et al., 1993) also induce the migration of NBT I1 rat bladder carcinoma cells.

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Similarly to what has been described for liver epithelial cells (Geimer and Bade, 19911, EGF and other growth factors that induce migration also cause a transient inhibition of DNA synthesis in NBT I1 rat bladder carcinoma cells (Muller et al., 1993a,b). EGF has been proposed to play an important role in intestinal regeneration by stimulating both epithelial cell proliferation and migration (Blay and Brown, 1985; Thompson et al., 1988, 1989; Saxena et al., 1992). Furthermore, EGF and TGF-a can also induce neurite extension in cell lines (Suardet et al., 1989; Zhang et a/., 1990), and in primary cultures of central nervous system (CNS) neurons. In the primary cultures, the factor was found to stimulate. in addition, the synthesis of neurotransmitters (Ferrari et a/., 1991), thus confirming that EGF can also be a positive modulator of differentiated functions. The EGF-induced migration of keratinocytes (Nickoloff et al., 1988; DeLapp and Dieckman, 1990) as well as liver epithelial cells (Bade and Feindler, 1988; Seebacher et al., 1988) is accompanied by an increased secretion of fibronectin. In the case of liver epithelial cells, the protein is deposited together with other extracellular matrix components [laminin; Seebacher et a/. (1991) and tenascin; S. Feindler and E. G. Bade (unpublished results)] into the ECM tracks on which the cells migrate (Bade and Nitzgen, 1985; Bade and Feindler, 1988; Manske et al., 1990; Seebacher et al., 1992) and consists of the cellular splicing variant (Seebacker et al., 1988). Substratum-bound plasma fibronectin, however, but not laminin or collagen IV inhibits the EGF-induced migration (Bade and Nitzgen, 1985). This migration-inhibiting effect of fibronectin suggested a highly specific inhibitory cell-ECM interaction, an interpretation that has been recently substantiated by the demonstration that the overexpression of the fibronectin integrin can also inhibit migration (Giancotti and Ruoslahti, 1990). In the liver epithelial cell system (Bade and Feindler, 1988; Manske et al., 1990) as well as in endothelial cells (Mawatari et a/., 1991; Matsuda et al., 1992), the induction of cell migration by EGF or TGF-a correlates with the early induction of plasminogen activator inhibitor PAI-I (EIP-I ; Bade and Feindler, 1988; Manske et a/., 1990), which is also deposited into the migration tracks (Manske, 1991; Seebacher et a/., 19921, thus suggesting that it is directly involved in the migratory process (Manske et al., 1990; Manske, 1991). Ha ras-transformed, malignant liver epithelial cells which migrate constitutively (Zoller, 1990) also produce the inhibitor (Seebacher et al., 1992)and can be induced by EGF to express the metalloproteinase, stromelysin-1 (Bauhofer et al., 1993). Metalloproteinases are also expressed by cells induced to migrate by other growth factors (Tsuboi et al., 1990; Bade et al., 1993b). The exact mechanism by which EGF and TGF-a induce cell migration has not yet been established, but protein synthesis is required (Muller et a/., 1993a). The proteins induced by the factors are assumed to be directly

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required for the process (Bade and Feindler, 1988; Chen et al., 1993), but the extent to which they are essential remains to be determined. The EGF-induced migration is also repressed by CAMP and cholera toxin, which in addition inhibit the expression of EIP-l/PAI-1 (Manske et al., 1990). Since the inhibitor is also coregulated with migration in other cells and situations (Seebacher et al., 1992; Feindler et al., 1993), it has been proposed to be an essential component of the migratory reaction (Bade and Feindler, 1988). Very recently the analysis of the signal transduction initiated at the EGF receptor has taken an interesting turn (Chen et al., 1993) as mutants of the carboxy terminal have been produced which can induce proliferation but not migration. Further work along these lines may be expected to allow a detailed dissection of the intracellular divergence of the two pathways. This approach might also help to determine the mechanisms involved in the transient inhibition of DNA synthesis associated with the induction of migration (Geimer and Bade, 1991; Miiller et al., 1993a).

6. Fibroblast Growth Factors The fibroblast growth factors (FGFs), also known as heparin binding growth factors (HBGFs) constitute a still-growing family of growth factors and (proto)-oncogenes (Burgess and Maciag, 1989),that at present consists of seven distinct polypeptides (Basilico and Moscatelli, 1992). For the best-known members of the family-acid FGF (aFGF/FGF-1) and basic FGF (bFGFIFGF-2)-a large number of functions, including multiple roles in development, have been described (Burgess and Maciag, 1989). Most of these functions require a modulation of cell migration. The four currently known FGF receptors have variable binding specificities (Dionne et al., 1991;Partanen et al., 1991),but are all transmembrane glycoproteins with extracellular immunoglobulin-like domains and a cytoplasmic tyrosine kinase domain. The signal transduction mechanisms that ultimately lead to proliferation, to stimulation or inhibition of differentiation, or to the complex morphogenetic processes that depend on cell migration are being analyzed in a number of laboratories.

1. Acidic Fibroblast Growth Factor (aFGF, FGF-1, HBGF-1) FGF-1 (aFGF) was first identified as an acidic brain-derived protein possessing mitogenic activity for fibroblasts (Gospodarowicz er al., 1978; Thomas et al., 1980). Structural analysis demonstrated a 55% identity

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with bFGF at the level of the polypeptides (Thomas, 1987). Human and bovine aFGFs consist of 154 amino acids (Burgess et al., 1986; Crabb e? al., 1986), but amino terminally truncated forms have also been described (Burgess, 1991).Their affinity for heparin (Maciag e? al., 1984)has allowed the efficient purification of this family of factors. Binding to heparin also increases biological activity, both by preventing proteolytic degradation (Rosengart et al., 1988; Sommer and Rifkin, 1989), and by facilitating binding by the specific high-affinity receptor(s) (Schreiber e? al., 1985; Yayon et al., 1991). Recent evidence (Kan et al., 1993) showing a direct, sequence-specific interaction of the FGF- 1 receptor with heparidheparan sulfate proteoglycan emphasizes the role of glycosaminoglycans in signaling by this receptor. In addition to stimulating cell proliferation, FGF- 1 efficiently induces the migration of lens epithelial cells (Chamberlain and McAvoy, 1989), and various types of endothelial cells (Herbert e? al., 1988; Klein-Soyer e? al., 1989, 1990; Stokes et al., 1990); it has also been described as stimulating the migration of bladder carcinoma cells ( Jouanneau e? al., 1991). The action on NBT-I1 cells, however, appears to depend on additional factors or culture conditions (Feindler et al., 1993).For microvascular endothelial cells, the optimal FGF- 1 concentration for chemotaxis and random migration was determined to be 10-I0M (Stokes e? al., 1990). Alone or in combination with heparin, FGF-1 but not bFGF, stimulates the migration of human vascular endothelial cells (Herbert e? al., 1988) and enhances the repair of a lesion in a cell monolayer by migration and proliferation (Klein-Soyer e? al., 1989, 1990). FGF-1 also increases the healing rate of corneal wounds more efficiently than bFGF (Fredj et al., 1987). Furthermore, in regenerating rat corneas, the expression of FGF1 by the epithelial cells was found to correlate with their migration into the wound (Dabin and Courtois, 1991). Determination of the mechanism by which expression of FGF-1 by migratory cells is increased may yield basic information on the regulation of cell renewal. In addition to stimulating cell migration, and similarly to EGF (Ferrari et al., 1991), FGF-1 was also found to induce neurite outgrowth. Embryonic spinal cord neurons (Sweetnam et al., 1991) as well as PC 12 pheochromocytoma cells (Neufeld et al., 1987;Rydel and Greene, 1987)extend neurites under FGF influence, but for PC 12 cells, the neurite-inducing potency of FGF-1 cells is lower than that of NGF or FGF-2 (Neufeld et al., 1987).

2. Basic Fibroblast Growth Factor (bFGF, FGF-2, HBGF-2) FGF-2/bFGF is a potent angiogenesis factor (Folkman and Klagsburn, 1987; Schweigerer, 1989; Risau, 1990), neurotrophic agent (Walicke,

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1988a) and regulator of eye development and corneal wound healing (Burstein, 1987; McAvoy et al., 1991; Tripathi et al., 1991). The factor was first detected in pituitary extracts as an efficient mitogen for fibroblasts (Armelin, 1973), and was later found to also stimulate the proliferation of several other cell types (Burgess and Maciag, 1989). The 18-kDa FGF-2 form is localized primarily in the cytosol, and 22 kDa, 22.5 kDa, and 24 kDa forms are found in the nucleus (Renko et al., 1990). Since FGF-2 lacks a signal peptide, it has been assumed that the soluble variant might be released by dead or injured cells (Schweigerer et al., 1987), but recently a specific exocytosis mechanism has been described (Mignatti et al., 1992). FGF-2 stimulates the motility of several cell types. A proliferationindependent induction of migration was reported for rat lens epithelial cells (Chamberlain and McAvoy, 1989; McAvoy and Chamberlain, 1989). The factor has also been described as accelerating wound healing (Broadley er al., 1989) and stimulating keratinocyte migration (Parsons and Wikner, 1989), but other authors concluded that the positive effect upon wound healing is due to a promotion of mitogenic activity (Sarret et af., 1992). It might be interesting to test if these apparently conflicting results are partly due to differences in the time of sampling or in cell density, as reported for other systems (Geimer and Bade, 1991; Muller et af., 1993a). Human teratocarcinoma cells are stimulated to proliferate at low concentrations (1-10 ng/ml) of FGF-2, while 100 ng/ml stimulate cell migration (Schofield et al., 1992). The migration of NIH 3T3 cells transfected with FGF-2 cDNA depends on the amount of factor released (Mignatti et a!., 1991), and the spontaneous migration of bovine vascular smooth muscle cells is also dependent on FGF-2. Interestingly, the PDGFstimulated migration of these cells correlates with the induction of FGF-2 mRNA, and can be blocked with anti FGF-2 antibodies (Sato et al., 1991). Conversely, FGF-2 sensitizes oligodendrocyte-type 2 glial precursor cells to the migration-inducing influence of PDGF (McKinnon et al., 1991). Thus, in at least two different cell systems, the migrationrelevant action of FGF-2 is part of a relatively complex regulatory circuit. FGF-2 is a very potent angiogenesis factor. In uitro, the factor is a chemoattractant for bovine aortic and human umbilical vein endothelial cells (Herbert et al., 1988), and the spontaneous migration of several types of endothelial cells is blocked by anti-FGF-2 antibodies (Sato and Rifkin, 1988; Tsuboi et al., 1990; Odekon et al., 1992; Pepper et a/., 1992). Thus, in the two-dimensional situation of the cell culture dish, the angiogenic capacity of FGF-2 can be separated into a mitogenic and a migration component. In more complex culture systems, for example, with collagen gel, the angiogenic capacity is expressed as morphogenetic invasion, with

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the formation of characteristic tubules (Montesano et af., 1986; Tweden et al., 1989). Similarly to other growth factors (see the sections on EGF, NGF), FGF-2 can also enhance viability and neurite outgrowth of nerve cells. However, the efficiency of FGF-2 as a trophic and morphogenetic factor of the nervous system is significantly superior to that of most of the other growth factors (Burgess and Maciag, 1989). Rat cerebral cortical neurons (Morrison et al., 1986), mouse cerebellar granule neurons (Hatten et al., 19881, rat fetal hippocampal neurons (Walicke et al., 1986), embryonic mesencephalon neurons (Ferrari et al., 1991) as well as rat embryonic spinal cord neurons (Sweetnam et al., 1991)have all been found to respond to the factor with neurite outgrowth. FGF-2 also stimulates neurite outgrowth from human IMR 32 neuroblastoma cells (Ludecke and Unsicker, 1990), rat adrenal chromaffin (Stemple et al., 1988), and pheochromocytoma PC 12 cells (Neufeld et al., 1987; Rydel and Greene, 1987; Rogelj et al., 1989; Altin el al., 1991). In agreement with the binding properties of FGF-2 to glycosaminoglycans, the factor was shown to stimulate neurite extension when bound to surfaces coated with heparin, heparan sulfate, or hyaluronic acid, but not with chondroitin or dermatan sulfate (Walicke, 1988b). In the light of recent evidence [Section III,B,I, and Kan et al. ( 1993)], this stimulatory effect of glycosaminoglycans might also involve the receptor directly. The mechanism by which FGF-2 influences cell motility is being gradually solved. For neurite formation, the ras pathway (Altin et al., 1991) and tyrosine phosphorylation are involved: in PC 12 cells, the induction of neurite outgrowth by FGF-2 can be inhibited by a monoclonal anti-p21 antibody (Altin et al., 1991), and neurite outgrowth can be induced by activated Ha ras and by src (Thomas et al., 1992). The FGF-2-induced tubule formation by capillary endothelial cells correlates with the induction of urokinase-type plasminogen activator (Montesano et al., 1986), and inhibition of the spontaneous migration of aortic endothelial cells by antibodies to FGF-2 is accompanied by a reduction of plasminogen activator activity (Sato and Rifkin, 1988). Antibodies to uPA inhibit the FGF-2-induced migration of aortic endothelial cells, which could also be stimulated by an enzymatically inactive, but receptor-binding fragment of the activator (Odekon et al., 1992). In addition to uPA and its receptor, metalloproteinases might also be involved in FGF-2-induced migration since in migrating and invasive bovine capillary endothelial cells the metalloproteinase activity correlated with FGF-2 levels (Tsuboi et al., 1990). Thus, both for endothelial cell migration and neurite extension, FGF-2 is a powerful inducer and mediator. The signal transduction mechanisms involve tyrosine phosphorylation and ras, and appear to share some crucial steps with other factors.

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C. Beta Transforming Growth Factors

The beta transforming growth factors were initially discovered as the fraction of tumor cell-conditioned media that complemented either TGF-a or EGF for the induction of anchorage-independent growth of mesenchymal cells (see also Section 111, A). Interestingly, TGF-/3 was also discovered as an inhibitor of epithelial cell proliferation (Holley et al., 1980)and mediator of immunosuppression (Fontana et al., 1987). The TGFs are a large family of polypeptides with autocrine, paracrine, and endocrine functions; they are secreted by a variety of cells and modulate both cell proliferation and differentiation (Massaguk, 1990b). In general terms, the beta TGFs stimulate the proliferation of mesenchymal cells, but inhibit the proliferation of hematopoietic, epithelial, and endothelial cells. The three human TGF-/3 isoforms currently known are 70-80% homologous at the amino acid level and exert similar biological effects. Of the several cell surface proteins with TGF-/3 binding capacity, type I1 belongs to the new family of serinehhreonine kinase receptors (Massague, 1992). The dual action of the beta TGFs on cell proliferation is also observed in their effects on cell migration. They stimulate the migration of a variety of cell types, including keratinocytes (Hebda, 1988; Nickoloff et al., 1988) and smooth muscle cells (Bell and Madri, 1989), and act as a chemoattractant for smooth muscle cells (Koyama et al., 1990), dermal fibroblasts (Postlethwaite et al., 1987), osteoblast-like cells and osteosarcoma cells (Pfeilschifter et al., 1990), monocytes (Wiseman et al., 1988), fibroblasts, and epithelial cells of the chorioallantoic membrane (Yang and Moses, 1990), as well as for Walker 256 carcinoma cells (O’Neill et al., 1985; Orr et al., 1990). The induction of directed cellular migration and tissue repair by TGF-P has also been described in the organism (Pierce et al., 1988). TGF-P can also inhibit the spontaneous or induced migration of endothelial cells (Heimark et al., 1986; Miiller et al., 1987; Bell and Madri, 1989; Coomber, 1991; Kojima et al., 1991;Sutton etal., 1991),dermal fibroblasts (Adelmann et al., 1990),smooth muscle cells (Koyama et al., 1990; Kojima et al., 1991), trabecular cells (Borisuth et al., 1992),and osteoclasts (Dieudonne et al., 1991). TGF-/3 is thus, for the time being, the only factor with a widespread capacity to inhibit cell migration. It remains to be determined if the migration-inhibitory action of members of the TGF-/3 family extends beyond the currently reported cases, or if other growth factors are also endowed with a similar capacity. In at least one case, the action of TGF-/3 was determined to be even more complex: it enhanced the chemotactic migration of cultured rat aortic smooth muscle cells (Koyama et al., 1990) but it inhibited their PDGF-induced migration. Analysis of this bifunctional action of the factor might yield important information

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about the interaction of the signal transduction pathways involved, which is expected to have relevance for in uiuo situations. The pathways by which the beta TGFs stimulate or inhibit cell migration are only partially known, but the identification of the new class of ser/ thr kinase receptors for this growth factor family (Massague, 1992) will facilitate the analysis. Some data obtained with white blood cells point to the possible involvement of other components. For human neutrophils, TGF-P is an extremely powerful chemoattractant. The chemotactic response is associated with a polymerization of actin, and requires protein synthesis (Reibman et al., 1991). The cytoskeleton is also involved in the inhibition of migration, as shown for endothelial cells (Coomber, 1991). TGF-P may also influence cell migration by altering the expression of ECM proteins. The production of fibronectin by keratinocytes is stimulated when they are induced to migrate by TGF-P, and part of the migration could be inhibited with specific antibodies (Nickoloff et al., 1988; DeLapp and Dieckman, 1990). However, the inhibition of the FGF-induced migration of bovine endothelial cells by TGF-P has also been reported to be associated with an increased fibronectin network (Muller et al., 1987). These results should not be interpreted as being in contradiction with the data obtained with keratinocytes. Instead, cell specificity and the influence of other ECM components, for example, antiadhesive proteins (ChiquetEhrismann et al., 1988), proteinases and their inhibitors, or differences in culture conditions and the splicing isoform of fibronectin to which the cells are exposed should also be considered. In bovine aortic endothelial cells, TGF-P, induces fibronectin mRNAs alternatively spliced in the IIICS region, and recombinant polypeptides with a spliced IIICS segment reduced migration (Kocher ef al., 1990).

D. Platelet-Derived Growth Factor As the name indicates, PDGF was originally identified as a growth-

promoting activity from blood platelets that was capable of restoring proliferation-inducing capacity to plasma-derived serum (Ross et al., 1974; Westermark and Wasteson, 1976). The PDGF isolated from human blood platelets consists mainly of the 30-kDa disulfide-bonded AB heterodimeric glycoprotein (Raines et al., 1990; Heldin, 1992). The discovery that the B-chain is highly homologous to p28sis, the transforming protein of simian sarcoma virus (SSV) (Doolittle et al., 1983) may be considered another landmark in the study of cell proliferation and oncogenesis. The PDGF family now consists of a triad (AA, BB, and AB) of factors that can bind to three combinations of receptor polypeptide dimers (Raines et al., 1990;

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Heldin, 1992). Both receptor polypeptides are structurally similar, with five extracellular immunoglobulin-like domains and an intracellular segment that contains a split tyrosine kinase domain and the protein-binding SH2 and SH3 domains. Binding of PDGF to the receptor causes its dimerization and the activation of the intracellular tyrosine kinase, which is essential for signal transduction. The cell and tissue specificity of PDGF actions results largely from the various combinations of ligands and receptors (Heldin and Westermark, 1990; Raines et al., 1990; Heldin, 1992). In addition to their effects on cell proliferation, the PDGFs are also capable of stimulating the migration of a variety of cells, including endothelial (Bell and Madri, 1989) and osteoblast-like cells (Gilardetti et af.,19911, smooth muscle cells (Nakao et al., 1985; Sat0 et af., 1991), glomerular mesangial cells (Barnes and Hevey, 1990), and glial cells (Noble et a/., 1988; McKinnon et af.,1991).The growth factor can also act as a chemoattractant for fibroblasts and fibrosarcoma cells (Wach et af., 1987; Adelmann and Cully, 1990), as well as for osteoblast-like cells (Tsukamoto el al., 1991), smooth muscle cells (Nomoto et al., 1988; Bell and Madri, 1989), retinal pigment cells (Campochiaro and Glaser, 1985, 1986; Hackett et a!., 1986; Hackett and Campochiaro, 1988), and glial cells (Bressler er al., 1985; Armstrong et al., 1990; Uchihori and Puro, 1991). PDGF or PDGF-related peptides also facilitate the migration of mesenchymal cells into the alveolar air space during acute lung injury (Snyder et af., 1991). The migration of teratocarcinoma-derived visceral endodermal cells (Liapi et al., 1990) and OA-2 glial precursor cells (McKinnon et al., 1991) is also stimulated. However, PDGF can also inhibit both the migration of dermal fibroblasts toward epidermal growth factor (Adelmann et al., 1990) and the spontaneous migration of aortic smooth muscle cells (Bell and Madri, 1989). In summary, and similarly to the other growth factors, cell-specific expression and sensitivity to PDGF is also multiply involved in the regulation of cell migration, both in the adult organism and during development. Further complexity is introduced into the migration modulating actions of this growth factor by its remarkable species- and isoform-specific effects (Siegbahn et al., 1990):both PDGF-AB and PDGF-BB can induce chemotaxis of fibroblasts and monocytes in a dose-dependent manner, whereas PDGF-AA does not stimulate chemotaxis, but even inhibits the chemotactic response to the other isoforms. A chemotactic response mediated by the type p PDGF receptor requires an intact protein-tyrosine kinase domain, and involves a reorganization of actin (Westermark et al., 1990). Modification of cellular actin was also proposed to be involved in the PDGF-induced migration of the cells of the trabecular meshwork of the eye (Tamura and Iwamoto, 1989). The chemotactic action of the growth factor has been described to be calciumdependent (Nomoto et al., 1988; Uchihori and Puro, 1991) and to involve

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protein carboxylation (Hackett and Campochiaro, 1988). In some cases the stimulation of cell migration by PDGF was found to be associated with an increased synthesis of other extracellularly acting molecules. The PDGF-induced migration of smooth muscle cells is indirect (Sato et al., 1991) and can be abolished by anti-FGF-2. For the PDGF-stimulated migration of human aortic smooth muscle cells, an essential role was suggested for the tissue collagenase/matrix metalloproteinase 1, the precursor of which is induced by the growth factor (Yanagi et al., 1991).

E. Insulin and the Insulin-like Growth Factors [IGF-I and IGF-II/MSAI The two approximately 7-kDa insulin-like growth factors (IGF-I and IGFI1 / MSA) are highly homologous to, but genetically different from insulin (Cohick and Clemmons, 1993). Both IGFs are good examples of growth factors with endocrine actions and are found in blood and tissue fluids associated with specific binding proteins (BPs) as acid-labile complexes. The factors bind with high affinity to specific receptors, and have a different profile of expression and actions. The 70-amino acid (aa) polypeptide IGF-I (somatomedin C ) mediates some actions of growth hormone (GH/ STH) on which its expression depends, while the 67 aa polypeptide IGFIIlMSA is growth hormone independent and is required during fetal development. IGF-I exerts its actions primarily through a heterodimeric tyrosine kinase receptor that is similar to but genetically and functionally distinct from the insulin receptor. IGF-II/MSA binds to the mannose-6phsophate receptor (M6P-R), and its actions appear to be mediated to some extent by a G-protein-dependent signaling pathway. IGF-I1 and to a lesser extent IGF-I have been found to be expressed in high levels in several tumors (see later discussion). Recently it has become evident that both IGF-I and IGF-I1 exert their typical actions through the IGF-I receptor (Cohick and Clemmons, 1993). Both IGFs can act as growth and as migration-stimulating factors. A variety of normal and malignant cells can be stimulated to migrate by insulin or IGFs. IGF-I induces a chemotactic response from retinal pigment epithelial cells (Grant et al., 1990) and from bronchial epithelial cells (Shoji el ul., 1990). These cells and vascular smooth muscle cells (Nakao et al., 1985) also migrate under the influence of insulin (Shoji et al., 1990).Insulin, IGF-I, and IGF-I1 also stimulate the migration of A2058 human melanoma cells. The response to IGF-I and insulin is predominantly chemotactic (Stracke et a / . , 1988). The stimulation of migration via the IGF-I receptor is insensitive to pertussis toxin, while the AMFinduced migratory behavior is inhibited by it (Kohn et al., 1990a). For

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human rhabdomyosarcoma cells, IGF-I1 acts as an autocrine motility factor only through the M6P receptor, while proliferation could also be stimulated via the IGF-I receptor (Minniti et al., 1992).Ha ras-transformed rat embryo fibroblasts 5R respond to insulin with both chemotaxis and chemokinesis (Kohn et al., 1990a). F. Hepatocyte Growth Factor / Scatter Factor

HGF/SF is the most recently discovered migration-inducing growth factor. Independently isolated as growth factor for cultured hepatocytes (Nakamura et al., 1984, 1989) and as a migration-inducing (scattering) factor (Stoker et al., 1987; Gherardi et al., 1989)for MDCK epithelial cells, both activities were determined to be identical (Furlong et al., 1991; Naldini et al., 1991; Weidner et al., 1991; Mizuno et al., 1992) and are now designated by convention as HGF/SF. Besides its proliferation-inducing and scatter factor activity, HGF/SF can also inhibit the proliferation of some tumor cells (Tajima et al., 1991). HGF/SF is a heparin binding protein that is processed from its single-chain secreted precursor into the biologically active 69/34-kDa heterodimer (Furlong, 1992). The biological effects of the factor are mediated by its tyrosine kinase receptor, the proto-oncogene MET (Naldini et al., 1991). In addition to MDCK cells, a number of other epithelial cells (Stoker et a / ., 1987), including epidermal keratinocytes (Stoker, 1989; Matsumoto et a!., 1991), BSLC monkey kidney cells (Stoker, 1989), endothelial cells (Morimoto et al., 1991; Rosen et al., 1991b), rat liver epithelial cells (Feindler et al., 1993; see also later discussion), and bladder carcinoma cells (Feindler et al., 1993) can also be induced to migrate by HGF/ SF. The factor also stimulates the invasion of MDCK cells into collagen matrices, with the formation of tubular structures (Montesano e t a / . , 1991) and facilitates the invasion of some human carcinoma cells into collagen gels (Behrens et al., 1991). Fibroblasts produce the factor but have been found to be insensitive to it (Stoker, 1989). Hepatocyte growth factor/ scatter factor has been demonstrated in the exocrine pancreas, in some neurons, and in interfollicular cells of the thyroid (Zarnegar et al., 1990); it is also produced by nonparenchymal liver cells and by some tumor cells. The in uiuo role of HGF/SF as growth factor for hepatocytes has been established beyond doubt, but its role as a migration-inducing factor in the liver is less well defined. Recently the suggestion that HGF/SF might also participate in reorganization reactions in the liver was inferred from data showing that liver epithelial cells are induced to migrate by HGF/SF (Bade et al., 1993a). A requirement of protein synthesis for the SF/HGF-induced migration

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has been confirmed by showing that it is inhibited by cycloheximide (Rosen et al., 1990). The HGF/SF-induced migration of liver epithelial cells, as well as bladder carcinoma cells (Feindler et al., 1993) and endothelial cells (Morimoto et al., 1991) correlates with the induced expression of PAI-1 (EIP-1; Bade and Feindler, 1988; Manske et al., 1990; Seebacher et al., 1992). Similarly to what had been previously reported for EGF/ TGF-a (Manske et al., 1990), both the HGF/SF-induced migration and PAI-1 expression in these liver epithelial cells and in bladder carcinoma cells are inhibited by CAMP and cholera toxin (Bade et al., 1993a). As for the liver epithelial cells, a similar reaction was demonstrated for FGF1 (Bade et al., 1993a). We have proposed that induction of a migratory phenotype by activation of tyrosine kinase receptors proceeds through a common signal transduction pathway and with the induction of a similar set of genes. PAI-1 appears to be a reliable indicator of the reaction and is assumed to be essential for it (Manske et al., 1990; Manske, 1991; Bade et al., 1993a). Thus, migratory reactions induced through tyrosine kinase growth factor receptors appear to be basically different from the pertussis toxin-sensitive migration (Liotta et al., 1986) induced by factors of the AMF group. Furthermore, the migration induced by HGF/SF (Muller et al., 1993b), as well as by other growth factors (Geimer and Bade, 1991), is associated with a transient inhibition of DNA synthesis, with accumulation of cells in the G1 phase (Muller et a l . , 1993b). G. Migration-Stimulating Factor and Autocrine Motility Factors

During the past few years several still incompletely characterized factors capable of stimulating cell migration have been demonstrated in media conditioned by cultured cells. Migration-stimulating factor (MSF; Schor and Schor, 1987; Schor et al., 1988) is produced by fibroblasts from fetal tissue and from cancer patients but not by the cells from normal adult tissues. The 70-kDa, proline-rich protein binds to heparin and cationexchange resins (Grey et al., 1989), and induces the migration of fibroblasts in collagen gels, with changes in the hyaluronic acid synthesized (Schor et al., 1989). An autocrine motility factor (AMF; Liotta et al., 1986) isolated from conditioned medium of A2058 melanoma cells is a protein of apparent M , 55,000 (nonreduced) with a number of essential intrachain disulfide bonds ( M , 64,000 reduced). AMF-mediated chemotaxis involves a pertussis toxin-sensitive signal transduction pathway (Kohn et af., 1990b). A 55/64-kDa AMF was also isolated from conditioned medium of human HT1080 fibrosarcoma cells, and its interaction with responsive

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cells correlates with the phosphorylation of a 78-kDa membrane receptor (Watanabe et al., 1991). A similar AMF is produced by B16 melanoma cells and also binds to a 78-kDa cell surface glycoprotein (Nabi et al., 1990; Silletti et al., 1991). Structural and functional comparisons are desirable to determine how the 55/64-kDa AMF is related to other similarly acting proteins (Seiki et al., 1991; Yoshikawa and Sakuda, 19911, and how the phosphorylation of the 78-kDa receptor is involved with the pertussis toxin-sensitive signal transduction pathway. Another AMF, with an apparent M , of 30,000, was recently isolated from a conditioned medium of Dunning R-3327 rat prostate adenocarcinoma cells (Evans et al., 1991). The action of this AMF is inhibited by cholera toxin or forskolin, but not by pertussis toxin, and by this criterion more closely resembles the migration-stimulating action of EGF (Manske et al., 1990; and Section 111, G), FGF-1, and HGF (Bade et al., 1993a), which act through tyrosine kinase receptors, than the 55/64-kDa A2058-AMF. Determination of the structure and of the effect on cell proliferation of the differently isolated AMFs should make it possible to decide if they constitute a separate group or if they should be considered as additional members of the family of “typical” growth factors.

H. Other Factors In addition to the factors already mentioned, cytokines and other differentiation factors have also been described as influencing cell migration. Endothelial cells can be induced to migrate by endothelial cell growth factor (ECGF) (Fischer et al., 1990), as well as by erythropoietin (Anagnostou et al., 1990), granulocyte macrophage-colony stimulating factor (GM-CSF) and by granulocyte-colony stimulating factor (G-CSF) (Bussolino et al., 1989, 1991). Nerve growth factor, the neurite outgrowthinducing ability of which is well characterized (Levi-Montalcini and Angeletti, 1968), was found to stimulate the migration, not only of pheochromocytoma cells and primary cultures of the central nervous system (Jacobs and Stevens, 1987),but also of embryonal carcinoma cells (Kahan and Kramp, 1987) and even leukocytes (Boyle et al., 1985; Abramchik et al., 1988). Interleukins in turn, besides modulating the motility of leukocytes (Wankowicz et al., 1988), can stimulate the migration of osteoblasts (Gilardetti et af.,1991),fibroblasts (Postlethwaite and Seyer, 1991),epidermal cells (Martinet et al., 1988), smooth muscle cells (Nomoto et al., 1988), keratinocytes (Sauder et al., 1989), endothelial cells (Rosen et al., 1991b), and various tumor cells (Orr et al., 1988; Wang et al., 1990; Krueger et al., 1991).Furthermore, tumor necrosis factor-alpha can stimulate the migration of some fibroblasts and epithelial tumor cells (Postleth-

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waite and Seyer, 1990; Rosen et al., 1991c), while interferon beta has been found to inhibit the migration of human fibroblasts (Melchiori et al., 1987) and interferon gamma that of keratinocytes (Nickoloff er ul., 1988).

IV. Conclusions and Outlook

The analysis of cell migration has come of age. Biochemical and molecular biology studies are beginning to reveal a relatively common pattern of changes in gene expression associated with migration and to provide information on the specific signal transduction mechanisms involved. The apparent uniformity of some changes for different cell types and growth factors, and their conservation under in uirro conditions seem to indicate their very basic, essential nature for the migratory reaction. The development of migration systems of increasing complexity, and the application of continuously refined analytical tools for the analysis of single-cell behavior should allow a clarification of those changes which define cell and situation specificity at the level of the organism. Once that stage has been reached, attempts to modify the course of abnormally regulated migration processes, especially during tumor development, may become feasible. In the meantime, however, the further study of cell migrations in uitro and the related extrapolations to and analysis of in uiuo situations will provide significant descriptive information on the control mechanisms operating during development, regeneration, wound healing, and tumor development. This improved knowledge will suggest alternatives for better handling of the many disease situations in which the regulation of cell migration reactions is altered.

Acknowledgments We thank Nuri Giiven for Fig. 3. Susanne Feindler for Fig. 7. Heike Schramke for Fig. 8, and Bettina Nusser for Fig. 10. The work described from the authors' laboratory was supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 156.

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cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature (London) 309, 418-424. Verschueren, H . , Dekegel, D., and De Baetselier. P. (1986). Development of a monolayer assay (MLA) for the discrimination and isolation of metastatic lymphoma cells. Invasion Metastasis. 7 , 1-15. Wach. F.. Hein, R.. Adelmann-Grill. B. C., and Krieg, T. (1987). Inhibition of fibroblast chemotaxis by superoxide dismutase. E m . J . Cell Biol. 44, 124-127. Walicke. P. A. (1988a). Basic and acidic fibroblast growth factors have trophic effects on neurons from multiple CNS regions. J . Neurosci. 8, 2618-2627. Walicke. P. A. (1988b). Interactions between basic fibroblast growth factor (FGF) and glycosoaminoglycans in promoting neurite outgrowth. Exp. Neurol. 102, 144-148. Walicke. P. A., Cowan, W. M., Ueno. N., Baird. A., and Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. U.S.A. 83, 3012-3016. Wang. J. M.. Taraboletti, G., Matsushima. K.. Van, D. J., and Mantovani. A. (1990). Induction of haptotactic migration of melanoma cells by neutrophil activating protein/ interleukin-8. Biochem. Biophys. Res. Commun. 169, 165-170. Wankowicz,, Z., Megyeri, P., and Issekutz, A. (1988). Synergy between tumour necrosis factor alpha and interleukin-l in the induction of polymorphonuclear leukocyte migration during inflammation. J . Leukoc. Biol. 43, 349-356. Watanabe, H.. Carmi, P . , Hogan. V., Raz. T., Silletti, S . , Nabi, 1. R., and Raz, A. (1991). Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor. J . Biol. Chem. 266, 13442-13448. Weidner. K . M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart, S . , Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuhara, Y., e t a / . (1991). Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Natl. Acad. Sci. U . S . A . 88, 7001-7005. Westermark. B., and Wasteson, A. (1976). A platelet factor stimulating human normal glial cells. Exp. Cell Res. 98, 170-174. Westermark, B.. Magnusson, A., and Heldin. C.-H. (1982). Effect of epidermal growth factor in membrane motility and cell locomotion in cultures of human glioma cells. J . Neurosci. Res. 8, 491-507. Westermark, B . , Siegbahn, A.. Heldin, C. H.. and Claesson-Welsh, L. (1990). B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase. Proc. Natl. Acad. Sci. U . S . A . 87, 128-132. Westermark, K., Nilsson, M.. Ebendal, T., and Westermark, B. (1991).Thyrocyte migration and histiotypic follicle regeneration are promoted by epidermal growth factor in primary culture of thyroid follicles in collagen gel. Endocrinology (Baltimore)129, 2180-2186. Wiseman. D. M.. Polverini, P. J., Kamp, D. W., and Leibovich, S. J. (1988). Transforming growth factor-beta (TGF beta) is chemotactic for human monocytes and induces their expression of angiogenic activity. Biochem. Biophys. Res. Commun. 157, 793-800. Yanagi, H . , Sasaguri. Y.. Sugama, K.. Morimatsu, M., and Nagase, H. (1991). Production of tissue collagenase (matrix metalloproteinase I ) by human aortic smooth muscle cells in response to platelet-derived growth factor. Arherosclerosis (Shannon,Ire/.)91,207-216. Yang. E. Y., and Moses, H. L. (1990). Transforming growth factor beta I-induced changes in cell migration, proliferation, and angiogenesis in the chicken chorioallantoic membrane. J . Cell B i d . 111, 73 1-741. Yayon, A., Klagsbrun, M . . Esko. J. D., Leder, P., and Ornitz, D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell (Cambridge, Muss.) 64, 841-848.

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Physiological and Biochemical Aspects of Cytoplasmic Streaming Teruo Shimmen and Etsuo Yokota Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-12, Japan

I. Introduction Cytoplasmic streaming has been reported in various plant species ranging from algae to higher plants and fungi. In addition, cytoplasmic streaming can be observed in cells of various plant organs. For example, when a tomato seed germinates, a young root appears first and a large number of root hairs appear at the subapical region of the root. The surface area of the root increases remarkably by generating root hairs and this increase in surface area is important in absorbing inorganic nutrients. These nutrients must be transported to conducting tissue, such as xylem. If they were transported by passive diffusion, it would take a long time. However, since the cytoplasm circulates in the longitudinal direction of root hairs at the speed of several micrometers per second, the absorbed nutrients are effectively transported. There are also large numbers of hairs at the surface of the stem, petioles, and leaves of the tomato plant and active cytoplasmic streaming occurs in these hair cells as well. When the tomato plant matures, flowers develop. Pollen germinates on the stigma of the pistil and the pollen tube extends to the ovule. Active cytoplasmic streaming is also observed in the pollen tube. It is assumed that cytoplasmic streaming is responsible for the intracellular transport of molecules and cell organelles in such long cells. After fertilization, the ovary develops into a fruit. The mature tomato fruit is composed of large, loosely packed cells that contain carotenoid pigments. In these cells, transvacuolar strands of cytoplasm develop and active cytoplasmic streaming is also observed. The pattern of cytoplasmic streaming is variable among cells in various species and has been classified into many types: (1) agitation, (2) circulation, (3) rotation, (4) fountain streaming, ( 5 ) cytoplasmic streaming along Inrernationol Reuien, of Cvrologv. V d . 155

97

Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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definite tracks, and (6) tidal streaming (Kamiya, 1959). The velocity of streaming is also variable. The most rapid and well-organized streaming is observed in cells of the Characeae algae, sometimes reaching more than 100 pm/sec. Recent studies have revealed that the motive force for cytoplasmic streaming in most plant cells is generated by the actin-myosin system, which is also responsible for generating the motive force in muscle contraction and ameboid movement, etc. The velocity of organelle movement in plasmodium of the slime mold reaches more than I mm/sec and organelle movement in the plasmodium has been sometimes classified as a seventh type of cytoplasmic streaming (shuttle streaming). Although the motive force is generated by the actin-myosin system in both plasmodium and plant cells surrounded by a cell wall, the organization of the cytoskeletal proteins that produce the motive force is different (Shimmen, 1992). It is believed that the mechanism that generates motive force in plasmodium is similar to that in ameboid movement (Shimmen, 1992). This chapter deals solely with organelle movement observed in cells surrounded by a cell wall. Since the mechanism of cytoplasmic streaming has been elucidated mainly using cells of Characeae, we will focus on results obtained in characean cells.

II. Mechanism of Motive Force Generation A. Site of Motive Force Generation

Until the elegant work by Kamiya and Kuroda (1956), the mechanism for generating the motive force in cytoplasmic streaming was controversial. It was unclear whether the motive force was generated by the flowing endoplasm itself or produced by a protoplasmic system surrounding the streaming endoplasm (Kamiya and Kuroda, 1956). In solving this problem, experiments using characean cells were indispensable. In most plant cells, streaming direction and velocity vary with time. In such materials, measuring streaming velocity per se is difficult. In cells of Characeae, the cytoplasm streams in a fixed direction with a constant velocity (rotation). Therefore, measurement of the streaming velocity is much easier in characean cells. In addition, the internodal cells or branchlet cells of the Characeae are exceedingly large. These cells are cylindrical, and the diameter and length sometimes reach 1 mm and 10 cm, respectively. Therefore, various operating techniques can be applied; for example, ligation with thread, cutting, and intracellular perfusion. Owing to these advantages,

CYTOPLASMIC STREAMING

99

the most important information about cytoplasmic streaming has been obtained from experiments using characean cells. The outermost layer of these cells is the cell wall, which is composed of cellulose and various hemicelluloses. The plasma membrane is attached to the inside of the cell wall (Fig. 1). The gel ectoplasm is located just inside the plasma membrane (not shown in Fig. 1) and the chloroplasts are attached to the ectoplasm. The sol endoplasm actively streams inside the chloroplast layer with a constant velocity. The central part of the cell is occupied by a very large vacuole surrounded by the tonoplast. Kamiya and Kuroda (1956) unequivocally demonstrated the mechanism of motive force generation by simply analyzing the distribution of velocity. There is no significant velocity gradient in the endoplasm, that is, endoplasm flows as a mass. On the other hand, a gradient of velocity is observed in the vacuole. The velocity is highest just inside the tonoplast and decreases toward the central part. Kamiya and Kuroda (1956) prepared an internodal cell that was completely filled with endoplasm and lacked the vacuole by centrifuging and subsequently ligating the cell. They then analyzed the velocity distribution. The velocity was highest at the peripheral layer and gradually decreased toward the central part. Based on these observations, Kamiya and Kuroda (1956) concluded that interaction of organized gel surface and sol phase produces the shearing force which brings about the interfacial slippage (“sliding theory”). That is, the sliding force is generated just inside the gel layer and consequently the bulk endoplasm and cell sap are passively transported. Endoplasm streams as a mass, since the viscosity of the endoplasm is significantly higher than that of the cell sap. This theory is also supported by the fact that an endoplasmic drop isolated from characean cells that lack any ectoplasm does not stream, indicating that endoplasm alone cannot generate the motive force (Kamiya and Kuroda, 1956). The sliding theory proposed by Kamiya and Kuroda corresponds to the sliding theory proposed for

FIG. 1 Longitudinal section of an internodal cell of Characeae. w, cell wall; p. plasma membrane; t , tonoplast; v, vacuole; e, endoplasm; a, actin cable; c , chloroplast layer. Arrow indicates direction of cytoplasmic streaming.

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TERUO SHIMMEN AND ETSUO YOKOTA

the mechanism of muscle contraction (Huxley and Niedeergerke, 1954; Huxley and Hanson, 1954). Later it was established that the motive force of both cytoplasmic streaming and muscle contraction is generated by the same molecular mechanism: the sliding between actin and myosin molecules. 6 . Involvement of Actin-Myosin System

1. Morphological and Physiological Approach in Characeae The sliding theory proposed by Kamiya and Kuroda (1956) explicitly showed the importance of the boundary between the gel layer and the sol layer (sol-gel interface). Attention has been focused on the elucidation of the entity that generates the motive force at the sol-gel interface. Kamitsubo (1966) found cables at the inner surface of chloroplasts where the motive force is generated (Fig. 2). Each cable was found by electron microscopy to be composed of about 100 microfilaments (Nagai and Rebuhn, 1966). When a small part of an internodal cell of Characeae is strongly illuminated under a microscope, the chloroplast of the area is removed together with the cables. In the chloroplast (actin cable)-free area (i.e., window), cytoplasmic streaming is strongly inhibited. After a few days, cables are regenerated at the chloroplast-free area and cytoplasmic streaming recovers concomitantly with the regeneration of the

FIG. 2 Actin cables of an internodal cell, Cham corallina. Bar

=

20 pm.

CYTOPLASMIC STREAMING

101

cables, indicating that the cables are involved in the generation of the motive force (Kamitsubo, 1972). Cables regenerate from the end of the delivering endoplasm to the damaged area and not from cables downstream from the window (Williamson et al., 1984). The microfilaments composing the cables were identified as actin filaments using heavy meromyosin (HMM), which is prepared by digesting myosin by trypsin (Williamson, 1974; Palevitz e? al., 1974; Palevitz and Hepler, 1975; Kersey and Wessels, 1976)and fluorescently labeled phallotoxin (Barak et al., 1980; Nothnagel e? al., 1981) or an antibody raised against actin (Owaribe et al., 1979; Williamson and Toh, 1979). The involvement of actin filaments in motive force generation was also supported by pharmacological studies, that is, cytochalasin, a specific inhibitor of actin-based motility, reversibly inhibits cytoplasmic streaming of characean cells (Williamson, 1972; Bradley, 1973; Nagai and Kamiya, 1977; Nothnagel el al., 1981; Shimmen and Tazawa, 1982a, 1983). The involvement of actin in cytoplasmic streaming necessarily evoked the idea that, by analogy to muscle, myosin is also involved in generating the motive force. Kato and Tonomura (1977) purified myosin from Nitella JIexilis. The chemical properties of characean myosin were similar to skeletal muscle myosin. It was speculated that myosin is localized in the flowing endoplasm, since the motive force of cytoplasmic streaming is generated by sliding between the gel surface (actin bundles) and the endoplasm. This possibility was supported by experiments done by Chen and Kamiya(1975, 1981)and Kamitsubo (1981). In skeletal muscle, it has been reported that myosin is much more labile to SH reagents or heat than actin is (Sibata-Sekiya and Tonomura, 1975; Tonomura and Yoshimura, 1962; Yamaguchi e? al., 1973; Hatano and Owaribe, 1977; Yasui et al., 1958). Chen and Kamiya (1975, 1981) prepared a special double chamber in which a cell was partitioned into two parts and could be centrifuged. By applying weak centrifugal force to the cell in the longitudinal direction, the flowing endoplasm was made to accumulate in one part of the cell (Fig. 3). In the first experiment, the cell part lacking endoplasm was treated with an SH reagent (NEM; N-ethylmaleimide) or heat (47.SoC,3 min) and the part containing the endoplasm was not treated. Then, the endoplasm was translocated to the NEM (or heat) treated cell part by applying a centrifugation force in the opposite direction. In this case, the endoplasm resumed streaming normally. In the second experiment, the cell part containing the endoplasm was treated with NEM or heat, and the treated endoplasm was then translocated to the untreated cell part. In this case, cytoplasmic streaming did not recover. Thus, it is evident that the endoplasm is NEM or heat labile and the gel layer (actin cables) is resistant to these treatments.

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Exp. 2

Exp. 1

........................

1 c

, . ....,.. .... ...

......

c

c

2

3

4

5

6 Normal streaming

Endoplasm stationary

FIG. 3 Differential treatment of gel ectoplasm and endoplasm of characean cell with NEM or heat. Exp. I: Treatment ofectoplasm. Exp. 2: Treatment ofendoplasm. ( I ) First centrifugation, (2) NEM or heat treatment, (3) 0-5 min after treatment, (4) second centrifugation, ( 5 ) 5-10 min after second centrifugation, (6) 15-20 min after second centrifugation. For further explanation, see the text. (Modified from Chen and Kamiya, 1981. with permission.)

Kamitsubo (1981) treated half of a noncentrifuged Nitelfa cell with heat. Cytoplasmic streaming stopped in the treated part of the cell but it continued in the untreated half. When the endoplasm of the untreated half streamed into the treated half, it continued to stream along the gel layer of the treated half. This experiment also supports the above conclusion. It is supposed that myosin is the component labile to NEM or heat in the endoplasm. The next question that arises concerns the localization of the myosin in the endoplasm. Nothnagel and Webb (1982) and Yoneda and Nagai (1988) proposed the importance of the association of myosin with organelles. This idea has been also supported by a morphological approach. Using tonoplast-free cells, which will be discussed later, the intracellular ATP can be completely depleted by intracellular perfusion. By analogy to skeletal muscle, it is assumed that myosin makes a so-called rigor crossbridge with the actin cables. In practice, Williamson (1975) and Nagai and Hayama (1979) found that in ATP-depleted cells, the streaming organelles stopped moving when they associated with actin cables. Nagai and Hayama (1979) found that rod or horn-like protuberances were associated with the organelles, and

CYTOPLASMIC STREAMING

103

Streaming

-

FIG. 4 Postulated mechanism for the generation of motive force for cytoplasmic streaming in characean cells. C, chloroplast; A, actin cables; M, myosin; 0, streaming organelle. (Reproduced from Jackson, W. T. (1992). Actomyosin, I n “The Cytoskeleton in Plant Growth and Development” (C. W. Lloyd, ed.). p. 19. Academic Press, London.)

small globular bodies 20-30 pm in diameter were arranged on the surface of the protuberances. The small globular bodies were associated with actin cables. The authors concluded that these bodies may be a part of myosin. The organelles have not been identified yet (Fig. 4). Williamson (1979) found that filaments 44 nm in diameter and up to 3 pm in length were associated with the endoplasmic reticulum and suggested that myosin is contained in the filament. Kachar and Reese (1988) also showed the possible association of myosin with the endoplasmic reticulum. Since in tonoplast-free cells, the endoplasm is drastically disorganized, large organelles such as endoplasmic reticulum might be disorganized. In onion epidermis cells, the close relation between actin filaments and the endoplasmic reticulum also evokes the possibility of an association of myosin with the endoplasmic reticulum (Quader et al., 1987). 2. Identification of Myosin in Various Plants Using HMM tagging, it was found that the cytoplasm streams from the pointed (minus) end to the barbed (plus) end of the actin filaments in characean cells (Kersey et al., 1976). In addition, pollen tube organelles slide along characean actin cables from the pointed ends to the barbed ends in reconstitution experiments, which will be discussed later (Kohno et al., 1990). The polarity of movement of plant organelles along actin filaments is the same as that of skeletal muscle myosin. At this point, it is reasonable to call the endoplasmic factor responsible for cytoplasmic streaming “myosin.” A biochemical approach has been used to find plant myosin (Table I). In most cases, it has been reported that plant myosin forms bipolar thick filaments in low ionic strength solution (Kato and Tonomura, 1977;Oksuka and Inoue, 1979; Sokolov et al., 1985). In addition, a cycle of high and low ionic strength treatment was applied during purification on the assump-

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TERUO SHIMMEN AND ETSUO YOKOTA

TABLE I Heavy Chain of Plant Myosins Studied by Biochemical Purification or lmmunoblotting

Plant species

Molecular weight (kDa)

Nitella

200

Egeria

180

Tomato Pea Lilium Allium

I00 165 170 200

Chara

110. 200

Alabidopsis

170

Mung bean

158. 170

Nicotianu

I75

Ernodesmis

85, 220

Methods

References

Purification Purification Purification Purification Purification Immunoblotting Immunoblotting Immunoblotting

Kato and Tonomura (1977) Ohsuka and Inoue (1979) Vahey et al. (1982) Ma and Yen (1989) Yokota and Shimmen (1994) Parke et al. (1986) Grolig et al. (1988) Qiao et al. (1989) Qiao et al. (1989) Tang et al. (1989) La Claire (1991)

Immunoblotting Immunoblotting Immunoblotting

tion that plant myosin is solubilized in high ionic strength solution and forms thick bipolar filaments in low ionic strength solution, as in the case of skeletal muscle myosin. Immunoblotting is a convenient method to identify protein and has recently been applied to some plant species using antibodies raised against heavy chains of myosin I1 of animals or cellular slime molds. The molecular weight of the components identified as the heavy chain varied significantly among plant species (Table I). Cytochemical localization using an antibody against animal myosin reveals an association of myosin with organelles in pollen tubes (Tang et ul., 1989; Heslop-Harrison and Heslop-Harrison, 1989), and characean internodal cells (Grolig et al., 1988). However, it has been reported that myosin antibody does not always cross-react with myosin of other species (Cote et al., 1985; Hagen et al., 1986; Carboni et al., 1988; Fukui et al., 1989; Collins et al., 1990). Recently, we found that an antibody raised against the heavy chain of lily pollen tube myosin does not cross-react with the heavy chain of skeletal muscle myosin (Yokota and Shimmen, 1994). Thus, it seems dangerous to use antibodies raised against animal myosin when identifying plant myosin. Given this situation, we tried purifying myosin using a completely different strategy. Since most plant cells are occupied by a large central vacuole, it is suspected that myosin may be digested by proteolytic enzymes or may be denatured by phenolic substances contained in the vacuole due to disruption of intracellular compartmentation when homogenizing cells.

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CYTOPLASMIC STREAMING

We used lily pollen tubes, since the vacuole does not develop much in the first stages of germination. In addition, we found that myosin retains its sliding activity after homogenization of pollen tubes (Kohno and Shimmen, 1988a). The most important strategy in our experiment was developing a method to check the activity of the myosin. Since myosin is a mechanochemical enzyme that generates a motive force by hydrolyzing ATP, ATPase activity is sometimes used as a marker during purification steps (Oksuka and Inoue, 1979; Vahey et al., 1982). However, plant cells contain various kinds of ATPase activities and it may be difficult to distinguish between myosin ATPase activity and the activities of other ATPases. In the study of microtubule-based motility, a novel motor enzyme, kinesin, was discovered in nerve cells (Vale er al., 1985). In this study, the authors ignored the peak of ATPase activity and collected fractions showing sliding activity between microtubules (Vale et al., 1985). Sliding between actin filaments is also the most specific activity of myosin. In a study of skeletal muscle, an in vitro motility assay was developed (Kron and Spudich, 1986), in which myosin is fixed on the glass surface and actin filaments fluorescently labeled with rhodamine (or F1TC)-phalloidin are applied together with Mg-ATP. Under such conditions, the sliding of actin filaments along myosin can be observed with an epifluorescence microscope equipped with a high-sensitivity video camera (Fig. 5). Thus, we can check the activity of myosin using an in vitro motility assay during the purification steps. We could partially purify myosin from plant materials while maintaining sliding activity between actin filaments for the first time (Kohno et al., 1992). By SDS-PAGE analysis, it was found that the plant myosin contains a heavy chain of 120 kDa. The velocity of sliding between pollen tube

actin filament myosin

FIG. 5 Motility assay of myosin in uitro. Myosin molecules are fixed at the surface of a cover glass. Actin filaments labeled with rhodamine (or F1TC)-phalloidin slide on the surface using ATP energy. The movement of actin filaments can be observed with an epifluorescence microscope equipped with a video camera of high sensitivity.

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TERUO SHIMMEN AND ETSUO YOKOTA

myosin and actin filaments was 1-2 pmlsec, which is one-third of the velocity of cytoplasmic streaming observed in the pollen tubes in uiuo. Later, however, we found that the 120-kDa polypeptide was a proteolyzed fragment of the heavy chain during purification. It is conceivable that even young pollen tubes also contain certain amounts of proteolytic enzymes. We further modified the purification procedures by adding casein to protect the myosin from proteolysis. Finally, we isolated myosin containing a 170-kDa heavy chain (Yokota and Shimmen, 1994).The velocity at which the actin filaments slid along pollen tube myosin in an in uitro motility assay was about 7 pmlsec, which is almost the same as that of cytoplasmic streaming in pollen tubes in uiuo. A polyclonal antibody was raised against the 170-kDa heavy chain and immunoblotting was carried out using a crude extract from pollen tubes. It was found that only a 170-kDa component cross-reacted with the antibody, indicating that the 170-kDa component is not a proteolytic product that is formed during purification, but a native form. The Mg-ATPase activity was enhanced up to 60-fold by actin filaments of skeletal muscle. This is also one of the most important properties of myosin. Recently, at least two types of myosin have beeen reported in animal and ameba cells-conventional myosin (myosin 11) and an unconventional myosin (myosin I) (Pollar and Korn, 1972, 1973; Maruta et al., 1979; Pollard et al., 1978, 1991; Maruta and Korn, 1977). Myosin I1 has been generally reported in various animals, typically in skeletal muscle. This myosin has two heads and forms bipolar thick filaments in low ionic strength solution. On the other hand, myosin I has a single head and does not form bipolar filaments. The question arises whether pollen tube myosin belongs to myosin I or myosin 11. To get a final answer, the head structure of pollen tube myosin must be examined with an electron microscope. Myosin I isolated from Acanthoamoeba has a domain that binds to the membranes and is capable of transporting organelles along actin filaments (Adams and Pollard, 1986, 1989a,b; Pollard et al., 1991). From this point of view, plant myosin may belong to myosin I, since the motive force of cytoplasmic streaming is generated by myosin bound to the surface of organelles. The formation of thick filaments is important for contraction in muscle or ameboid cells (Shimmen, 1992). In generating the motive force of cytoplasmic streaming, which is apparently different from muscle contraction, the formation of thick filaments may not be necessary. The myosin of pollen tubes can be extracted without increasing the ionic strength (Kohno etal., 1991,1992; Yokota and Shimmen, 1994),indicating that pollen tube myosin does not form thick filaments (or the filaments are very small). The velocity of cytoplasmic streaming of many plant cells is in the range of a few micrometers per second to a several micrometers per second

107

CYTOPLASMIC STREAMING

(Kamiya, 19591, which is close to the velocity of actin-myosin sliding in skeletal muscle (Crowder and Cooke, 1984). However, the velocity of cytoplasmic streaming of characean cells is extraordinarily high and sometimes reaches 100 pmlsec. When the surface of the cover glass used in an in uitro motility assay was coated with a crude extract of characean

0

a

.

*

I

Vacuolar perfusion

b

Disintegration of tonaplast

x*

~

X ' X

xfi;i;zx.Xx

I

Inactivation and removal of endoplasm

~

~

x

x

d

1

Perfuslon with organelles or myosin-coated beads

e Procedure for preparation of tonoplast-free cells and reconstitution of myosin movement on characean actin cables. (a) intact cell, (b) vacuole-perfused cell, (c) tonoplast-free cell, (d) endoplasm-free cell, (e) reconstituted movement. The arrow in the cell shows the direction of cytoplasmic streaming (a, b, c) or reconstituted movement (d). (Adapted from Shimmen, 1988b. with permission.) FIG. 6

~

108

TERUO SHIMMEN AND ETSUO YOKOTA TABLE II Velocity of Reconstituted Sliding Using Actin and Myosin of Skeletal Muscle and Characeae in Various Combinations

Origin of actin

Origin of myosin

Velocity

Skeletal muscle Skeletal muscle Characeae

Skeletal muscle Characeae Skeletal muscle

Slow Rapid Slow

Characeae

C haraceae

Rapid

cytoplasm, actin filaments of skeletal muscle slid at a velocity close to that of cytoplasmic streaming in characean cells (this experiment has been carried out in four laboratories: Fujime group, Shimmen group, Yamamoto group and Ogihara group). Using reconstitution experiments based on characean actin (Fig. 6) and in vitro motility assays (Fig. 5 ) , it is possible to reconstitute the sliding movement between actin and myosin of skeletal muscle and Characeae in various combinations (Table 11). As is evident, the velocity is dependent on the origin of the myosin but not the actin. Thus, characean myosin has a capacity to move along actin filaments at a very high velocity. Further elucidation of plant myosin, including genetic analysis, is required for understanding the mechanism of cytoplasmic streaming.

3. Identification of Actin in Various Plants Studies of actin have progressed further than those concerned with myosin. In various kinds of plant cells, the presence of actin filaments has been reported using electron and light microscopy, and actin has also been purified (reviewed in Staiger and Schliwa, 1987). A dynamic change in the organization of actin during the cell cycle has been also reported (Clayton and Lloyd, 1985; Heslop-Harrison and Heslop-Harrison, 1986; Mole-Bajer et al., 1988;Seagull et al., 1987;Traas et al., 1987). In addition, genetic analysis has progressed significantly and it was found that the amino acid sequence of plant actin is conserved and is similar to those of animal, fungal, or protist actins (see Meagher, 1991). Purified plant actin can polymerize to form filaments (Vahey et al., 1982; Andersland et al., 1992) and activates myosin ATPase (Vahey et al., 1982; Liu and Yen, 1992), as do animal actins. In addition, it was found that actin purified from yeast can slide along muscle myosin (Kron et al., 1992).

CYTOPLASMIC STREAMING

C. Cytoplasmic Streaming Supported by a Microtubule System

In the majority of plant cells, cytoplasmic streaming is supported by the actin-myosin system. However, this is not always the case. In all plant cells, microtubules are generally observed just inside the plasma membrane. This microtubules are called cortical microtubles and are believed to be concerned with the orientation of cell wall microfibrils (Lloyd, 1982). In some algae, large numbers of microtubules are organized not only in the periphery but also in an area distant to the plasma membrane and they are supposed to be concerned with cytoplasmic streaming. Such microtubules were found in Caulerpa by Sabnis and Jacobs (1967) and it was suggested that they may influence cytoplasmic streaming directly or indirectly. Kuroda and Manabe (1983) found that cytochalasin did not inhibit cytoplasmic streaming but colchicine reversibly inhibited it in Caulerpa. In addition, cytoplasmic streaming was found to be inhibited by 3-(2-hydroxynonyl) adenin (EHNA) which is an inhibitor of dynein, a motor protein which slides along microtubules. Thus, it is assumed that the motive force of cytoplasmic streaming in Caulerpa may be produced by the microtubule-dynein system. Later, Schliwa ef al. (1984) reported that EHNA also inhibits actin-based motility, including cytoplasmic streaming of Chura. This must be examined further. The inhibition of cytoplasmic streaming by antimicrotubule agents and the ineffectiveness of cytochalasin was also reported in Bryopsis (Mizukami and Wada, 1981) and in Dichotornosiphon (Maekawa et al., 1986). Menzel and Elsner-Menzel (1989) analyzed organelle movement in the extruded cytoplasm of Bryposis. They found that the tracks along which particles moved were identical with the arrays of microtubules and that an antimicrotubule agent inhibited the particle movement but cytochalasin did not. This supports the idea that the microtubule system is concerned with cytoplasmic streaming in Bryopsis. In Acetabularia, the situation seems more complex. In this alga, two types of streaming are observed; one is observed along definite tracks of thin filaments and another consists of headed streaming bands. Along the definite tracks, chloroplasts move at a velocity of 1-2 pm/sec. In headed streaming bands, the small vesicles, polyphosphate granula, and secondary nuclei are transported at a velocity of 3-1 1 pm/sec (Koop and Kiermayer, 1980a). When the algal cell was treated with cytochalasin, both types of streaming were inhibited (Koop and Kiermayer, 1980b; Nagai and Fukui, 1981). However, antimicrotubule agents inhibited headed streaming but did not inhibit streaming along the definite tracks of thin

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filaments (Koop and Kiermayer, 1980b). It is suggested that not only actin but also microtubules are concerned with headed streaming in Acetabula ria.

111. Demembranated Cell Models of Characeae and Characteristics of Cytoplasmic Streaming A. Preparation of the Demembranated Cell Model

Various functions of cells are manifested by the action of corresponding biomolecules such as enzymes. The elucidate the function of an enzyme, in uitro experiments are useful, in which the targeted enzyme is isolated and characterized. The method which complements in uitro experiments is the in uiuo method, in which the function of the enzyme is studied in living cells. Each of these two methods has its advantages and disadvantages. In in uitro experiments, the function of the enzyme is analyzed in a well-characterized test solution but there is no guarantee that the function studied is really expressed in the cell. In in uiuo experiments, the function of the enzyme can be studied in situ but further detailed analysis is difficult, since the surrounding medium cannot be easily modified because of the presence of the plasma membrane, which acts as a diffusion barrier between the exterior and interior. To overcome the disadvantages of above two methods, a demembranated cell model was developed. In this model the plasma membrane is removed or its semipermeability is drastically altered, while intracellular structure is maintained. This technique was first developed in a study of cell motility and it is now used in various fields of cell biology.

1. Plasma Membrane-Permeabilized Cell Model Since the cell exterior and interior are separated by the plasma membrane, the most direct access to the cell interior is obtained by removing the plasma membrane or increasing its permeability. Such a model was first developed in the study of muscle contraction. Szent-Gyorgy (1949) prepared a glycerinated model in which the skeletal muscle was treated with glycerin solution at low temperature. Later, various detergentpermeabilized models were developed in which the phsopholipid of the plasma membrane was extracted with detergent. One of the most famous detergent models is the so-called Triton model developed by Gibbons and Gibbons (1972) to study the motility of sperm.

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A plasma-membrane permeabilized model of plant cells was first developed in characean cells (Shimmen and Tazawa, 1982a) by treating the cell with saponin. However, in the model thus prepared, the reactivated streaming continued for less than 10 min (Shimmen and Tazawa, 1982a). The model was later modified as follows without using detergent (Shimmen and Tazawa, 1983):The cell was first treated in an ice-cold ethylene glycolbis (@-aminoeth ylether)N,N ,N' ,"-tetraacetic acid (EGTA) medium and then transferred into an ice-cold EGTA medium whose osmolarity was made more hypertonic than that of the cell. This resulted in plasmolysis. By treating the cell with EGTA, a Ca2+ chelator, the integrity of the plasma membrane was partially impaired. Low temperature caused the fluidity of the membrane to decrease. Thus, upon detachment of the plasma membrane from the cell wall during plasmolysis under these conditions, the semipermeability of the plasma membrane was completely lost (Shimmen and Tazawa, 1983; Shimmen and MacRobbie, 1987). It was assumed that the plasma membrane splits upon detachment from the cell wall. In the model thus prepared, the reactivated streaming continued up to 60 min if the treatment was successful. Later, we noticed that the plasmolysis method is successful only in young characean cells and that for unknown reasons, in old cells the plasma membrane does not lose its semipermeability with this treatment. The model prepared by the plasmolysis method corresponds to skinned fibers in which the plasma membrane of the skeletal muscle is mechanically removed (Natori, 1954). In skinned muscle fibers, the membranes of organelles such as the endoplasmic (sarcoplasmic) reticulum remain intact and can be used to study excitationcontraction coupling (Endo, 1975a,b; Thorens and Endo, 1975). In permeabilized characean cells, the tonoplast remains intact and can be used to study ion transport through the tonoplast (Shimmen and MacRobbie, 1987; Tester et af., 1987). La Claire (1984) succeeded in permeabilizing the plasma membrane by treating the cell of Ernodesmis with saponin in a study of wound healing. 2. Tonoplast-Free Cells

A unique model was developed by taking advantage of the morphology of plant cells, especially Characeae (Williamson, 1975;Tazawa et al., 1976) (Fig. 6). Since the internodal cell of Characeae is large and cylindrical, both cell ends can be easily removed by cutting them with a scissors. After both cell ends are removed, a perfusion medium is introduced at one end and effused out from the other. At the beginning of the perfusion, only the cell sap is replaced with the perfusion medium. After replacement of the cell sap with the medium containing EGTA, the tonoplast disintegrates

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because of the Ca”-chelating action of EGTA. After the disintegration of the tonoplast, the chemical composition of the cytoplasm can be controlled by intracellular perfusion (tonoplast-free cell). Cytoplasmic streaming was also analyzed in Acetubuluriu using tonoplast-free cells (Fukui and Nagai, 1985). In characean cells, this model can be used for reconstituting streaming with exogenous myosin. By further perfusing the cell interior in the tonoplast-free cells, the flowing endoplasm can be completely removed but the actin bundles remain at the inner surface of the chloroplasts (endoplasm-free cell). When exogenous myosin attached to beads is introduced into the endoplasm-free cell, the myosin slides along characean actin bundles, resulting in the streaming of the beads (Shimmen and Yano, 1984; Shimmen, 1988b). Streaming can also be reconstituted by introducing organelles isolated from other species (Adams and Pollard, 1986; Kohno and Shimmen, 1988a). From such experiments, it is concluded that myosin is bound to the organelles.

3. Longitudinally Cut Model Other models have been developed to reconstitute the movement of actinmyosin (Fig. 7). In one case, an internodal cell is longitudinally cut open and attached on a glass surface, so that the inner surface of the cytoplasm is exposed. After the endogenous endoplasm is washed away, exogenous myosin is introduced (Kuroda, 1983; Sheetz and Spudich. 1983). B. Energy Requirements

Using tonoplast-free characean cells, it was demonstrated that cytoplasmic streaming is fueled by ATP (Williamson, 1975; Tazawa et al., 1976). If ATP is depleted by intracellular perfusion, cytoplasmic streaming completely stops and organelles strongly bind to the actin cables (Williamson, 1975; Nagai and Hayama, 1979). It was suggested that myosin bound to the organelles formed so-called rigor cross-bridges in the absence of ATP (Williamson, 1975). Using tonoplast-free cells, Shimmen (1978) quantitatively analyzed the relation between ATP concentration and the velocity of cytoplasmic streaming and found that it showed a hyperbolic curve, indicating that an enzymatic reaction is involved in the energization of cytoplasmic streaming by ATP. Maximum velocity was attained at ATP concentrations greater than 200 p M and Shimmen (1978) suggested that in intact cells of Characeae the ATP concentration (0.5-1.6 mM) is saturated with respect to supporting

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C

FIG. 7 Longitudinally cut model of Characeae. (a) An internodal cell was cut open at the sites shown with dotted lines. (b) The cell was separated into two halves. (c) Cell fragments were attached on the cover glass so that cell wall contacted the cover glass. (d) Side view of c. s, glass slide; w, cell wall; m, plasma membrane: ch, chloroplasts; mf, actin cable; en, endoplasm; t , tonoplast: md, bathing medium; N , nucleus. (Reproduced from Kuroda. 1983, with permission. Copyright of The Proceedings of the Japan Academy.)

cytoplasmic streaming. Reid and Walker (1983) treated characean cells with inhibitors of energy metabolism and analyzed the relation between the ATP concentration and the streaming velocity. They found a linear relationship between the ATP concentration and the velocity, and suggested that an increase in ADP caused by inhibiting energy metabolism was inhibitory to streaming. Shimmen (1988a) analyzed the effect of ADP, AMP, Pi, and pyrophosphate using tonoplast-free characean cells. He found that ADP, Pi, and pyrophosphate inhibit streaming in a manner competitive with ATP (Fig. 8). When a medium containing 1.6 m M ATP,

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I

0.2

I

I

I

I

0.4

0.6

0.8

I 1.0

CATPI (mM)

FIG. 8 ATP-dependence of cytoplasmic streaming and competitive inhibition by Pi in Nitellopsis. A tonoplast-free cell of Nitellopsis was perfused with media containing ATP of various concentrations with ( 0 )or without (0) 10 mM Pi. (Reproduced from Shimmen, 1988a. with permission.)

0.6 mM ADP, 0.8 mM AMP, 14.7 mM Pi, and 2 mM pyrophosphate (the concentration of these phosphates in the cytoplasm of intact cells) was introduced into the tonoplast-free cells, the velocity of cytoplasmic streaming was 96% of that of tonoplast-free cells containing only 1.6 mM ATP. When tonoplast-free cells were perfused with the medium containing ADP but lacking ATP, streaming was not observed at the very beginning but the velocity increased with time (Shimmen, 1978, 1988a). It was hypothesized that ADP was converted to ATP by the action of adenylate kinase bound to chloroplast. This was demonstrated by an experiment in which addition of an inhibitor of adenylate kinase to the ADP medium inhibited the increase of the streaming velocity after perfusion (Shimmen, 1988a).

C. Mg2+ Requirement It is suggested that Mg2+is required as a cofactor for the ATPase reaction of myosin in transducing the chemical energy of ATP into the sliding force, as is the case in muscle contraction. This was also demonstrated in tonoplast-free cells of characean alga (Williamson, 1975). Shimmen (1978, 1988a) also analyzed relation of the Mg2+ concentration and the streaming velocity. He found that at a constant ATP concentration, cytoplasmic streaming was severely inhibited, when Mg2+concentration was lower than the ATP concentration. In addition, when tonoplast-free cells

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were perfused with a medium containing a strong Mg2+ chelator, cytoplasmic streaming was irreversibly inhibited. This was also the case in plasma-membrane permeabilized cells (Shimmen and Tazawa, 1983). It is suggested that Mg2+ is necessary not only as a cofactor of myosin ATPase reaction but also as a factor responsible for maintaining the streaming system. We ordinarily used MgCI, to add Mg2+to the perfusion medium. When we used MgSO, instead of MgCl,, cytoplasmic streaming was inhibited, especially at lower ATP concentrations (Shimmen et al., 1990). By kinetic analysis, it was found that SO:- inhibits streaming in a manner competitive with ATP. D. pH Dependence

Since the motive force of cytoplasmic streaming is generated by the proteins, actin and myosin, it is expected that the streaming strongly depends on the intracellular pH. The effect of pH on cytoplasmic streaming was studied in tonoplast-free cells of Characeae (Fuji et al., 1979; Tazawa and Shimmen, 1982). After the tonoplast was removed by perfusing the vacuole with an EGTA-containing medium of neutral pH, the cell interior was perfused with EGTA media of various pHs and the velocity of the cytoplasmic streaming was measured. This velocity was highest at neutral pH and decreased at both lower and higher pH regions. The pH dependence obtained in tonoplast-free cells made it possible to elucidate the mechanism of inhibition of cytoplasmic streaming by a fatty acid, myrmicacin, which is secreted by a leaf-cutting ant (Schildknecht and Koob, 1971). It was reported that the fatty acid inhibits cytoplasmic streaming (Iwanami el al., 1981). However, the mechanism of inhibition remained unsolved. It was found that cytoplasmic streaming of characean cells is inhibited by myrmicacin only when the external medium is acidic. Tonoplast-free cells were treated with an acidic medium containing myrmicacin. After the velocity of cytoplasmic streaming was measured, the cell content of the tonoplast-free cell was collected by intracellular perfusion and its pH was measured. It was found that the extent of inhibition of cytoplasmic streaming in tonoplast-free cells was proportional to the acidification of the cell interior. It was also found that not only myrmicacin but other fatty acids inhibit cytoplasmic streaming in acidic external medium by lowering the intracellular pH. The relationship between intracellular pH and the velocity obtained in tonoplast-free cells treated with fatty acids, and that obtained in tonoplast-free cells whose pH was changed with intracellular perfusion, was completely consistent (Shimmen and Tazawa, 1985).

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E. Analysis of Cytoplasmic Streaming by Reconstitution Experiments The mechanism of cytoplasmic streaming has been mainly elucidated by physiological studies using characean cells, i.e. the motive force of cytoplasmic streaming is generated by sliding of myosin along actin filaments. Although actin filaments can be easily observed as a cable, even with a light microscope, information concerning myosin is scanty. Physiological studies suggested the presence of myosin in the flowing endoplasm as a NEM, a heat-sensitive component (Chen and Kamiya, 1975, 1981; Kamitsubo, 1981). The presence of myosin in the flowing endoplasm was also supported by reconstitution experiments (Shimmen and Tazawa, 1982a). A tonoplast-free cell was prepared by perfusing an internodal cell of Nitella axillif'rmis and its putative myosin was irreversibly inactivated by treating the cell with NEM. On the other hand, about 40 tonoplastfree cells were prepared by perfusing internodal cells of Chara curaffina. Flowing endoplasm was collected from tonoplast-free cells of C. coralfina by intracellular perfusion. The organelles were separated from the cytosolic components by centrifugation. When the organelles of C . coralfina, suspended in a medium containing Mg-ATP, were introduced into the endoplasm-free cell of N. axillifurmis, the organelles moved along the actin cables at 16-50 pm/sec, indicating that myosin is associated with organelles and that Chara myosin can interact with Nitella actin cables to generate the motive force (Shimmen and Tazawa, 1982b). Although this reconstitution experiment suggested an association of myosin with organelles, this association was not directly demonstrated. It is assumed that such an association is important for generation of the motive force, as suggested by mathematical modeling (Nothnagel and Webb, 1982; Yoneda and Nagai, 1988). We tried to mimic the situation in characean cells by using identified myosin. We fixed myosin isolated from skeletal muscle of rabbit on the surface of latex beads coated with poly-L-lysine. When myosin-coated beads suspended in Mg-ATP medium were introduced into the endoplasm-free cell, they moved along the characean actin cables at a velocity of 1-10 pm/sec (Shimmen and Yano, 1984, 1986), supporting the hypothesis of the association of myosin with organelles. In this reconstitution experiment, the skeletal muscle myosin corresponds to characean myosin and the latex beads correspond to characean endoplasmic organelles. Prior to our publication (Shimmen and Yano, 1984), Sheetz and Spudich (1983), using the longitudinally cut system, also succeeded in reconstituting a sliding movement between characean actin cables and skeletal muscle myosin fixed on the surface of beads. As early as 1975, Kudoda and Kamiya showed that skeletal muscle myosin can interact with characean actin to generate a motive force. They

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isolated endoplasmic drops from an internodal cell in which chloroplasts actively rotated. The rotation of chloroplasts is caused by the interaction of actin cables fixed around the chloroplast and myosin in the endoplasm surrounding the chloroplast. After the surface membrane of the endoplasmic drop was mechanically broken in a medium containing Mg-ATP, the rotation of chloroplasts was irreversibly inhibited by treating the demembranated endoplasmic drop with NEM (inactivation of characean myosin). When HMM prepared from rabbit skeletal muscle myosin was applied, the chloroplasts began to rotate slowly. In 1983, Kuroda also succeeded in restoring characean streaming with skeletal muscle HMM using the longitudinally cut system. After longitudinally cutting open the internodal cell, she inactivated the streaming with NEM. The characean myosin was irreversibly inactivated by NEM, and organelles remained fixed to the actin cables. When Kuroda added skeletal muscle HMM, the characean organelles began to move. In the reconstitution experiments performed by Kuroda and Kamiya (1975) and Kuroda (1983), solubilized myosin was applied directly. HMM might generate the motive force by spontaneously associating with the characean organelles, or the soluble HMM could generate a motive force without an association with organelles. Since actin filaments are generally observed in various plant cells, and cytoplasmic streaming in these cells is inhibited by cytochalasins (Staiger and Schliwa, 1987),it is suggested that the actin-myosin system is responsible for generating the motive force of streaming in plant cells. In analogy to characean cells, it is assumed that the motive force is generated by the sliding of myosin associated with organelles along filaments of actin. The plausibility of this assumption is also supported by reconstitution experiments. Kohno and Shimmen (1988a) isolated organelles from lily pollen tubes. When a suspension of pollen tube organelles was introduced into an endoplasm-free characean cell, the organelles moved along the characean actin cables, supporting the idea that myosin is associated with organelles and the motive force of streaming in lily pollen tubes is generated by the sliding of myosin along actin filaments. Pollen tube organelles moved from the pointed end (minus end) to the barbed end (plus end) of characean actin cables, as does skeletal muscle myosin and characean “myosin” (Kohno et af., 1990). During the experiment, we noticed that the association of myosin with the organelles was weak, since the motility of the organelles was drastically impaired by repeatedly washing them before introducing them into an endoplasm-free characean cell. This possibility was also supported by the observation that the pollen tube myosin could be extracted without drastically increasing ionic strength of the extraction medium (Kohno et af., 1991, 1992; Yokota and Shimmen, 1994).

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F. inhibition of Cytoplasmic Streaming of Ca2+

The effect of intracellular Ca2+on cytoplasmic streaming can be observed by treating plant cells with A23187, a Ca2+ionophore, in the presence of extracellular Ca2+.This treatment resulted in the inhibition of cytoplasmic streaming in pollen tubes (Herth, 1978; Koho and Shimmen, 1988b), in stamen hair cells of Tradescantia (Doree and Picard, 1980),and in trichome cells of Lycoperisicon (Woods et al., 1984b). The effect of Ca2+ can be more directly tested by microinjecting Ca2+into the cytoplasm. Hayama and Tazawa (1980) isolated endoplasmic drops from internodal cells of Chara, in which chloroplasts were rotating as a result of the sliding between actin cables and myosin. They inserted two microelectrodes into the endoplasmic drop, one filled with KCl solution and other with CaCl, solution, and applied a voltage between the electrodes. When the voltage was applied so that K + was iontophoretically injected, the rotation of chloroplasts was not affected. On the other hand, the injection of Ca2+ resulted in a reversible inhibition that was proportional to the applied voltage. Mg2+ did not affect chloroplast rotation at all. Kikuyama and Tazawa (1982) directly inserted a microelectrode filled with CaCl, solution into the cytoplasm of an intact internodal cell of Nitella and showed that Ca2- reversibly inhibits cytoplasmic streaming. Inhibition of cytoplasmic streaming was also shown in stamen hair cells of Tradescantia by microinjection (Hepler and Callahan, 1987). Although microinjection experiments unequivocally demonstrated that Ca2+inhibits cytoplasmic streaming, quantitative analysis of Ca2+concentration and velocity of streaming is difficult because of difficulty in calculating intracellula Ca2 concentration after microinjection. The development of a demembranated model of characean cells made it possible to study the relation between Ca2+concentration and the velocity of cytoplasmic streaming. In tonoplast-free cells of Characeae, it was shown that Ca2+inhibits cytoplasmic streaming (Williamson, 1975). Hayama er al. (1979) studied the quantitative relation of Ca2+concentration and streaming velocity using tonoplast-free cells of Chara. Cytoplasmic streaming was inhibited at Ca2+ concentrations higher than 1 pM. At 1 mM Ca” , cytoplasmic streaming stopped completely. It recovered only partially when Ca2+ was removed. Tominaga and Tazawa (1981) reexamined the relation between Ca2+concentration and streaming velocity using tonoplast-free cells of Chara. When the velocity was measured 1-3 min after perfusion with a Ca2+-containingmedium, cytoplasmic streaming was scarcely affected even at 0.1 mM Ca2+.At 1 mM, it was partially inhibited but still continued. After 1 hr of perfusion, it was inhibited at 10 p M but inhibition was only partial. Recovery after washing out Ca2+was also partial. +

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CYTOPLASMIC STREAMING

Thus, results obtained with tonoplast-free cells were not physiological in the sense that rather high concentrations of Ca2+ were necessary to inhibit streaming and that reversibility was only partial. This problem was overcome by developing a plasma membrane-permeabilized cell (Shimmen and Tazawa, 1983) in which cytoplasmic streaming was drastically inhibited at 1 p M Ca2+ and completely inhibited at 10 p M (Tominaga et al., 1983) (Fig. 9). Cytoplasmic streaming inhibited by 1 p M or 10 pwM Ca2+ completely recovered when the Ca2+concentration has lowered. Although the flowing endoplasm disorganized in tonoplast-free cells, it retained its organization in permeabilized cells, since the endoplasm was sandwiched by the tonoplast and cell wall (or permeabilized plasma membrane). Therefore, it was concluded that the difference in Ca2+ sensitivity of cytoplasmic streaming in tonoplast-free cells and that of plasma membranepermeabilized cells is caused by a difference in the extent of organization of the endoplasm after preparation of demembranated cell models. This possibility was supported by the development of Ca2+-sensitive tonoplast-free cells. Tominaga et al. (1987) prepared tonoplast-free cells in which larger amounts of endoplasm were retained by slowly perfusing the vacuole with a slightly hypotonic solution. They proposed that the inhibition of cytoplasmic streaming by Ca2+ in characean cells is caused by phosphorylation of myosin (Tominaga ef al., 1987). The association of a Ca2+-dependentprotein kinase with actin cables supports this hypothesis (McCurdy and Harmon, 1992). Inhibition of the cytoplasmic streaming

6o

r

c

30, 20

O L

8

7

6

5

4

3

PCo

FIG, 9 Dose-response curve of Ca2 concentration and velocity of cytoplasmic streaming in permeabilized (0) or tonoplast-free characean cells ( 0 ) (Reproduced . from Tominaga er ul., 1983, with permission.) +

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by phosphorylation of myosin is discussed later in relation to regulation by an action potential (Section IV,A). In lily pollen tubes treated with A23 187, cytoplasmic streaming was irreversibly inhibited when extracellular Ca2+ was higher than 10 p M (Kohno and Shimmen, 1988b). The mechanism was analyzed using reconstituted experiments. Since characean actin bundles have no Ca2+-sensitivity, it is possible to analyze the Ca2+ sensitivity of exogenous myosin applied into endoplasm-free characean cells. Organelles of pollen tubes were suspended in Mg-ATP medium supplemented with Ca2+of various concentrations and introduced into endoplasm-free characean cells. The movement of organelles along characean actin cables occurred only when the Ca2+ concentration was lower than 1 p M (Kohno and Shimmen, 1988b). Since characean actin bundles have no Ca2+ sensitivity, it was concluded that myosin associated with pollen tube organelles is Ca2+ sensitive. Organelles were isolated from pollen tubes in which cytoplasmic streaming had stopped due to treatment with A23187 and Ca2+.When they were suspended in Mg-ATP medium lacking Ca2+ and were introduced into endoplasm-free characean cells, they moved along characean actin cables (Kohno and Shimmen, 1988b). Thus, inhibition of pollen tube myosin by Ca2+ is reversible. The question arises of why cytoplasmic streaming of pollen tubes is irreversibly inhibited by treatment with A23 187 and Ca2+. It was found that the actin filaments in the pollen tubes are irreversibly fragmented by such treatment (Kohno and Shimmen 1988b). Thus, pollen tubes are equipped with dual mechanisms for inhibiting cytoplasmic streaming by Ca2+.

IV. Extracellular Factors Affecting Cytoplasmic Streaming A. Action Potential

In characean cells, an action potential is generated at the plasma membrane upon the application of an electrical or mechanical stimulus. The action potential is generated by the activation of some ion channel(s), as it is in animal cells. At present, it is assumed that Ca2+ and C1- channels are involved. According to Shiina and Tazawa (1987a,b), Ca2+channels first open and Ca2+ions flow into the cytoplasm, resulting in membrane depolarization. In the next step, Ca2+ activates CI- channels and induces a further membrane depolarization.

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When an action potential is generated, cytoplasmic streaming quickly stops and recovers within several minutes (Kishimoto and Akabori, 1959) (excitation-cessation coupling). This clearly contrasts with muscle, in which a contraction is induced upon generation of an action potential (excitation-contraction coupling). The involvement of Ca2+in excitation-cessation coupling in characean cells was first suggested by observations of the effects of various extracellular divalent cations (Barry, 1968). When either Mg2+or Ba2+was added to the external medium of N . axillaris, a prolonged action potential was generated. Furthermore, it was found that cytoplasmic streaming did not stop at all upon generation of an action potential (uncoupling of excitation and cessation). Even in the presence of these divalent cations, the addition of Ca2+ relieved the uncoupling. When Ca2+ was added to a medium containing Mg2+ or Ba2+,cytoplasmic streaming was partially inhibited by membrane excitation. A further increase in the Ca2+ concentration completely recovered the coupling. Similar results were obtained using Pb2+(Kamitsubo, 1980) or Mn2+ (Shimmen and Nishikawa, 1988). Thus, extracellular Ca2+seems to play an important role in the coupling between excitation and cessation of streaming. The question arises of whether Ca2 concentration in the cytoplasm increases upon membrane excitation. Williamson and Ashley (1982) microinjected aequorin into the cytoplasm of Nitella and Charu. At the resting state, cytoplasmic Ca2+ concentration remained at a very low level. Upon generation of an action potential, the light emission of aequorin drastically increased, indicating that cytoplasmic free Ca2+ increased (Fig. 10). They estimated cytoplasmic Ca2+ concentration at the peak to be 43 p M and 6.7 p M in Nitella and Chara, respectively. As mentioned earlier, the inhibition of cytoplasmic streaming of Characeae by Ca2+has been demonstrated by microinjection and experiments using demembranated cell models. Quantitative analysis of Ca2+ concentration and the velocity of cytoplasmic streaming obtained in the plasma membrane-permeabilized model shows that 1-10 p M Ca2+ is sufficient to stop the streaming. At present, it is believed that cessation of cytoplasmic streaming upon membrane excitation is caused by an increase in cytoplasmic free Ca2+. By analogy to the actin-myosin system in animal cells, it is possible that cessation of cytoplasmic streaming by Ca2+ is mediated by either myosin or actin. Reconstitution experiments gave clues as to the mechanism of cessation. In skeletal muscle, control by Ca2+ is mediated by the troponin-tropomyosin system incorporated into the actin filaments (Ebashi, 1976) (actin-linked regulation). Therefore, myosin of skeletal muscle per se has no Ca2+ sensitivity. When latex beads coated with skeletal muscle myosin were introduced into the endoplasm-free chara+

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a

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5

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I,

10”

200

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Time (sec)

FIG. 10 Temporal relation between (a) action potential (b) aequorin luminescence, and (c) cessation of cytoplasmic streaming in Characeae. The ordinate of each figure represents (a) the membrane potential, (b) intensity of aequorin luminescence, and (c) velocity of cytoplasmic streaming, respectively. [Reproduced from Shimmen, T. (1992). Inhibitory regulation of cytoplasmic streaming by CaZ+in plant cells. In “Calcium Inhibition: A New Mode for Ca2+Regulation” (K. Kohama, ed.), p. 76. Japan Scientific Societies Press, Tokyo, Japan, with permission.]

cean cells, a sliding movement could be induced irrespective of Ca2+ concentration. However, when the troponin-tropomyosin complex isolated from skeletal muscle was incorporated into the characean actin bundles, the sliding movement between muscle myosin and characean actin cables became Ca2+sensitive, that is, the movement occurred only at Ca2+concentrations higher than 1 p M , as is the case in muscle contraction (Shimmen and Yano, 1986).This indicates that the actin filaments composing the cables in characean cells have no Ca2+-sensitizing machinery and that the mechanism by which Ca2+causes cessation of cytoplasmic streaming is associated with myosin (myosin-linked regulation). This possibility is also supported by the fact that Ca2+ sensitivity is lost in

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endoplasm-poor tonoplast-free cells but is retained in plasma membranepermeabilized cells or endoplasm-rich tonoplast-free cells (Tominaga et al., 1983, 1987). Ca2+-sensitivemovement of myosin along characean actin cables was effected by using Ca2+-sensitivemyosin (Kohama and Shimmen, 1985). Myosin has been isolated from the plasmodium of the slime mold Physwum polycephalum. This myosin binds Ca2+, and its actin-activated ATPase activity is inhibited at higher Ca2' concentrations (direct myosinlinked regulation; Kohama and Kendrick-Jones, 1986). When latex beads coated with Physarum myosin were introduced into endoplasm-free characean cells, Ca2+-sensitivemovement could be reconstituted (Kohama and Shimmen, 1985). Myosin-linked but Ca2+-activatablemovement could be also reconstituted. In the muscle of scallop, Ca2+ regulation is also caused by direct binding of Ca2+to the myosin molecule (direct myosin-linked regulation) but this movement is activated by Ca" (Kendrick-Jones et al., 1976). Reconstituted movement between scallop myosin and characean actin bundles was activated by Ca2+ (Vale et al., 1984). In smooth muscle, Ca" does not bind to myosin or actin. It binds to calmodulin and the Ca" -calmodulin complex accelerates the phosphorylation of the light chain of myosin by activating the myosin light chain kinase. Following phosphorylation, myosin is converted to the active form (indirect myosinlinked regulation) (Cassidy et al., 1979;Sellers et al., 1981).In this system, once myosin is phosphorylated, it shows activity irrespective of the Ca" concentration. In accordance with a previous report, phosphorylated smooth muscle myosin fixed on the surface of latex beads actively moved on characean actin cables irrespective of Ca2+concentration, but dephosphorylated myosin was inactive irrespective of Ca2+concentration (Sellers et al., 1985). Thus, all kinds of Ca2+ regulation could be reconstituted using characean actin cables, since characean actin cables have no Ca" sensitivity. Tominaga et al. (1987)showed the possibility of the involvement of myosin phosphorylation-dephosphorylation using plasma membranepermeabilized model cells and endoplasm-rich (Ca2+-sensitive) tonoplastfree cells. They postulated that phosphorylated myosin is in an inactive form, dephosphorylated myosin is in an active form, and that myosin is phosphorylated as the result of an increase in cytoplasmic Ca2+concentration, based on the following experiments. When plasma membrane-permeabilized models were treated with ATPy-S [adenosine-5'-0-(3-thiotriphosphate)]in the presence of Ca*+,cytoplasmic streaming reactivated by ATP was irreversibly inhibited (Fig. 11). It has been reported that ATP-y-S cannot be a substrate of ATPase but can be used as a substrate for protein kinase (Gratecos and Fischer, 1974;

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- 60 -

0 Caz+

0 v) al

Y

.-F?

5 zal

20

c

0

al

c

m

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-

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Morgan et al., 1976). Once protein is thiophosphorylated, it cannot be dephosphorylated by phosphatase. Therefore, it is supposed that characean myosin was irreversibly thiophosphorylated in the presence of ATPy-S and Ca2+.When an inhibitor of phosphatase 1 was applied, reactivated streaming was inhibited even in the absence of Ca2+.On the other hand, addition of phosphatase 1 activated streaming irrespective of Ca2+.Recently, it has been reported that Ca2+-dependentprotein kinase is associated with actin cables of Characeae (McCurdy and Harmon, 1992), supporting the possibility of indirect myosin-linked regulation in the Ca2+ regulation of cytoplasmic streaming. At present, the involvement of calmodulin in Ca2+inhibition of cytoplasmic streaming has not been reported. Rather it has been postulated that calmodulin is involved in the recovery process (Tominaga ef al., 1985). With respect to the mechanism of cessation, some questions remain. Based on perfusion experiments, Tazawa and Kishimoto (1968) reported that cytoplasmic streaming stops due to a loss of the motive force. On the other hand, Kamitsubo et al. (1989) and Kamitsubo and Kikuyama (1992), using the centrifuge microscope, reported that some cross-bridges are formed between “myosin” and actin cables. The discrepancy between the two observations might be caused by the difference in their objective lenses. Tazawa and Kishimoto (1968) used an objective lens of lower magnification and it is supposed that they observed only the bulk endoplasm. On the other hand, Kamitsubo et al. (1989) used an objective lens

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of higher magnification and observed the peripheral endoplasm very close to actin cables. According to the emission of aequorin, the Ca2+ concentration increased by membrane excitation returns to the level of the resting state within 10 sec. However, it takes several minutes for the streaming to recover its original velocity (Williamson and Ashley, 1982). It is supposed that dephosphorylation of myosin is a rather slow process. Since the phosphorylation hypothesis is based only on pharmacological studies, further biochemical support is awaited.

B. Light In most plant cells, cytoplasmic streaming occurs continuously (primary streaming). However, in Vallisneria or Egeria, cytoplasmic streaming is induced by light or chemicals such as histidine (secondary streaming). The mechanism by which light regulates secondary cytoplasmic streaming has been extensively studied and it was found that the pigment responsible is phytochrome (Takagi and Nagai, 1983, 1985). Recently. evidence suggesting the cooperative action of phytochrome and photosynthetic pigments was reported (Takagi et al., 1990). It has been suggested that Ca2+ is involved in the control by cytoplasmic streaming by light (Takagi and Nagai, 1986). Since this aspect was extensively reviewed by Nagai (1993), it is not considered further in this article. It has been reported that the velocity of cytoplasmic streaming in characean cells is enhanced to some extent by illumination (Barr and Broyer, 1964). The acceleration of cytoplasmic streaming by light in characean cells is inhibited by treating cells with 3-(3’,4’-dichlorophenyl)-lt l-dimethylurea (DCMU) (Plieth and Hansen, 1992). Miller and Sanders (1987) reported that Ca2+concentration in cytoplasm decreases on illumination in Chara. They proposed that this decrease is caused by the activation of Ca2+uptake into chloroplasts as a result of the activation of photosynthesis. This is based on the fact that the response was inhibited by DCMU. Since cytoplasmic streaming is inhibited Ca2+(Tominaga et al., 1983), it is expected that lowering the cytoplasmic Ca2+concentration causes an acceleration of cytoplasmic streaming. C. Low Temperature

Sachs first reported in 1864 that cessation of cytoplasmic streaming is one of the most rapid responses to low temperature (cited in Woods et a/., 1984a). Since that time, the effect of low temperature on cytoplasmic

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streaming has been studied by various authors. Lewis (1956) reported that the cytoplasmic streaming response is very variable among plant species. In cold-sensitive plants-tomato, watermelon, honeydew melon, tobacco, and sweet potato-cytoplasmic streaming ceased or was drastically inhibited after 1 or 2 min at 10°C. In most cases, streaming stopped at 5 or 0°C. On the other hand, in cold-tolerant plants-radish, carrot, and alfilaria (Erodium cicutarium), cytoplasmic streaming was observed even at 0 or 2.5"C. Woods et al. (1984a,b) analyzed the mechanism by which low temperatures affect cold-sensitive plants. Decreasing the temperature drastically inhibited cytoplasmic streaming, transvacuolar strands disappeared, and cytoplasm vesiculated. Upon rewarming, the original morphology returned. Such effects were not observed in cold-tolerant plants. These authors observed similar effects on cytoplasmic streaming and cytoplasmic structure by treating cells with cytochalasin B or uncoupling agents (Woods er al., 1984b). Woods et af. (1984b) found that intracellular Ca2+ is redistributed by cold treatment. When cells were treated with A23187 in the presence of extracellular Ca2+ in order to increase cytoplasmic Ca2+ concentration, cytoplasmic streaming stopped and transvacuolar strands disappeared, as in the case of cold treatment. Based on these observations, they concluded that cold treatment inhibits energy metabolism and disturbs the intracellular Ca2+homeostasis. They postulated that the distrubance of the Ca+ homeostasis affects the polymerization or bundling of actin filaments, which is responsible for generation of the motive force of cytoplasmic streaming and the maintenance of cytoplasmic structures, such as transvacuolar strands. Minorsky (1985) suggested a possible increase in cytoplasmic Ca2+as a primary physiological transducer of chilling injury. Quader et af. (1989) found that in epidermal cells of cold-tolerant onion bulb scales, the cisternae and long tubular strands of the endoplasmic reticulum disintegrate into short tubules upon cold treatment. However, the array of actin filaments remained similar to that observed in untreated cells. Upon rewarming, the original structure recovered, with slight relocation. The recovery process was not affected by a microtubule-destabilizing agent but was severely inhibited by cytochalasin. The authors concluded that cold treatment does not affect the integrity of the actin filaments but affects myosin or the force-generating system necessary for the reorganization of the endoplasmic reticulum. The relation between temperature and the velocity of cytoplasmic streaming has been reported by many authors (Tazawa, 1968; Ding and Tazawa, 1989; Mustacich and Ware, 1977; Tsuchiya et af., 1991; Takamatsu et al., 1993). We also analyzed the temperature dependence of cytoplasmic streaming velocity by taking advantage of characean cells

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(Shimmen and Yoshida, 1994).The temperature was lowered from culture temperature (25°C) linearly to 0.5"C on a computer-regulated cryomicroscope, and velocity was measured at various temperatures. In some cells, the relationship between temperature and streaming velocity was almost linear in the range between 25 and 0.5"C. In other cells, however, the velocity changed linearly in the range of 25 to 15°C but inhibition became significant at temperatures lower than 10°C. The same experiment was also carried out using tonoplast-free cells which contain strong pH and Ca2+-buffering capacities [piperazine-N,N'-bis(2-ethanesulfonicacid) (PIPES) and EGTA]. In all tonoplast-free cells tested, the velocity changed linearly at 25-0.5"C. We assumed that the intrinsic temperature dependence of the actin-myosin system responsible for generating the motive force is linear. The deviation from the linear curve in some intact cells might be caused by a disturbance of the intracellular pH and Ca'+ homeostasis. It is hypothesized that low concentrations of free Ca2+and neutral pH in the cytoplasm are at least partly maintained by active transport processes and that homeostasis is disturbed at temperatures lower than a critical level (less than 10°C). Ding and Tazawa (1989) reported that the ATP concentration does not change at low temperature, indicating that depletion of ATP, an energy source, is not the cause of inhibition of cytoplasmic streaming. Our study shows the possible involvement of homeostasis of Ca2+ and H t in the inhibition of cytoplasmic streaming. Inhibition by Ca2+ and the strong dependence of cytoplasmic streaming on intracellular pH has been already demonstrated in characean cells using demembranated cell models (Tominaga et al., 1983; Fujii et al., 1979; Tazawa and Shimmen, 1982).

0. Gravity Since all organisms living on earth are always exposed to the force of gravity, cytoplasmic streaming is necessarily affected by gravity. It is expected that gravity exerts a force ( F ) on cytoplasmic streaming of plant cells whose central part is occupied by a large vacuole according to the following equation:

F = a(D, - D,)g where a, D , , D , , and g represent thickness of cytoplasm, density of endoplasm, density of vacuolar sap, and gravitational acceleration, respectively. When a cell is placed in a vertical direction, it is expected that the endoplasm going up would be slowed down and the endoplasm going down would speed up from the force. A difference in velocity between upward and downward streaming has in fact been observed in algal cells

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(Ewart, 1903; Hejnowicz et al., 1985; Wayne et al., 1990) and in Auena coleoptile (Bottlier, 1934, cited in Buchen et al., 1991). Recently, the effect of gravity on cytoplasmic streaming was extensively studied using internodal cells or rhizoid cells of Characeae. In rhizoid cells, the velocity of acropetal streaming is 37 pmlsec and that of basipetal streaming is 32 pm/sec in the normal vertical position. Interestingly, some of this difference was maintained even after the cell was placed in an inverse vertical position. It was suggested that the difference in velocity in the two directions is mainly caused by an endogenous difference in the force, and a small part is attributed to gravity (Hejnowicz et al., 1985). This hypothesis was supported by experiments that measured velocity under reduced gravitational field during the parabolic flight of rockets (Texus 21 and Texus 25; Buchen et al., 1991). When a cell was treated with cytochalsin, basipetal streaming stopped earlier than acropetal streaming, suggesting some difference in the actin cables (Hejnowicz et al., 1985) (or that the tip is more permeable to cytochalasin than the flanks of the cell). In contrast to rhizoid cells, the cytoplasmic streaming velocity of internodal cells is very sensitive to gravity. Wayne et al. (1990) found a difference in the velocity of upward and downward streaming in vertically oriented cells. The same result was obtained irrespective of the orientation of the morphological top and bottom of the cell. In the horizontal position, the velocities of both streams were the same, indicating that a difference in velocity is caused by gravity alone. However, it was found that this difference due to gravity is not just physical; some kind of signal processing is involved in the perception of gravity. Polarity was lost with various treatments of the vertical cell. To get normal polarity, external Ca” higher than 1 p M was necessary. A calcium channel inhibitor, nifedipine, reversibly inhibited the gravitational response. When either the proximal or the distal end of the cell was separated by ligating the cell, the gravitational response was lost. Similarly, cells irradiated with UV at their cell end lost gravitational sensitivity but the cells irradiated at their middle part maintained their sensitivity (Wayne et al., 1990). When the density of the bathing medium was increased by adding bovine serum albumin (BSA), the polarity of streaming under gravity stimulation was lost, and a further increase in the density of the external medium reversed the polarity. In the horizontal position, hydrostatic pressure could take the place of gravity. The cell was separated into two halves using an apparatus modeled after Kamiya’s double chamber. When positive pressure was applied to one half, the velocity of streaming away from the applied pressure increased and that of streaming toward the applied pressure decreased. On

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application of negative pressure, the opposite result was obtained. The response of cytoplasmic streaming to hydrostatic pressure was similarly affected by ligation, UV irradiation, external Ca*+, and nifedipine, etc. (Staves et al., 1992). Based on these results, these authors postulated that under gravitational stimulation, the cell recognizes gravity through compressive or tensile forces that occur at the cell ends as a result of the settling of the entire protoplast (Wayne et al., 1990; Staves et al., 1992). Based on pharmacological studies, the importance of the plasma membrane and extracellular matrix in signal transduction has been suggested (Wayne et al., 1992).

V. Concluding Remarks Cytoplasmic streaming may play an important role not only in intracellular transport but also in other cell functions. In characean cells, it may affect photosynthesis by controlling the transport of ions or substrate through the plasma membrane (Lucas and Dainty, 1977; Lucas and Shimmen, 1981). In addition, it has been reported that cytoplasmic streaming is responsible for intercellular transport in Characeae (Bostrom and Walker, 1976; Box et al., 1984; Ding and Tazawa, 1989) and in the stem of some higher plants (Worley, 1968). It has been suggested that cytoplasmic streaming brings solute to the mouth of the plasmodesmata (Bostrom and Walker, 1976). In leaf cells of Egeria (Erwee and Goodwin, 1983) and in stamina1 hair cells of Sectreasea (Tucker, 1987), however, it was reported that cytoplasmic streaming has no significant effect. Cytoplasmic streaming may be involved in geotropism by affecting the positioning of amyloplasts as statoliths (Sack and Leopold, 1985). The mechanism of cytoplasmic streaming was elucidated mostly by experiments using characean cells. It is reasonable to say that the motive force of cytoplasmic streaming in other plant cells is also generated by the same mechanism as that in characean cells, that is, the sliding of myosin associated with organelles along actin filaments using ATP energy. In many plant cells, however, the tracks and velocities are always changing. In characean cells, the actin filaments are fixed at the inner surface of the chloroplasts. On the other hand, it is postulated that the organization of actin filaments is dynamic and will always change in many plant cells. To fully understand the mechanism of cytoplasmic streaming, it is necessary to elucidate the mechanism of the dynamics of actin filaments in the cell, including actin binding proteins.

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Studies of myosin from plant cells have just started. To understand the mechanism of cytoplasmic streaming at the molecular level, the elucidation of the myosin molecule is necessary. It has been suggested that the plant myosin responsible for cytoplasmic streaming should have two important functions. One is to hydrolyze ATP and to transduce the chemical energy into the mechanical energy needed to slide along actin filaments. The other one is to bind to organelles. The elucidation of the molecular structure of plant myosin by electron microscopy is awaited. Plant myosin can slide along animal actin, and animal myosin can do the same along plant actin. Both animal and plant myosins slide on actin filaments from the pointed end (minus end) to the barbed end (plus end). Thus, the active site for the interaction between actin filaments seems fundamentally similar in both plant and animal myosin. However, characean myosin seems to be different from animal myosin, in that characean myosin can slide on animal and plant actin filaments with a very high velocity (Table 11). Higashi-Fujime et al. (1993) made a nick in skeletal muscle actin by treating it with proteinase. The actin with a nick could make filaments but its function was modified. In an in uitro motility assay, the motility of the defective actin filament on a glass surface coated with skeletal muscle myosin was signficantly inhibited, but on a glass surface coated with characean “myosin” (crude extract), it was almost normal. Thus, there should be some difference in the active site among these myosins. In general, the sliding velocity of myosin along actin filaments is almost proportional to the activity of actin-activated ATPase (Shimmen, 1988b). It must be elucidated whether characean myosin can move by consuming many ATP molecules or by using the energy economically. In animal systems, motility based on actin-myosin is generally activated by Ca2+.Inhibition of cytoplasmic streaming by Ca2+in plant cells shows a clear contrast with animal systems. In plant cells, the Ca2+ inhibition of cytoplasmic streaming has been studied in in uiuo systems and by using demembranated models. In pollen tubes, the Ca2+regulation of streaming was studied using purified myosin for the first time (our unpublished data). In characean cells, cytoplasmic streaming stops upon the generation of an action potential caused by electrical or mechanical stimulation. At present, the physiological function of cessation of cytoplasmic streaming on membrane excitation is not known. In pollen tubes, there is an intracelMar gradient of free Ca2+and it is highest at the very tip, where the cell is actively elongating. The Ca2+inhibition of cytoplasmic streaming may have some important implications for the control of tip growth (Reiss and Nobiling, 1986; Nobiling and Reiss, 1987),although this is just speculation. Further biochemical and genetic studies are needed.

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Acknowledgment The authors wish to express their cordial thanks to Dr. Randy Wayne for kindly reading our manuscript.

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Cell and Molecular Biology of Flagellar Dyneins David R. Mitchell Department of Anatomy and Cell Biology and Program in Cell and Molecular Biology, SUNY Health Science Center, Syracuse, New York 13210

1. Introduction Few biological machines rival eukaryotic cilia and flagella for compact size, continuous high-speed motility, and exquisitely fine control. Beat patterns of these organelles can vary from the symmetric undulations of spermatozoa to the highly asymmetric sweep of epithelial cilia, with frequencies from the 60 beats per second of Chlamydomonas flagella to the slow “walking” bends of Stylonichia cirri, and both waveform and beat frequency can change in response to appropriate stimuli on a millisecond time scale. With this wonderful variability comes a structural and biochemical complexity that presents a constant challenge to the study of cell motility. Dyneins are the ATPase motors that power these machines, the motors whose activity must be regulated to produce the precise patterns of microtubule sliding necessary to propagate bending waves of an appropriate frequency and waveform. As ATPase enzymes and as molecular motors, dyneins are unique for their large size, variety, and subunit complexity. Genetic and biochemical dissection of flagellar dyneins has revealed that several similar but unique types are present in any given cilium or flagellum, and that some dyneins contain as many as 3 catalytic heavy chains, each greater than 500 kDa, and up to 12 smaller proteins, while others appear to have a single heavy chain and 2 or 3 smaller subunits. In just one organelle, the Chlamydomonus flagellum, at least 1 1 dynein heavy-chain isoforms are present, distributed among 5 or more unique enzyme complexes. In addition to the large family that populates ciliary organelles, dyneins are found in association with many forms of cytoplasmic motility, including axonal transport (Paschal and Vallee, 1987; Vallee et al., 1989),centripetal localization of Golgi vesicles (Corthesy-Theulaz et al., 1992), and mitosis (Pfarr el al., 1990; Steuer et al., 1990), and are present in such seemingly simple, nonflagellated organisms as Dictyostelium discoideum (Koonce et ul., 1992) and Saccharomyces cereuisiae (Eshel et al., 1993). Inrrrnarional Reuirw, of Cyroloxv. Vol. I55

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Copyright 0 1994 by Academic Press. Inc. All rights of reproduction in any form reserved.

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In most of these systems, dyneins act as motors that transport themselves and any attached cargo in a single direction, toward the minus ends of microtubules. The only exception reported so far is the motor for vesicular traffic in a giant ameba, Reticufomyxa, in which a single type of dynein apparently supports bidirectional motility along cytoplasmic microtubules (Schliwa et a f . , 1991). However, the search for members of the dynein superfamily through means that have revealed the prolific superfamily of kinesins, that is, amplification of conserved sequences with degenerate oligonucleotide primers (Endow and Hatsumi, 1991; Aizawa et a f . , 1992) and production of antibodies specific for conserved domains (Sawin et a f . , 1992), is just in its infancy. Since the exact number, location, and functional abilities of dynein motors have not even been fully explored within the narrow confines of cilia and flagella, the full range of their functional repertoire as cytoplasmic motors is undoubtedly broader than currently realized. Dyneins were first described nearly 30 years ago as ATPases that could be extracted from cilia (Gibbons and Rowe, 1965), and in the intervening time they have been the objects of numerous papers and several reviews. For reference to much of the literature and many excellent summaries from workers in the field, volume one of the two-volume book Cell Movement (Warner et a f . , 1989) should be consulted, along with reviews by Porter and Johnson (1989) and Omoto (1991). This chapter describes some of the unique attributes of flagellar motors and their potential significance for flagellar function, and highlights some structural, enzymatic, and motility properties of flagellar dyneins that may be common to all family members. Recent progress in the molecular characterization of dyneins is summarized, and some attempt is made to reconcile conflicting views of dynein structure with the available molecular, biochemical, genetic, and ultrastructural data. I have made no attempt to be comprehensive, nor to give equal attention to all areas of current research on flagellar dynein motors, but rather to bring into focus areas where recent advances have clarified older concepts, and to identify areas of uncertainty that may warrant further study.

II. Dyneins in Flagellar Motility

A. Flagellar Structure and Doublet Sliding Most motile cilia and flagella from organisms across a wide phylogenetic spectrum are built to the same complex design specifications. Since this high level of structural conservation must exist for a good reason, it

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requires brief review, even if the functional significance of many of these details remains undetermined. Cilia and flagella are unique among motile systems in the ease with which they can be removed from the rest of the cell and reactivated, allowing ready application of biochemical methods to dissect their structure and function. Many cells, after appropriate treatment, will detach their flagella at a unique transition zone between the anchoring basal body and the flagellar shaft. Flagella can then be purified by differential centrifugation and extracted with a mild detergent to strip away the cell membrane and wash out a soluble flagellar cytoplasm or matrix. This leaves an axoneme, a cylinder of 9 doublet microtubules (which consist of 13 complete protofilament A-tubules and 1 1 protofilament B-tubules) surrounding 2 singlet microtubules, all interconnected by numerous microtubule-associated proteins and protein complexes (Fig. 1). Upon addition of Mg-ATP, under appropriate buffer conditions, axonemes beat with waveforms and frequencies typical of flagella in living cells. Dyneins are attached to outer doublets in two rows of projections or “arms” along each A-tubule. These arms extend toward the B-tubule of an adjacent doublet (Fig. l ) , where they form rigor-like cross-bridges in the absence of ATP, and transient, force-generating attachments when

FIG. 1 Thin-section electron micrograph of a Trrruhymenu ciliary axoneme. A, outer doublet A-tubule; B. outer doublet B-tubule; oa, outer row dynein arms; ia, inner row dynein arms; rs. radial spoke; cm. central pair microtubules; nl. nexin link. Bar = 50 nm.

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Mg-ATP is present. The outer row consists of a single variety of dynein with a 24-nm periodicity, whereas the inner row contains several dyneins arrayed in a 96-nm repeat pattern. Major recent advances in flagellar dynein research, summarized in this chapter, include sequence analysis of several outer row dynein subunits, and correlation of the biochemical, structural, and functional changes accompanying mutations that alter structure or prevent assembly of one or more flagellar dyneins. Brief exposure of axonemes to proteases before addition of Mg-ATP prevents beating, but releases the potential for doublet microtubules to slide, one along the next, until all nine doublets lie end to end. Such sliding is the direct result (and an easily measured consequence) of forces produced by dyneins. This simple observation, first reported by Summers and Gibbons (1971), not only reveals the basic sliding mechanism that underlies flagellar motility, but also suggests that a protease-sensitive regulatory system is required to transform generalized dynein-powered sliding into localized flagellar bends. No dynein regulatory system has been clearly proven to be responsible for conversion of sliding into bending, but two different protein complexes associated with outer doublet microtubules, radial spokes, and a “dynein regulatory complex,” do influence dynein activity. The potential function of each in bend propagation, beat frequency regulation, and waveform regulation is considered further in later paragraphs. Axonemes can be structurally divided into two systems: the central pair complex and the outer doublet complex. The two single microtubules of the central pair are interconnected by short bridges at a periodicity of 16 nm. Other structures project off of these microtubules with periods of 16 or 32 nm to encircle the microtubules in a cylindrical cage (Murray, 1989), and in some organisms the entire central pair complex has been observed to rotate relative to the surrounding cylinder of doublet microtubules (Omoto and Kung, 1979; Kamiya, 1982; Hosokawa and MikiNoumura, 1987). The protein constituents of some central pair-associated structures have been identified through the analysis of Chlamydumunas mutations that prevent their assembly (Warr et al., 1966; Witman et al., 1978; Adams er al., 1981; Dutcher et al., 1984) but no phenotypic differences are seen among these mutants (all have nonmotile flagella), and the precise functions of each projection have not been determined. Because flagellar paralysis caused by mutations in the central pair complex can be by-passed by suppressor mutations without restoration of the central pair defect (Huang et al., 1982; discussed further later), the central pair complex may regulate dynein activity but must not be essential for either dynein-induced doublet sliding or bend propagation. Doublets are also interconnected at 96-nm intervals by bridges termed nexin links located in the same region as inner row dyneins. They have

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been distinguished from dyneins by their resistance to conditions that extract dyneins, but as yet their molecular composition has not been determined. Shear displacement between adjacent outer doublets during a normal beat cycle may reach a maximum of more than 100 nm, so that these bridges must slide at their B-tubule attachment sites (Bozkurt and Woolley, 1993). It is tempting to speculate that nexin links maintain interdoublet connections without generating any sliding resistance, and that the stiffness of ATP-depleted (rigor) axonemes is entirely due to rigor dynein cross-bridges, but it is equally possible that nexins perform an active role in modulating sliding resistance during bend propagation. Radial spokes project from each outer doublet in toward the central pair complex, where they appear to form contacts with central pair projections. Spokes are found in groups of two or three (depending upon the organism) at a repeat interval of 96 nm, with the S2 spoke in each group located just proximal (toward the base of the organelle) to a nexin link (Goodenough and Heuser, 1989; Mastronarde et a / ., 1992). Numerous spoke assembly mutations have been isolated (Huang e l a!., 1981); 17 proteins have been identified as spoke components, and 3 have already been cloned and sequenced (Williams et al., 1989; Curry et al., 1992). Defects in radial spoke assembly inhibit flagellar motility, but once again bypass suppressor mutations have been characterized that can restore motility without restoring the spoke structures (Luck et a / . , 1977; Huang et a / . , 1981, 1982). Most suppressors of central pair defects also suppress radial spoke mutations, highlighting the obvious transmission through radial spokes of some regulating signal generated in the central pair complex. This signal must in turn be passed to the two rows of dyneins along each doublet, where it may control the temporal and spatial range of active sliding.

6. Conversion of Sliding t o Bending The basic geometry of flagella requires that dynein-induced microtubule sliding be regulated at two levels. The experiments of Sale and Satir (19771, Fox and Sale (1987), and others have shown that all flagellar dyneins tested are minus-end directed motors and induce sliding of an adjacent doublet toward the tip of the axoneme. Thus, conversion of sliding between any doublet pair into a bend can only cause bending in one direction, with a force vector at a tangent to the cylinder of outer doublets. In order to bend, the axoneme must have active dyneins between doublets on one side, while dyneins on the opposite side slide passively toward the plus end of the adjacent doublet. In addition to regions of active and passive sliding, conversion of sliding into bends requires regions of “active hold-

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ing,” proximal to the bend, in which sliding is prevented, perhaps by rigor-like dynein or nexin link attachments. Once initiated, bends usually propagate spontaneously toward the tip of the axoneme (see Omoto, 1991, for a discussion of bend propagation). The second level at which sliding must be regulated is the level of spatial and temporal regulation required to propagate bending waves of a particular shape and beat frequency. The change from a highly asymmetric ciliary type of beat to a more symmetric flagellar waveform that accompanies loss of radial spoke/central pair interactions in Chlamydomonas (Brokaw and Luck, 1985) suggests that modulation of bending parameters may be a fundamental role of radial spokes, but the ability of flagella to beat in the absence of radial spokes shows that the basic underlying regulation of dynein activity required simply for bend formation and propagation must reside entirely within the outer doublet system. Similar conclusions can be drawn from the unusual flagella of some organisms, which completely lack a central pair complex (Gibbons et al., 1983).

111. Outer Row Dyneins

A. Purification and Subunit Composition Axonemal outer arm dyneins have been more thoroughly studied than dyneins from any other source. They are more abundant than inner row species, are easily extracted from most other axonemal components in solutions of high ionic strength, support microtubule gliding in uitro, and under most conditions display higher ATPase activities than inner row dyneins. The identification of extracted proteins as outer row dynein components has relied on several approaches, including analysis of proteins coextracted under conditions that remove outer row dynein structures (as monitored by electron microscopy), analysis of extracted proteins that copurify with ATPase activity, and analysis of proteins missing from extracts of outer arm assembly mutants. Since at least two such criteria must be met to make an unambiguous identification of a dynein as an inner vs an outer row component, convincing analysis has been limited to a few sources. Those most studied include spermatozoa of some sea urchin species, from which outer row dyneins can be selectively extracted with 0.6 M KCl or NaCl (Yano and Miki-Noumura, 1981; Gibbons and Gibbons, 1973); the single-celled biflagellate alga Chlamydomonas reinhardtii, in which dynein assembly mutants have been isolated (Kamiya, 1988); and the ciliate Tetrahymena, in which outer row dyneins have been identified by their selective extraction and reassociation (Warner et al.,

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1985). Structural and immunological similarities have been used to identify the outer row dyneins of trout spermatozoa (Gatti et al., 1989)and Paramecium cilia (Walczak et al., 1993). The subunit composition of outer row dynein varies slightly among these organisms, but all contain 10-15 proteins spanning a remarkable range of molecular weights. Outer row dyneins of Chlamydomonas contain three ca. 500-kDa catalytic heavy chains (alpha, beta, and gamma), two intermediate chains of 78 kDa and 70 kDa (IC78 and IC70), and eight light chains of 22 kDa to I 1 kDa (Piperno and Luck, 1979a; Pfister et al., 1982). Clones for both intermediate chains and for two of the three heavy chains have now been sequenced (King et al., 1992b; Mitchell and Kang, 1991; Mitchell and Brown, 1994; Wilkerson et al., 1994). Paramecium outer row dynein, like that of Chlamydomonas, has three heavy chains, two intermediate chains (88 kDa and 76 kDa) and eight light chains (Travis and Nelson, 1988; Walczak et al., 1993). Tetrahymena outer row dynein differs from that of Chlarnydomonas and Paramecium in having three intermediate chains (100 kDa, 85 kDa, and 70 kDa) rather than only two (Porter and Johnson, 1983).In sea urchin sperm, outer row dynein consists of only two heavy chains (alpha and beta), three intermediate chains of 112 kDa, 79 kDa, and 70 kDa (ICI, IC2, and IC3), and at least six light chains of 23 kDa to 6 kDa (Tang et al., 1982;Moss et al., 1992a). Sea urchin p-chains were the first dynein heavy chains to be cloned and sequenced (Gibbons et al., 1991; Ogawa, 1991); sequence data on the other subunits are not available. Light chain stoichiometry has not been reported, but densitometry measurements of proteins separated by SDS gel electrophoresis has shown that all five larger proteins in both the Chlamydomonas and sea urchin enzymes are present at one copy per complex (Moss et al., 1992a; Mitchell and Rosenbaum, 1986). There is a clear split between protozoans and metazoans in the number of heavy chains, but no obvious pattern to the number of intermediate and light chains in each outer row dynein. 1. Light Chains Little is known of the structure or function of any of the dynein light chains, but some information is available about their probable locations within the enzyme. In Chlamydomonas, high salt extraction dissociates a smaller complex containing the gamma heavy chain and two light chains (22 kDa and 18 kDa) from a complex containing the remainder of the enzyme (Piperno and Luck, 1979a; Pfister et al., 1982). Further dissociation with detergent (Mitchell and Rosenbaum, 1986) or low ionic strength dialysis (Pfister and Witman, 1984) can be used to purify subcomplexes of the a-chain and 16-kDa light chain, and the p-chain and 18-kDa light

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chain. The functional significance of tight association between each heavy chain and either one or two light chains has not been determined, nor has the generality of this arrangement in outer row dyneins from other organisms been confirmed. Under the conditions tested, the remaining light chains either fractionated with a heterodimer of the two Chlamydomonus intermediate chains, or dissociated completely from other subunits of the enzyme. In similar dissociation studies, sea urchin outer row dynein can be split into three fractions consisting of the a-chain alone; the pchain and ICl; and a complex of 1C2, IC3, and several light chains (Tang er al., 1982; Moss er al., 1992b). There is no evidence for tight association of heavy chains and light chains in sea urchin dyneins. 2. Intermediate Chains Both antibody cross-reactivity and sequence analysis have shown that outer row dynein intermediate chains from many organisms retain at least limited homology. Antibodies raised against the smaller Chlamydomonus intermediate chain (IC70) react with sea urchin IC3 and trout sperm IC2 intermediate chains, and a monoclonal antibody specific to Chlamydomonus IC78 also detects trout sperm IC2, indicating that the two Chlamydomonas sequences are themselves related (King er al., 1985, 1990). A weak similarity between IC78 and IC70 can indeed be observed in sequence alignments (King er al., 1992b) and IC70 also shows homology to a 74-kDa intermediate chain of mammalian cytoplasmic dynein (Paschal et al., 1992). As indicated in Fig. 2, the Chlamydomonas IC70 protein can be divided into three regions based on comparison with the rat brain cytoplasmic dynein intermediate chain: an N-terminal region with no obvious sequence similarity, a central region that retains approximately 28% similarity to the C-terminal portion of the cytoplasmic dynein subunit, and a short C-terminal region that has a high probability of forming an ahelical coiled-coil. In the 74-kDa brain dynein, a coiled-coil structure is

oda6 revertants

cytoplasmic dynein homology domain

coiled coil domain

FIG. 2 Domain structure of the 567 aa Ch/arnydornonos 70-kDa intermediate chain. The region between residues 30 and 65 that is altered in the oda-6 revertants analyzed by Mitchell and Kang (1993) may be important for outer arm function. The cytoplasmic homology domain is a region showing sequence similarity with a 74-kDa rat brain dynein intermediate chain (Paschal et a / . , 1992). The coiled-coil domain has a high probability of forming an a-helical coiled-coil according to the algorithm of Lupas cr a / . (1991).

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predicted as an N-terminal rather than a C-terminal domain (Paschal et al., 1992). The functional significance of these predicted a-helical structures is not known, but by analogy with other protein complexes, one might expect them to participate in subunit interactions, perhaps between the two intermediate chains or between an intermediate chain and a heavy chain. Dissociation of either Chlamydomonas (Mitchell and Rosenbaum, 1986; Pfister and Witman, 1984) o r sea urchin (Sale et al., 1985; Moss et al., 1992b) outer row dynein results in subcomplexes of intermediate chains (IC78 and IC70 in Chlamydomonas, IC2 and IC3 in sea urchin) that remain tightly bound to each other. The regions of each intermediate chain involved in this interaction have not been determined. Under mild conditions, the Chlamydomonas intermediate chain heterodimer copurifies with the beta heavy chain, and a determinant recognized by anti-IC70 monoclonal antibody C11.4 is masked (Mitchell and Rosenbaum, 1986). Higher detergent concentrations separate the P-chain from this heterodimer, and expose the C 11.4 determinant, which is located within the C-terminal half of IC70 (Mitchell and Kang, 1991, 1993). The Chlamydomonas outer row dynein assembly mutation oda-6 was found to be closely linked to the IC70 gene by restriction fragment length polymorphism (RFLP) mapping (Ranum et al., 1988; Mitchell and Kang, 1991) and was identified as an IC70 structural gene defect by transformation studies (Mitchell and Kang, 1991) and sequence analysis (Mitchell and Kang, 1993). While it is clear that oda-6 prevents assembly of outer row dyneins, it is not known whether IC70 is directly involved in dynein binding to outer doublet microtubules, or is required at an earlier step in the formation of a complex that is competent to bind. In further molecular and genetic studies, Mitchell and Kang (1993) sequenced several intragenic pseudorevertants of one oda-6 allele and analyzed their effects on flagellar motility and dynein assembly. Those studies identified an N-terminal domain (see Fig. 2) that is not required for correct assembly of outer row dynein, but that is important for a normal outer arm contribution to motility. When sequences in this region were altered by frame-shift mutations, assembly was not greatly affected but the flagellar beat frequency remained reduced to levels typical of mutants completely lacking outer arms. Since dynein light chains were not analyzed in that study, the possibility remains that one or more light chains failed to assemble in these mutants. Further work will be needed to understand the role of the 70-kDa intermediate chain in outer row dynein function. Chemical cross-linking studies have implicated the Chlamydomonas IC78 protein in microtubule attachment. King et af. (1991) treated axonemes with a “zero-length” cross-linker, and found that IC78 became linked to a-tubulin, as well as to IC70. Preliminary studies using IC78

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fragments expressed in vitro from cDNA clones also indicate that IC78 can bind to microtubules, and have identified probable microtubule binding regions in the IC78 sequence (King et al., 1992b).Other structural evidence (see later discussion) supports a role for intermediate chains in attachment of outer row dynein to A-tubule binding sites. Somewhat surprisingly, the Chfamydomonas78-kDa intermediate chain can become labeled in the presence of Mg (but not Mn) by the photoactivatable affinity analogs 8-N,-ATP, 2-N3-ATP, and 2-N3-ADP (Pfister et al., 1985; King et al., 1989). The significance of this observation is not clear, since the intermediate chain dimer has no measurable ATPase activity, and no recognizable nucleotide binding sites have been reported in intermediate chain sequences. Because the labeling reactions were performed on complexes that contained light, intermediate, and heavy chains, these experiments would not have distinguished between the presence of a nucleotide binding site on the 78-kDa intermediate chain itself, or on some other closely juxtaposed protein. In spite of considerable evidence that these Chfamydomonas intermediate chains reside at an A-tubule attachment site, far from the heavy chain domains thought to be involved in catalysis and mechanochemistry, the possibility remains that part of the 78-kDa intermediate chain makes contact with a nucleotide binding site (catalytic or otherwise) on one or more heavy chains.

3. Heavy Chains Three experimental approaches have been used to define regions within the large heavy chains that are functionally important: proteolytic fragmentation, mapping of sites that react with active site-directed probes, and primary sequence determination. Two types of active site probes-nucleotide adducts and the phosphate analog vanadate-have been used in combination with proteolytic cleavage and detection of specific fragments with monoclonal antibodies to map the relative location of reactive sites on linear maps of each molecule. Comparison of these cleavage maps with maps based on the predicted amino acid sequence has provided an initial framework for proposing relationships between chemically reactive sites and conserved sequence motifs common to the catalytic sites of other nucleotide metabolizing enzymes, and between quaternary structures observed by electron microscopy and secondary structures predicted from the sequence. Major structural features of the Chfamydomonus beta heavy chain are summarized in Fig. 3, where Vl and V2 indicate sites of vanadate-mediated photocleavage, and P1-P4 are sequences that match the P-loop consensus, a sequence implicated in binding the beta and gamma phosphates of ATP or GTP in a wide variety of nucleotide metabolizing enzymes (Walker et

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P1 P2 P3 P4

assembly domain

A

Vl

I

II I

A 1= alpha helical coiledcoil

v2

L

I

I

I

0

1,000

2,000

3,000

I

4,000 aa

FIG. 3 Domain structure of the Chlamydomonas beta heavy chain. The N-terminal about 160 kDa is designated as an assembly domain based on data of Sakakibara ef a / . (1993). PI-P4 are sequences conforming to the P-loop consensus of Saraste et al. (1990). V1 and V2 are vanadate photocleavage sights mapped by King and Witman (1987).

a/., 1982; Fry et ul., 1986; Saraste er al., 1990). Regions predicted to form a-helical coiled-coils and an N-terminal domain sufficient to support dynein assembly are also shown. Descriptions of the experiments used to generate these maps, and conclusions based upon them, are presented in the following sections. To map sites in heavy chains that are sensitive to proteolytic cleavage, purified outer arm dynein has been treated with different proteases for varying lengths of time, or at different concentrations, and the resulting fragments separated by SDS-PAGE. Fragments generated from a single heavy chain were identified by following the size of fragments recognized by chain-specific monoclonal antibodies, measuring apparent fragment sizes, and calculating the pathway of fragmentation responsible for the observed patterns. Much of this work was aided by the observation of Gibbons (Lee-Eiford er al., 1986) that dynein can be cleaved at a unique site by UV irradiation in the presence of vanadate and ATP. Although vanadate can act as a phosphate analog and inhibit a wide spectrum of enzymes, vanadate-mediated cleavage appears more restricted and has become a diagnostic test for both flagellar and cytoplasmic dynein ATPases. Vanadate-dependent UV-induced cleavage of sea urchin outer arm dynein requires ATP or ADP and a divalent cation in addition to vanadate. Under these conditions, both of the sea urchin heavy chains are cleaved and ATPase activity decreases in parallel with the extent of cleavage. The two fragments formed from each chain were designated heavy and light U V fragments (HUV and LUV), and the larger HUV fragment became labeled in the presence of the ATP photoaffinity analog CX-~~P-~-N,-ATP. In further work it was shown that cleavage could be induced at a second site (V2) in the absence of nucleotide if vanadate concentrations were increased to 0. I mM (Tang and Gibbons, 1987; reviewed in Gibbons et al., 1989). Cleavage at the V2 site is not correlated

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with enzyme inactivation, supporting the designation of V1, and not V2, as the catalytic site. King and Witman (1987, 1988) performed similar experiments to map the V1 and V2 sites in all three outer row dynein heavy chains of Chlamydomonas. Each of these algal heavy chains has a single V1 site cleaved in the presence of Mg-ATP and low concentrations of vanadate. However, when irradiation is performed at higher vanadate concentrations and in the presence of Mn but no nucleotide (V2 cleavage), two of the three Chlamydomonas outer row heavy chains are cleaved at more than one location. The Chlamydomonas P-chain has a single V2 cleavage site, located approximately 75 kDa from the V1 site (Fig. 3), whereas the ychain has two V2 sites, 35 kDa and 70 kDa from V1, and the a-chain has three V2 sites, approximately 60 kDa, 90 kDa, and 100 kDa, from V1. Since detailed photocleavage studies have not been performed on dyneins from other organisms, there is unfortunately no way to know the significance of differences in V2 cleavage between sea urchin and Chlamydomonas dyneins. One other chain-specific vanadate photocleavage difference should be noted. The Chlamydomonas a-chain, unlike the other Chlamydomonas and sea urchin outer arm dynein heavy chains, can be cleaved at V1 in the absence of nucleotide when irradiation occurs in the presence of 0.1 mM vanadate and 5 mM Mg (King and Witman, 1987). While the functional significance of these differences remains unknown, further analysis may strengthen the correlation between UV cleavage reactivity and the different functions of each heavy chain suggested by in uitro motility studies and by analysis of mutations affecting individual heavy chains (discussed further later). Both 8-N, and 2-N, analogs of ATP have been used as active site probes of outer row dynein heavy chains (Pfister et al., 1985; Tang and Gibbons, 1987; King et al., 1989), but the data have not yielded clear-cut answers. For example, Chlamydomonas a- and P-chains can both be labeled with either analog after V1 photocleavage, but alpha labels with both analogs on the larger (C-terminal) V1 fragment, whereas beta labels predominantly on the C-terminal fragment with 8-N3-ATPand the N-terminal V1 fragment with 2-N,-ATP. Much of the labeling results support a model of dynein heavy chains in which the active site is formed by two or more distant regions of the primary structure that are juxtaposed in the tertiary structure, and in the case of P-dynein, these two distant regions form portions of the active site that are close to the 2 and 8 positions of the adenine rings (King et al., 1989). An alternative interpretation is that each heavy chain contains more than one nucleotide binding site. Although multiple sequences that fit the consensus for the phosphate binding loop portion of a nucleotide binding site are indeed present in dynein heavy chains, the participation of these sites in nucleotide binding has not been proven,

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and most of the labeling data support the presence of only a single highaffinity site on each heavy chain. At the time of this writing, two flagellar dynein heavy chain sequences have been published, both of them outer row dynein p-chains from sea urchin embryonic cilia (Gibbons et al., 1991; Ogawa, 1991). These two sequences show dyneins to be unrelated to other motor enzymes such as myosin and kinesin, save for the presence of P-loop motifs. However, they are nearly identical to each other, and therefore provide little basis for comparisons that might reveal regions of sequence conservation among flagellar dyneins. Two cytoplasmic heavy chain sequences, from rat brain cDNAs (Mikami et al., 1993) and from the slime mold Dictyostelium (Koonce el al., 1992),give some insights into regions that may be common to all dyneins and regions that may be specific to dyneins designed for flagellar motility rather than cytoplasmic transport. Further insights into specialization within outer row dyneins are now available from the sequences of the three Chlamydomonas outer row dynein heavy chains (Wilkerson et al., 1994; Mitchell and Brown, 1994). Amino acid sequences of the y-chain (Wilkerson et al., 1994) and p-chain (Mitchell and Brown, 1994) are complete, while those of about half of the a-chain are also available for comparison (Mitchell and Brown, 1994). A striking feature of all dynein heavy chains is the presence of four nucleotide binding consensus sequences within the central third of the molecule (Fig. 3). While the first of these four sites (Pl) is thought to be the catalytic site for ATP hydrolysis, based on its location at the approximate site of V1 photocleavage (Gibbons et al., 1991), functions for the remaining three sites have not been determined. Alignment of each Ploop consensus across all available dyneins reveals greatest conservation at the P1 site, least conservation at P2 and P3 sites. The P4 site is nearly as conserved as PI among the flagellar dyneins (Mitchell and Brown, 1994), but is much less conserved in rat brain (Mikami et al., 1993) and Dictyostelium (Koonce et al., 1992) cytoplasmic dyneins. Fe(111)-mediated photocleavage near P3, and V2 photocleavage near P4 of urchin sperm flagellar dynein suggest that these sites participate in ligand binding rather than being merely cryptic evolutionary remnants (Gibbons et al., 1991), but the nature of the in uiuo ligands for these sites, and their role in dynein force generation, require further study. V2 cleavage sites and P-loops in the Chlamydomonas alpha and beta sequences do not appear to coincide (Mitchell and Brown, 1994) (Fig. 3). Although the overall level of sequence conservation among flagellar dynein heavy chains is not remarkable, dot-matrix comparisons between urchin and Chlamydomonas outer arm heavy chain sequences reveal a similarity that extends throughout each sequence, with least similar sequences found in the N-terminal third of each protein. When flagellar and

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cytoplasmic dyneins are compared, this trend continues, although the absolute levels of similarity are reduced to the point that N-terminal domains appear unrelated (Koonce er al., 1992). Because flagellar dyneins must form structural attachments to outer doublet A-tubule binding sites, whereas cytoplasmic dyneins bind to vesicles, kinetochores, etc., it has been hypothesized that the least-conserved N-terminal region is involved in structural binding, and the central and C-terminal regions are important for catalysis, ATP-sensitive microtubule binding, and force generation. Computer-generated predictions of dynein secondary structure have created more puzzles than answers. Like kinesin and myosin, dynein heavy chains appear as globular heads attached to extended tails (see the following section). Unlike those other motor enzymes, dynein tails do not dimerize through formation of a-helical coiled-coils. Many regions of each heavy chain are predicted to exist in a-helical structures, but few possess the heptad hydrophobic repeat typical of coiled-coils and none of these are very extensive. A graph depicting the relative probability that urchin dynein sequences could form a-helical coiled-coils (Fig. 4) instead shows several probability peaks clustered on either side of a large central domain that is free of peaks. A similar distribution of coiled-coil probability is seen in the Chlamydomonas P-dynein sequence (Fig. 3). This central domain spans all four P-loop sites, which is,consistent with a model in which N- and/or C-terminal regions form a structural tail and the central domain forms a globular catalytic head (King and Witman, 1989). Since each globular head of Chlamydomonas 18s dynein (which contains both alpha and beta heavy chains) has a molecular weight estimated by scanning transmission electron microscopy (STEM) as 375 kDa (Witman et al.,

2 3 4

0.5 U a,

.0

1000 2000 3000 amino acid number

4000

FIG. 4 Plot of the probability of sea urchin P-dynein sequences (Gibbons er d., 1991)forming a-helical coiled-coils. as determined by the algorithm of Lupas ei ul. (1991). Arrowheads indicate the location of P-loop sequences.

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1983), each head must contain not only this central domain (ca. 185 kDa for P-dynein) but an additional 190 kDa of C-terminal and/or N-terminal sequences. Most of the predicted coiled-coils are therefore associated with the globular heads, and could be involved in maintaining the tertiary structure of the motor domain.

6. Outer Arm Structure Much is known about the structure of extracted, purified outer row dyneins as they appear after adhesion to a surface and preparation for electron microscopy, and likewise many detailed studies have been made on the structure of flagellar outer arms in situ by negative stain, freeze-etch and thin-section electron microscopy, yet the relationship between these two views of the same dynein complex is still subject to several conflicting interpretations. In the following section I briefly summarize the data, including recent electron microscopy studies on Chlamydomonas mutants that lack only part of the outer arm complex (Sakakibara et al., 1991, 1993). 1. Isolated Outer Row Dynein Outer row dynein particles extracted from flagella by high ionic strength solutions or with high concentrations of ATP have been visualized as unstained molecules by STEM (Witman et al., 1983; Johnson and Wall, 1983), and as either negatively stained (King and Witman, 1990)or freezeetched rotary shadowed particles (Goodenough and Heuser, 1984) by conventional transmission electron microscopy. The images from all methods are largely in agreement, with STEM providing direct mass estimates, and freeze-etched images the greatest structural detail. Upon adhesion to a surface, dyneins usually spread into a fan-shaped bouquet in which globular domains (one for each heavy chain) are joined through thin extended structures to a common base. Because each globular domain is too small to contain an entire heavy chain, the thin extended structures must also be formed at least in part by heavy chains, as confirmed by images of the isolated Chlamydomonas gamma subunit (Witman er al., 1983; Goodenough and Heuser, 1984) and sea urchin a- and P-subunits (Sale et al., 1985). Immunoelectron microscopy with an antibody to the Chlamydomonas 70-kDa intermediate chain (King and Witman, 1990) has shown that an I U L C complex forms (part of) the common base. In freezeetched images it is especially clear that different globular heads may have unique shapes (round, oblate, pear-shaped) and that a short, thin, knobbed antenna projects from each head in a unique orientation (Goodenough

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and Heuser, 1984). A cartoon of the overall shape of outer arm dynein particles is given in Fig. 5A. As mentioned earlier, the heavy chain sequence domains that correspond to the extended tail, globular head, and knobbed antenna cannot be readily deduced from primary sequence data, but biochemical data are consistent with a model in which the extended tail of each heavy chain is formed from its N-terminal domain and the globular head contains the central catalytic domain with its four P-loop consensus sequences, and perhaps the C-terminal domain as well. Mocz and Gibbons (1993) treated intact sea urchin axonemes with proteolytic enzymes and determined that cleavage of the urchin p-chain produced a C-terminal fragment that could be extracted from the axoneme with low concentrations of ATP, conditions that disrupt only the “ATP-sensitive” or “active-end’’ microtubule attachment, whereas the N-terminal fragment was extractable with high salt, indicating that it is involved in “structural-end” attachment. From this it follows that the C-terminal fragment should encompass the globular enzymatic B tubule binding domain and the N-terminal fragment should contain the extended tail. In other studies, tryptic fragmentation of urchin p-dynein produced an N-terminal noncatalytic fragment with sedimentation properties of an extended molecule, and a larger, catalytically active fragment with properties of a globular particle (Ow et al., 1987; Gibbons et al., 1991), supporting the identity of globular heads with catalytic domains. Further refinements of this model of heavy chain structure have been deduced from analysis of a Chlamydomonas heavy chain mutant. Sakakibara et al. (1993) describe a p-dynein mutant (oda-4-s7)that synthesizes

A

B

FIG. 5 Cartoon of the likely location of Chlamydomonas outer row dynein subunits within the structure of (A) isolated outer row dynein particles and (B) in situ outer row arms viewed from outside the axoneme. In (B) the tip of the axoneme would be to the right and the gamma heavy chain is largely hidden behind the alpha and beta heavy chains. Small unlabeled circles represent light chains.

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a truncated P-chain with an estimated molecular weight of only 160 kDa. This mutant dynein retains its N-terminus, as determined by Western blot analysis with a monoclonal antibody, and supports assembly of all of the recognized Chfumydomonasouter arm dynein polypeptides, including the tightly associated IS-kDa light chain. When extracted from axonemes, this truncated P-chain retains its association with the m-chain and the ICI LC complex. The N-terminal portion of this P-dynein must include that portion of the extended tail that interacts with other subunits and is located at the A-tubule end of the in situ outer arm. This relationship between the primary and tertiary structure of heavy chains within isolated outer row dyneins, in which globular catalytic domains are formed by the Cterminal portion of each heavy chain, thin connecting tails are formed from N-terminal heavy chain sequences, and a basal attachment region is formed through association of N-terminal heavy chain domains with smaller proteins, is likely to hold true for other dyneins as well, since similar tertiary structures have been reported for both flagellar inner row (Goodenough et ul., 1987) and cytoplasmic (Vallee et al., 1988) dynein motors. 2. In Sifu Outer Row Dynein Arms

The unified model of dynein particle structure shown in Fig. 5A cannot be easily reconciled with the many disparate views of in situ outer arm structure derived from EM images of thin-sectioned, negatively stained, and freeze-etched material. The example of negatively stained Tetrahymenu outer doublets shown in Fig. 6 serves to illustrate several of the problems encountered. First, such images cannot clearly distinguish the contribution of one dynein from that of its nearest proximal and distal neighbors. Where gaps occur, as at the arrowheads, it is clear that individual dyneins tilt, and that each dynein overlaps its proximal neighbor (Avolio et al., 1984; Goodenough and Heuser, 1984). Direct interaction of adjacent overlapping arms may contribute to the cooperativity observed in outer arm binding to microtubules (Mitchell and Warner, 1980; Haimo and Fenton, 1984). Second, such two-dimensional images of a threedimensional object provide no information about the superposition of multiple subunits. Attempts have been made to extract three-dimensional information from stereoscopic or tilt-series images of negatively stained (Witman and Minervini, 1982), freeze-etched (Goodenough and Heuser, 1982), and thin-sectioned (Muto et ul., 1991) material, or by using Fourier transforms (Burgess er a f . , 1991) to circumvent this problem. Third, the overall dimensions of outer row arms are smaller than those of isolated particles, indicating that extraction is accompanied by a considerable change in conformation.

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FIG.6 Electron micrograph of negatively stained Tetrahyrnena outer doublet microtubules to which purified outer row dynein has been bound in the presence of MgATP. Outer row arms project from the A-tubule of each doublet. Arrows in (B) point to arms at the beginning and end of a gap in the row, where the true extent of arm overlap can be seen. Bar = 30 nrn.

Images of transversely sectioned flagellar axonemes reveal a roughly triangular outer row dynein arm, with an A-tubule structural attachment at one corner of the triangle and an electron-lucent gap between a side of the triangle and the adjacent B-tubule (Fig. 7). Images of flagellar axonemes from Chlamydomonas mutants missing the a-chain (Sakakibara et al., 1991) or the globular head of the p-chain (Sakakibara et al., 1993) suggest that each heavy chain head generates one third of the dynein arm density across the face of the triangle adjacent to the B-tubule, as indicated in Fig. 7. In Chlamydomonas, at least, the a-subunit is located farthest from the center of the axoneme and the p-subunit is in the “middle” of the arm in these cross sections of axonemes prepared in the absence of

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FIG. 7 Computer-averaged thin-section electron micrograph image of a doublet microtubule from a Chlumydomanus axoneme showing the probable locations of outer arm heavy chain globular domains. Bar = 10 nm. (Reproduced from Fig. 1 1 of Sakakibara et ul. (1993). by copyright permission of the Rockefeller University Press.)

ATP. The inner “corner” of the arm image may be contributed by gamma heavy chains, but this relationship must remain speculative at present because no y-chain assembly mutations have been characterized. In alpha and beta mutants, although portions of the outer arm appear missing, the remainder of the arm image has an apparently normal stain distribution. The absence of part or all of one subunit has not altered the conformation of remaining subunits, at least as observed in transverse sections, suggesting that interactions between heavy chain globular domains within each dynein complex are not essential for correct heavy chain positioning. The beautiful and detailed freeze-etched studies of Goodenough and Heuser (1982, 1984, 1989) revealed many additional details of in situ dynein structure. Fine bridges were seen connecting globular outer arms to adjacent B-tubules, and by comparisons with images of extracted particles, these bridges were equated with the antennae of the heavy chains. Similar structures have been observed in Tetrahymena, Chlamydomonas, and sea urchin axonemes (Goodenough and Heuser, 1982; Sale et al., 1985), and their presence accounts for the otherwise puzzling gap between the arm and the B-tubule in electron micrographs of sectioned material where this bridge is either too thin to be visible, or is too inconsistent in location to be enhanced through alignment of the multiple bridges present in one section. Outer row arms undergo a major conformational change when ATP is present, shifting from a more compact appearance to a more extended, tilted appearance (Goodenough and Heuser, 1982), but the thin bridge remains the only apparent connection between arms and B-tubules under all conditions examined.

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One unresolved aspect of these detailed freeze-etched images is the appearance of only a single thin bridge per arm, whereas each arm has either two (sea urchin) or three (Tetrahymena, Chfamydomonas)heavy chains. Does only one heavy chain form a bridge? Is the thin bridge formed by contributions from multiple heavy chains? Are additional bridges present, but obscured by other structures? It would be remarkable if multiple bridges were present, but rarely observed in any of the stereoscopic deepetched views that have been published, yet other evidence suggests that each heavy chain can independently form a cross-bridge. In rare cases, thin bridges between the arm and the B-tubule appear in cross-sectional images, usually after enhancement of heavy metal staining with tannic acid. Examples of thin bridges on in situ arms may be seen in Figs. 1A and 7 of Mastronarde et al. (1992) and Fig. 8 of Sakakibara et ul. (1993). Similar bridges are seen after reassociation of outer row dynein with doublet microtubules (Fig. 6B of Warner et al., 1985), or with microtubules assembled from brain tubulin (Haimo and Fenton, 1984, 1988). In these images, bridges do not project from a single region of the outer arm, but appear in some cases to be emanating from the a-subunit, in other cases from the p- or (presumed) y-subunits. Similar conclusions were reached from studies of ATP-induced dissociation of dynein from microtubules. The ATP concentration dependence and kinetics of ATP-induced dissociation of three-headed Tetrahymena outer arm dynein from brain microtubules (Shimizu and Johnson, 1983) suggest that each head forms a separate ATP-sensitive microtubule attachment. In the presence of vanadate, which allows binding and hydrolysis of only a single ATP molecule per active site before formation of an inhibitory dynein/ADP/V complex, three ATP molecules were required per dynein to achieve complete dissociation. Using a different approach, Moss et ul. (1992a,b) fractionated sea urchin outer row dynein into a , plIC1, and IC2/IC3/LC particles, and demonstrated that each fraction containing a heavy chain was able to form an “active end” association with microtubules. Binding properties were heavy chain-specific, but the data do support independent B-tubule binding by each heavy chain. Goodenough and Heuser have interpreted the bridges in their freezeetched images as either a bridge that extends from a single subunit while bridges from the other subunits remain hidden or are pointed toward the A-tubule (1984), or as a single composite bridge formed by antennae from all three heavy chain subunits (1989). The interpretation most consistent with all of the data would be that additional bridges between outer arm heavy chains and B-tubule sites exist, but are too deep between doublets to be viewed by the freeze-etching technique (Fig. 5B). New negative stain and freeze-etched studies must be done using the Chlamydomonas

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alpha (oda-ll) and beta ( o d a - 4 ~ 7mutants ) before these conflicting views can be fully resolved. C. Structure-Function Relationships

Contributions of individual heavy chains to outer arm dynein function have been deduced in part from studies of flagellar motility in the presence of dynein mutations and in part from studies of in uitro motility generated by intact and dissociated outer arm complexes. Unfortunately, much of the work with in situ outer arms involves Chlamydomonas mutants, while work with purified fractions has been most successful with sea urchin sperm dynein, making direct correlations difficult. The questions to be answered include whether both (all three) outer arm heavy chains participate equally in force generation, and whether any of the smaller proteins (intermediate and light chains) serve specific regulatory roles in modulating heavy chain activity. Available sequence data show that the three Chlamydomonas outer arm heavy chains retain only about 45% sequence identity, suggesting functional diversification within the outer arm (Mitchell and Brown, 1994). Direct evidence that each heavy chain contributes differently to flagellar motility is provided by the effects of Chlamydomonas alpha and beta heavy chain mutations. The oda-1 I mutation disrupts the entire a-subunit (alpha heavy chain and 16-kDa light chain) but does not prevent assembly of the other outer arm proteins (Sakakibara et a/., 1991). This mutation is likely to be an a-dynein structural gene defect, based on the close genetic linkage of oda-11 to the alpha dynein gene detected by RFLP analysis (Sakakibara er al., 1991) and on the absence of immunologically detectable alpha heavy chain in the cytoplasm of oda-11 cells (D. R. Mitchell, unpublished observations). Phenotypically, oda-I1 flagella beat with a frequency that is reduced by about 15% compared with the wild type. In addition, unlike wild-type flagella, the two flagella of oda-I1 cells show no tendency to beat at two different frequencies. In contrast, P-dynein mutations have a greater effect on average beat frequency, but no effect on the difference in frequency between cis and trans flagella. The oda-4-s7 mutation, which results in assembly of outer arms with a severely truncated beta heavy chain, reduces beat frequency by 50% (Sakakibara et al., 1993). Another P-dynein mutation, sup-pf-1, produces a P-chain of diminished size and reduces beat frequency by about 45% (Brokaw and Luck, 1985). Thesep-chain mutants have frequencies only slightly higher than those of Chlamydomonas flagella that completely lack outer arms, showing the central4mportance of P-dynein in

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Chlamydomonas outer arm function. Unfortunately, mutants that assemble defective y-chains have not as yet been reported, and oda-11, oda-4s7 double mutants assemble few outer arms (Sakakibara et al., 1993), so the contribution of y-chains to flagellar beating is not known. Urchin alpha and beta heavy chains differ in their microtubule binding properties, since a dissociated portion of sea urchin outer arms containing only the @chain and ICI is not capable of cross-bridging microtubules, whereas microtubules were bundled by the a-chain alone (Moss et al., 1992b). Microtubules assembled from purified calf-brain tubulin will bind to a glass surface coated with intact outer row dynein and glide when MgATP is present, as first reported by Paschal et al. (1987), and PlICl particles also supported gliding, but no microtubules could bind to glass coated with a-dynein (Sale and Fox, 1988; Moss et al., 1992a,b; Vale et al., 1989). Gliding velocity was dependent on Mg-ATP concentration, with the PlICl fraction producing an identical K , but a V,,, about 1.6fold greater than that of intact outer arm dynein. Depletion of ATP resulted in dissociation of microtubules from the outer arm orplIC1 coated surface. These in vitro results with sea urchin dyneins, while very different in nature from the in v i m results with Chlamydomonas mutants, nonetheless support the overall conclusion that each of the two or three heavy chain subunits within an outer row dynein arm provides a unique contribution to dynein force generation. IV. Inner Row Dyneins: 57 Varieties?

Although much confusion still exists concerning the number, periodicity, distribution, and subunit composition of inner row dyneins, great progress can also be reported; this is largely due to the selection and careful characterization of Chlamydomonas mutations that affect the assembly of dyneins. Advances in protein purification technology (high-performance liquid chromatography, HPLC) and electron microscopy (computer-aided image averaging) have also contributed to this rapidly advancing area. Current results derive almost entirely from the Chlamydomonas system, and must be confirmed in other systems as additional immunologic and molecular probes become available. A. Purification and Subunit Composition

The subunit composition of inner row dynein complexes has been determined by analyzing the proteins that copurify with ATPase activity and are retained in outer arm assembly mutants, and by noting which proteins

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are missing from mutants that lack inner arm structures (Huang et al., 1982; Kamiya et ul., 1991; Kagami and Kamiya, 1992; Goodenough et ul., 1987). Not all of the inner arm dynein heavy chains are well resolved by the more common gel electrophoresis techniques, and procedures developed in different laboratories to circumvent this problem have not always allowed direct comparisons of published results. By comparing migration in two different gel systems of heavy chains separated by HPLC, Kagami and Kamiya (1992) identified eight high-molecular-weight bands in high salt extracts from outer armless oda-1 axonemes, and demonstrated that all eight heavy chains are susceptible to vanadate-mediated photocleavage. These eight bands were distributed into seven fractions labeled a-g, with two heavy chains in fraction f and a single heavy chain in each of the other fractions. All fractions except f were capable of binding to glass and translocating microtubules, and in all cases but one (fraction b) the microtubules rotated during translocation. The two-headed dynein in fraction f is missing from flagella of mutants at the ida-1 ( = p f - 9 , =pf-30), ida-2, and ida-3 loci, and has been designated inner arm 1 (11) by Piperno et al. (1990). In addition to two heavy chains ( 1 alpha and 1 beta), this particle contains proteins of 140 kDa (Goodenough et al., 1987; Piperno et al., 1990) and 97 kDa (Smith and Sale, 1991; Kagami and Kamiya, 1992; 110 kDa in Porter et al., 1992). Lack of microtubule translocation by fraction f may have resulted from some alteration induced by HPLC fractionation, since I1 purified by sucrose gradient sedimentation supports translocation, albeit at a slow rate (Smith and Sale, 1991). Mutants missing I1 dynein swim slowly due to an abnormal waveform (Brokaw and Kamiya, 1987). Heavy chains of fractions a, c, d, and e are all missing from assembly mutant ida-5 (Kagami and Kamiya, 1992), and may correspond to a structure designated inner arm 2 (12) of Piperno et al. (1990) and Smith and Sale (l991), but because subsets of these are missing from mutants ida-4 (heavy chains a, c, and d) and ida-6 (heavy chain e; R. Kamiya, personal communication), it is not clear at this time whether all four heavy chains are subunits of one complex, or whether they reside in several different complexes that fail to assemble from lack of a common subunit. Structural studies (see later discussion) indicate that ida-4 is lacking three widely separated staining densities within each 96-nm repeating unit of inner arm dyneins, suggesting that each heavy chain resides in a different complex. Alternatively, Piperno and Ramanis (1991) suggest that 12 inner arms differ in composition between proximal and distal portions of the axoneme, so that two of the heavy chains identified as 12 dyneins (a and d) may reside in one region while the other two (c and e) have a complementary distribution. All four heavy chains copurify with a light chain of 42 kDa that has been identified as a form of actin (Piperno and Luck, 1979b);

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heavy chains a, c, and d are also associated with a 28-kDa light chain, while d has additional light chains of 44 kDa and 38 kDa. Thus it is entirely consistent that several heavy chains, present in different structures, might fail to assemble due to lack of a common light chain. Unfortunately, gene products have not as yet been identified for any of the IDA loci. No mutations have been identified that prevent the assembly of heavy chains b or g, which Kagami and Kamiya (1992) equate with the heavy chains of inner arm 3 (13) of Piperno et al. (1990). Of these, heavy chain b has been equated with inner arm 3' and heavy chain g with inner arm 3, which Piperno and Ramanis (1991) suggest are restricted to the proximal and distal portions of the axoneme, respectively.

B. In Situ Localization Inner row dynein structures have been much more difficult to image by electron microscopy than outer row dyneins, but through a combination of approaches a coherent picture is emerging. Goodenough and Heuser (1985) first described the 96-nm periodicity of inner row dynein density distribution as observed in deep-etched replicas. This departure from the 24-nm outer arm periodicity was confirmed by thin section and deepetched images of extracted and reassociated Terrahymena inner arm dynein (Warner et al., 1985) and has been refined by examining Chlamydomonas inner row dynein mutants in conventional (Piperno et al., 1990; Piperno and Ramanis, 1991), tilt-series (Muto et al., 1991), and computer-averaged (Kamiya et al., 1991; Mastronarde et al., 1992) images of thin sections. The particularly detailed analysis of thin sections by Mastronarde et al. (1992) reconciles several conflicting views of inner row dynein structures and highlights remaining uncertainties, and the reader is directed to that reference for a more complete understanding than can be gained by the following summary. In cross sections, the inner arm image can be divided into two densities-an inner lobe located closer to radial spokes and an outer lobe located closer to outer arms (Fig. 7 and 8). Each lobe does not correspond to a single dynein complex, however, since assembly mutants missing a single dynein show reductions in density that are not limited to the inner or outer lobe. Analysis of longitudinal sections and very thin serial cross sections were used to further define the location of each inner row dynein. In longitudinal views as many as 10 unique structures (staining densities) can be distinguished within the 96-nm spoke repeat (which contains only 2 spokes in Chlamydomonas, S l and S2, but 3 in many other organisms), and all but 2 or 3 of these 10 densities can be matched with inner row dyneins through mutant analysis. Inner arm 11, missing in ida-1 ( = p f - 9 ,

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=pf-30), idu-2, and idu-3 (see Fig. 8), corresponds to three densities clustered adjacent and proximal to the S l spoke. High salt extracts of outer-arm-deficient axonemes contain 11 particles that appear as two globular heads connected through flexible tails to a basal thickening (Goodenough ef ul., 1987), and the three domains in isolated I1 particles likely correspond to the three densities observed in situ. Heavy chains a, c, and d, missing in idu-4, are distributed among three densities, one located at the base of each spoke and one located in the gap between spoke groups (at approximately the location of spoke S3 in other organisms). As discussed above, these heavy chains (along with heavy chain e) were previously identified as subunits of a single complex, inner arm 12, but probably function as individual single-headed dyneins based on the size and distribution of their associated structures. Because no mutations that block assembly of heavy chains b, e , or g were analyzed, no correspondence could be made between any of these

FIG. 8 Computer-averaged thin-section electron micrograph image of doublet microtubules from Chlnmydomonns axonemes. ( A ) Wild-type inner and outer arms. (B) Inner arm assembly mutant gf-9-2. (C) Outer arm assembly mutant p f 2 8 . (D)gfi9-2 pf28 double mutant. Bar = 10 nm. (Reproduced from Fig. 7 of Porter P I nl. (1992). by copyright permission of the Rockefeller University Press.)

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dyneins and the remaining four densities observed by Mastronarde et al. (1992). One of these densities is reduced in pf-2, and thus may contain proteins of a “dynein regulatory complex” (see later discussion), and a second density is missing in the mutant bop-2 along with a 152-kDa protein (King et a/., 1992a). The bop3 mutation reduces swimming speed in a manner similar to mutations that affect inner row dynein assembly, but has the added phenotype of selective paralysis when cells are illuminated with wavelengths of light involved in phototaxis (Dutcher et al., 1988). Another structure present within the 96-nm repeat unit, and tentatively assigned to a position overlapping that of the pf-2 deficiency, is the nexin link. Since the connection between adjacent doublets formed by nexin links may require regulation coordinated with dynein activity, close juxtaposition of nexin links with a dynein regulatory complex would seem appropriate.

V. Distinct Roles for Outer and Inner Row Dyneins A. In Vivo Flagellar Motility For many years, no clear answer was available to the question of whether inner row and outer row dyneins contributed equally to doublet sliding forces. Axonemes of some unusual species have only a single row of dyneins (Baccetti et al., 1979; Gibbons et al., 1983; Hyams, 1985), but no distinctive motile properties were shared by these organelles. Genetic experiments with Chlamydomonas suggested that the absence of either inner row (pf-23)or outer row (pf-13, pf-22) dyneins resulted in flagellar paralysis (Piperno and Luck, 1979a; Huang et a / . , 19791, whereas selective extraction of outer row dyneins from sea urchin flagella merely reduced beat frequency by about 50% (Gibbons and Gibbons, 1976). Eventually, these Chlamydomonas and sea urchin results were reconciled by the selection of new Chlamydomonas mutants that were motile in spite of defects in assembly of either outer row (Mitchell and Rosenbaum, 1985; Kamiya and Okamoto, 1985; Kamiya, 1988) or inner row (Brokaw and Kamiya. 1987; Kamiya et a/., 1991) dyneins. The combined presence in one cell of mutations that block assembly of any two dyneins results in nonmotile flagella, but these double mutant flagella rarely assemble beyond half-length, suggesting that loss of dyneins may have a pleiotropic effect on other flagellar structures (Kamiya et al., 1991). The earlier results in nonmotile flagella, but these double mutant flagella rarely assemas the result of single gene mutations that alter assembly of more than one dynein (Piperno et al., 1990; Piperno and Ramanis, 1991). Many of

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FIG. 9 Dark-field stroboscopic images of Chlamydomoncls axonemes reactivated at lo-’ M Ca’t (A and C) beat with an asymmetric waveform, while those reactivated at M Ca” (B and D) beat with a symmetric waveform. A and B, axonemes from wild-type cells. C and D. axonemes form outer arm assembly mutant pf-28. Bar = 10 pm. (Reproduced from Fig. 6 of Mitchell and Rosenbaum (1989, by copyright permission of the Rockefeller University Press.)

the overall conclusions regarding distinct roles for inner and outer row dyneins are presented by Brokaw and Kamiya (19871, who show that in Chlamydomonas, as in sea urchin sperm, the absence of outer row dyneins reduces beat frequency but has little effect on waveform (see Fig. 91, whereas the absence of either of two inner row dyneins alters waveform without major effects on frequency. B. Microtubule Translocation in Vitro

In order to measure dynein force generation in the absence of the regulation needed to propagate bends, two types of simplified in uitro motility assays have been developed. Sliding of doublet microtubules can be measured following proteolytic treatment of axonemes, and gliding of doublets or of in uitro assembled singlet microtubules across a glass surface coated with dynein can also be used for qualitative and quantitative comparisons of purified flagellar dyneins. While doublet sliding allows an assessment of dynein activity under more normal circumstances (dyneins attached at one end to an A-tubule and bridging at the other to a B-tubule), it is transient and requires the availability of assembly mutants (at present only well characterized in Chlamydomonas) to sort out contributions of different dyneins. Kurimoto and Kamiya (1991) compared doublet sliding velocities of wild-type and mutant Chlamydomonas axonemes, and found that in some respects outer row dyneins are dominant over inner row dyneins. Although mutants missing all inner row dyneins (and hence retaining only outer

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row dynein) are not available, mutant combinations were constructed that blocked assembly of five of the eight different inner row dynein heavy chains. Maximum sliding velocity (VmaX)and K,,, for Mg-ATP dependence of sliding were essentially identical (25 pm/sec and 180 p M , respectively) for wild-type axonemes and for axonemes of inner arm mutants ida-2, ida-4, or double mutant ida-2,ida-4cells. In contrast, axonemes from oda1 , which lack outer row dyneins but retain a full complement of inner row dyneins, displayed a V,,, of only 5 pm/sec and a K,,, of 65 p M . This 5fold reduction in sliding velocity is reminiscent of the 2-fold reduction observed after outer arm extraction from sea urchin sperm axonemes (Yano and Miki-Noumura, 1981), and suggests that inner row dyneins have an intrinsically slower microtubule translocation rate than outer row dyneins. Studies of the rate of singlet microtubule gliding on glass surfaces coated with inner vs outer row dyneins from Tetrahymena cilia (Shimizu et al., 1991; Mori and Miki-Noumura, 1992) generate results similar to those seen with Chlamydomonas and sea urchin flagellar doublet sliding, but only if the Tetrahymena outer row dynein is activated by inclusion of 0.1% Triton in the assay. Direct comparison of singlet vs doublet microtubule gliding rates on Tetrahymena inner and outer row dyneins at varying concentrations of ATP (Mori and Miki-Noumura, 1992)reveal other differences. At ATP concentrations below 0.1 mM, both 22s (outer row) and 14s (inner row) dyneins support gliding of either singlet or doublet microtubules but at higher ATP concentrations, both dyneins support gliding of singlet microtubules while only 14s dynein supports gliding of doublets. When Ca2+ is added to a concentration of M , both dyneins support doublet gliding at high ATP concentrations, and the translocation rate is about 5-fold slower with inner than with outer row dynein. Differences in gliding rates generated by inner vs outer row dyneins therefore basically parallel the differences observed in doublet sliding rates and in flagellar beat frequency, but are sensitive to the concentrations of ATP and Ca2+ as well as to differences between singlet and doublet microtubules.

VI. The Cross-Bridge Cycle Analysis of the thermodynamic and kinetic parameters of the Tetrahymena outer row dynein ATPase, and the effects of in vitro assembled singlet microtubules on those parameters, have provided a framework for understanding the relationship between enzyme catalysis and motility (Holzbauer and Johnson, 1989a,b; Porter and Johnson, 1989). In the absence

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of microtubules, dynein binds ATP rapidly and with a large change in free energy, and ATP hydrolysis then proceeds with little free energy change. Inorganic phosphate is rapidly released, but ADP release is much slower and is the rate-limiting step of this reaction. Under steady-state conditions, most enzyme molecules are associated with ADP. In the absence of ATP, Tetrahymena dynein binds to singlet microtubules by its active (B-tubule) end and forms a tight dynein-microtubule complex, which is rapidly dissociated upon ATP addition (Porter and Johnson, 1983),and ATP hydrolysis takes place on the free enzyme. The rate-limiting release of ADP is accelerated by reassociation with microtubules, and it is this step that is likely to be accompanied by a free energy change that drives motility, perhaps through a conformational change in the microtubule-bound dynein. Is dynein motor activity dependent on a conformational change linked to the enzymatic cycle? Different outer row dynein conformations are indeed seen in electron micrographs of axonemes prepared in the presence and absence of ATP (Goodenough and Heuser, 1982). Based on kinetic considerations, the extended “ATP” conformation should be equated with the long-lived dynein-ADP intermediate. Since this intermediate has a high affinity for microtubules, the observed retention of thin B-tubule bridges in ATP or ATP + V treated axonemes would be entirely expected. Direct light microscopic observations of dynein-microtubule interactions in uitro also support some aspects of this cross-bridge model. Microtubules will not bind to glass coated with sea urchin dynein (or to just the PlICl fraction) unless nucleotide is present (Vale et al., 1989; Moss et ul., 1992a), but binding can be supported by the combination of ADP and vanadate (Vale et ul., 1989)which apparently locks dynein into a complex resembling the normal dynein-ADP-Pi intermediate. Surprisingly, although the microtubules do not actively glide, they are able to undergo one-dimensional diffusion while remaining tethered to the glass-bound dynein. Some caution should be used, however, in extrapolating in uitro kinetic data to in viuo mechanochemistry. Kinetic measurements support the presence of three ATPase sites on the three-headed Tetrahymena outer row dynein (Shimizu and Johnson, 1983),but all of the kinetic parameters can be fit to single discrete values, that is, all three heads appear to have identical kinetic properties and no allosteric behavior is observed (Holzbaur and Johnson, 1989a).This is in marked contrast to the allosteric behavior seen when Tetrahymena dynein is activated by doublet microtubules (Warner and McIlvain, 19861, and to the heavy chain-specific mechanochemical properties suggested by the effects of mutations in the a-(Sakakibara et al., 1991)and p- (Sakakibara et al., 1993)chains of threeheaded Chlamydomonas outer row dynein.

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VII. Dynein Regulation The obvious complexity of inner row dyneins. especially when compared with the simple 24-nm repetition of a three-headed outer row dynein, raises several questions. What unique functions are provided by so many different motors? What structural features create the underlying 96-nm period, with specific attachment sites for each dynein species? How are the activities of multiple inner row enzymes coordinated with each other and with outer row dyneins, and how are they regulated to propagate bends? The answers to many of these questions may involve the close relationship between inner row dyneins and two other structures that repeat at 96-nm intervals along doublet microtubules-radial spokes and a “dynein regulatory complex.” Radial spokes have been characterized genetically through the analysis of mutations that disrupt their structure and through the selection of revertants and suppressors of those mutations (Brokaw et al., 1982;Huang et al., 1981, 1982; Luck et al., 1977), and several radial spoke subunits have been cloned and sequenced (Williams et al., 1986, 1989; Curry et al., 1992;reviewed in Curry and Rosenbaum, 1993).Although radial spoke assembly mutants are paralyzed, several bypass suppressor mutations have been isolated that allow motility in the absence of spokes. One suppressor (sup-pf-I) is an outer arm P-dynein mutation (Huang et al., 1982; Porter et al., 1993), whereas mutations at five other suppressor loci (pf-2,pf-3, sup-pf-3, sup-pf4, and sup-pf-5) were all found to be missing a subset of the same six axonemal proteins. These proteins, which range in size from 108 kDa to 29 kDa (Huang et al., 1982; Piperno et af., 1992) were designated by Piperno et al. (1992)as the dynein regulatory complex or DRC. Although there is as yet no biochemical evidence that they coexist in a complex, these proteins have been tentatively associated with an axonemal structure positioned between the outer row of dyneins and one of the inner row I2 dyneins, at the level of spoke S2 in each 96-nm repeat (Mastronarde et al., 1992). The evidence that both radial spokes and the DRC play important roles in dynein regulation is clear and compelling, but the mechanisms through which that control is exerted remain largely unknown.

A. Radial Spokes and Sliding Velocity Although flagella missing radial spokes do not beat, their doublet microtubules will still slide if they are treated with trypsin in the presence of ATP (Witman et al., 1978). Is some link in the regulatory chain digested by

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trypsin, allowing dyneins that were inhibited by lack of spokes to generate force, or are interdoublet links digested by trypsin, allowing already active dyneins to cause sliding? A partial answer comes from careful measurements of trypsin-induced sliding rates of axonemes that either retain or lack radial spokes. If the doublet sliding rate of either wild-type axonemes, or axonemes from mutants missing outer row dynein arms, are compared with the sliding rates of comparable axonemes that also lack radial spokes, the absence of spokes consistently reduces doublet sliding velocities by about 60% (Smith and Sale, 1992a). Dyneins are thus only partially inhibited (or else only some dyneins are completely inhibited) by the absence of radial spokes. Inner row dyneins, at least, may be regulated by a spoke-dependent modification mechanism. When inner row dyneins are extracted from axonemes of outer arm assembly mutant pf-28, the axonemes will no longer slide; rebinding the extracted inner row dyneins restores normal inner arm structures and original sliding velocities (Smith and Sale, 1992b). Extraction and rebinding of inner arm dyneins to axonemes from pf-28, pf-14 mutants, which lack spokes, restores the slower sliding velocity typical of axonemes that lack spokes. However, if inner row arms from pf-28 axonemes, which retain spokes, are recombined with extracted pf28, pf-14 axonemes, which lack spokes, the reconstituted axonemes slide at the faster rate, as if spokes were present (Smith and Sale, 1992a). If inner arms from the slower sliding pf-28, pf-14 axonemes are recombined with extracted pf-28 axonemes, sliding also occurs at the faster rate, suggesting that the inner row dyneins are activated by the pf-28 spokes after recombination, perhaps through phosphorylation or some other covalent modification of an inner row dynein subunit. Further studies with mutants that selectively lack individual inner arm dyneins, along with biochemical analysis of the spoke-induced activation step, will be needed to further understand this aspect of dynein regulation.

B. Mutations Define a Dynein Regulatory Complex Present understanding of DRC function is derived from phenotypic analysis of the five suppressor mutations that define this complex. All five (pf2, p f - 3 , sup-pf-3, sup-pf-4, and sup-pf-5)suppress paralysis in combination with radial spoke assembly mutations, whereas only one, sup-pf-5, can also suppress defects in central pair assembly. If radial spokes are missing, then no signal will be transmitted to the DRC, and suppressors will only need to let dynein activity be controlled by bend parameters. If the central pair complex is absent, but radial spokes remain, the spokes might

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transmit an unmodulated signal that blocks bend propagation, but that the sup-pf-5 DRC modification can suppress. What can the phenotypes of each suppressor reveal of the suppression mechanism? By themselves (i.e., in the presence of wild-type radial spokes and central pair) the sup-pf-3 and sup-pf-4 mutations do not alter motility and sup-pf-5 shows only a slight reduction in swimming speed (Piperno et af.,1992), whereas pf-2 and pf-3 have motility phenotypes similar to inner row dynein assembly mutants (Brokaw and Kamiya, 1987). However, these motility effects may be an indirect consequence of reductions in inner arm assembly associated with sup-pf-5, pf-2, and pf-3 (Piperno et af., 1992) rather than a direct result of changes in inner arm function. If this is the case, then DRC function must be redundant during normal motility. In the combined presence of DRC suppressors and spoke or central pair defects, motility is quite abnormal, perhaps reflecting the contribution of only a limited subset of dyneins to bend propagation. Since sup-pf-5, pf-2, and pf-3 reduce the amount of heavy chains associated with I2 and 13, but not I1 inner arms (Piperno et af., 1992), and since the putative DRC structure is located farther from I1 than from the other inner row dynein structures (Mastronarde et af., 1992), it is possible that signals transmitted through the DRC regulate only a subset of the inner row dyneins, or that normal DRC function is to coordinate the activity of multiple dynein motors. Other suppressors of radial spoke and central pair mutations include one inner arm dynein mutation (pf-9-2;Porter et af.,1992). one outer arm dynein mutation (sup-pf-1;Huang et af.,1982), and one mutation that has not been associated with any recognizable loss or change of an axonemal protein (sup-pf-2; Huang et af., 1982). Suppression by pf-9-2 is specific to a temperature-sensitive allele of central pair mutation pf-16 (pf-16BR3), and is the only reported case of a mutation that suppresses a central pair defect, but not radial spoke defects. One interpretation might be that the structure disrupted by pf-16 (a set of proteins associated with the C1 central pair tubule; Dutcher et af., 1984) exerts a regulatory influence (through radial spoke SI?) limited to inner arm 1 1 , which is disrupted by pf-9-2, whereas disruptions of other spoke or central pair structures alter regulation of other inner row dyneins (through radial spoke S2?), such as the I2 and I3 dyneins affected by DRC suppressors. Suppression by sup-pf-1 and sup-pf-2, like that by sup-pf-5, is effective in the presence of either radial spoke or central pair mutations (Huang et af., 1982). Since sup-pf-1 is a gain-of-function mutation of an outer row dynein heavy chain (Huang et af., 1982), its effects cannot be related directly to an interaction between the mutant protein and radial spokes, but it could involve an interaction with an intermediate complex such as the DRC, which may reside between spoke S2 and outer row arms

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(Mastronarde et al., 1992). How would an outer arm dynein mutation suppress the paralysis caused by lack of radial spokes or the central pair? Outer arm dynein assembly mutations do not act as suppressors (Karniya, 1988; Sakakibara et al., 1991), so the effects of the sup-pf-f alleles are not simply to eliminate outer row dynein contributions altogether. Although the beat frequency of sup-pf-f flagella is only 33 Hz, compared with 60 Hz for flagella that have normal outer row arms and 28 Hz for mutants that lack outer row arms (Brokaw and Luck, 1985), the trypsininduced doublet sliding velocity of sup-pf-f axonemes, 13.0 pm/sec, is equivalent to that of wild-type axonemes, 12.1 pmlsec rather than that of outer armless pf-28 axonemes, 2.7 pm/sec (Smith and Sale, 1992a). Sup-pf-1 outer arms can thus support the higher sliding velocities typical of wild-type outer arms when sliding occurs under no-load conditions. In addition, the reduction in trypsin-induced sliding velocity caused by the absence of radial spokes is less severe in the presence of sup-pf-1 outer arms than in the presence of wild-type outer arms (Smith and Sale, 1992a). Perhaps this small difference in sliding velocity reflects the contribution of a mutant outer row dynein that escapes regulation by a radial spoke/ DRC system. VIII. Conclusions After 30 years, what have we learned about flagellar dyneins, and what new questions have been raised? Many details of dynein structure have been determined, including the sequence of several subunits and the relationship between sequence and quaternary structure. Much of this information can be applied broadly to cytoplasmic as well as flagellar dyneins, and may ultimately provide insights into general motor enzyme mechanisms. Some additional work in this direction will be needed to clarify protein-protein interactions that are important for maintaining dynein structure and for attaching dyneins to doublet microtubules, and much must be done to understand the role of smaller subunits (intermediate and light chains) in dynein structure and function. The determination of heavy chain sequence has provided a first look at the primary structures of these catalytic subunits, increased their estimated sizes from 350 kDa to over 500 kDa, shown that they are unrelated to other motor enzymes, and revealed the presence of multiple (nucleotide/ phosphate binding?) P-loops. The ability to modify Chlamydomonas dynein genes in uifro and express them in uiuo now gives us the opportunity to more directly test the function of conserved heavy chain motifs, and to clarify the unique functions of each heavy chain in this species. Because of their large size, however, dynein genes may yield their secrets more

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easily through traditional in uiuo mutagenesis followed by molecular analysis of the most phenotypically interesting mutants, rather than targeted in uitro mutagenesis and transformation. Most of the work to establish kinetic parameters of dynein ATPase activity and the effects of microtubules on those parameters has relied on flagellar dyneins. One surprising result of those studies, in light of more recent work, is the lack of data indicating unique catalytic properties of the multiple ATPase sites present in outer row dyneins. Do all heavy chains contribute equally to the kinetic parameters of isolated dynein complexes, or does a single subunit dominate and mask contributions of less active subunits? The field might benefit from studies (perhaps using mutant dyneins lacking individual catalytic sites) that test the enzymatic properties unique to each heavy chain. The importance of comparing the effects of singlet microtubules with those of doublet microtubules when testing either enzyme kinetics or in uitro motility properties of flagellar dyneins has also been largely overlooked. Present evidence suggests that cytoplasmic dyneins share more properties with outer row than with inner row dyneins. Both outer row and cytoplasmic dyneins are invariably two- or three-headed, and have evolutionarily related intermediate chains of 70-80 kDa, whereas most inner row dyneins are apparently single-headed and share a 42-kDa actin-related intermediate chain. Many inner row dyneins also have the unique property of causing simultaneous rotation and translocation of singlet microtubules, a property not reported for outer row or cytoplasmic dyneins. Are these inner row dynein characteristics related to their function as probable targets of radial spoke regulatory signals? Continued work is needed on dynein regulation by radial spoke-central pair interactions, on the coordination between inner and outer row dyneins, and on the structural basis of the complex 96-nm repeat unit that dictates specific attachment sites for each inner row enzyme along a doublet microtubule. The greatest advances in understanding the complex regulation of dyneins needed for flagellar bend propagation will likely come from exploitation of the powerful molecular and genetic approaches available in Chlarnydornonas, but a better understanding of basic dynein force-generating mechanisms will require exploitation of other lower eukaryotes such as Dictyosteliurn and yeasts, continued application of the broad range of materials and approaches currently used, and the continued curiosity of scientists about things that move. Acknowledgments I would like to thank Ian Gibbons. Ritsu Kamiya, Mary Porter, and George Witman for communicating results prior to publication; Chris Turner and Fred Warner for reading the

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manuscript; and the members of my own laboratory for their many contributions to this work. Portions of the work described in this review were supported by National Institutes of Health grant GM44228.

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Pfister. K. I(.. Haley, B. E., and Witman. G. B. (1985). Labeling of Chlumvdomonas 18s dynein polypeptides hy 8-azidoadenosine 5‘-triphosphate, a photoaffinity analog of ATP. J . Biol. Chem. 260, 12844-12850. Piperno, G.. and Luck, D. J. L. (1979a). An actin-like protein is a component of axonemes from Chlamydomonas flagella. J . B i d . Chem. 254, 2187-2190. Piperno, G., and Luck. D. J. L. (1979b). Axonemal adenosine triphosphatases from flagella of Chlamydomonas reinhardtii. J . Biol. Chem. 254, 3084-3090. Piperno. G., and Ramanis. Z. (1991). The proximal portion of Chlum.ydomonas flagella contains a distinct set of inner dynein arms. J . Cell Biol. 112, 701-709. Piperno, G.. Ramanis. Z.. Smith, E. F., and Sale. W. S. (1990). Three distinct inner dynein arms in Chlamydomonus flagella: Molecular composition and location in the axoneme. J . Cell B i d . 110, 379-389. Piperno. G.. Mead, K., and Shestak, W. (1992). The inner dynein arms I2 interact with a “dynein regulatory complex” in Chlarnydomonus flagella. J . Cdl B i d . 118, 1455-1463. Porter. M. E., and Johnson, K. A. (1983). Characterization of the ATP-sensitive binding of Tetrahymenu 30 s dynein to bovine brain microtubules. J . Biol. Chem. 258, 6575-6581. Porter, M. E., and Johnson, K. A. (1989). Dynein structure and function. Annrr. Reu. Cell B i d . 5, 119-151. Porter, M. E., Power, J.. and Dutcher. S. K. (1992). Extragenic suppressors of paralyzed flagellar mutations in Chlarnydomonas reinhardtii identify loci that alter the inner dynein arms. J . Cell Biol. 118, 1163-1 176. Porter, M. E.. Knott, J. A., Gardner, L. C.. Mitchell, D. R.. and Dutcher, S. K. (1993). Mutations in the structural gene for the dynein beta heavy chain reveal the location of a regulatory domain. Mol. B i d . Cell 4, 47a (abstr.). Ranum. L. P. W., Thompson. M. D.. Schloss, J. A , , Lefebvre, P. A,, and Silflow, C. D. (1988). Mapping flagellar genes in Chlumydoinonas using restriction fragment length polymorphisms. Generics 120, 109-122. Sakakibara. H., Mitchell, D. R.. and Kamiya, R. (1991). A Chlamydomonus outer arm dynein mutant missing the a heavy chain. J . Cell B i d . 113, 615-622. Sakakibara. H., Takada, S . , King, S. M., Witman, G. B., and Kamiya. R. (1993). A Chlamydomonas outer arm dynein mutant with a truncated p heavy chain. J . Cell Biol. 122, 653-661. Sale, W. S . , and Fox, L. A. (1988). Isolated P-heavy chain subunit of dynein translocates microtubules in vitro. J . Cell B i d . 107, 1793-1797. Sale, W. S., and Satir. P. (1977). Direction of active sliding of microtubules in Tetrahymena cilia. Pro<,.Nail. Acad. Sci. U . S . A . 14, 2045-2049. Sale. W. S . . Goodenough, U. W., and Heuser. J. E. (1985). The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella. J . Cell B i d . 101, 1400-1412. Saraste, M.. Sibbald, P. R., and Wittinghofer. A. (1990). The P-loop-a common motif in ATP- and GTP-binding proteins. Trends Biochem. Sc,i. 15, 430-434. Sawin, K. E., Mitchison, T. J.. and Wordeman. L. G. (1992). Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies. J . Cell Sci. 101, 303-313. Schliwa, M., Shimizu. T., Vale, R. D.. and Euteneuer, U. (1991). Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable. J . Cell B i d . 112, 1199-1203. Shimizu. T.. and Johnson. K. A. (1983). Kinetic evidence for multiple dynein ATPase sites. J . B i d . Chem. 258, 13841-13846. Shimizu. T., Furusawa, K.. Ohashi. S.,Toyoshima. Y. Y . . Okuno, M.. Malik. F., and Vale. R. D. (1991). Nucleotide specificity of the enzymatic and motile activities of dynein. kinesin. and heavy meromyosin. J . Cell B i d . 112, 1189-1 197. Smith, E. F.. and Sale, W. S. (1991). Microtubule binding and translocation by inner dynein l Cytoskel. 18, 258-268. arm subtype I I . C ~ l Motil.

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Smith, E. F.. and Sale, W. S . (1992a). Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes. J. Cell B i d . 117, 573-581. Smith, E. F.. and Sale, W. S . (1992b). Regulation of dynein-driven microtubule sliding by the radial spokes in flagella. Science 257, 1557-1559. Steuer, E. R., Wordeman, L.. Schroer, T. A,, and Sheetz. M. P. (1990). Localization of cytoplasmic dynein to mitotic spindles and kinetochores. Nature (London)345, 266-268. Summers, K. E., and Gibbons. I. R. (1971). Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm. Proc. Natl. Acad. Sci. U . S . A . 68, 3092-3096. Tang, W. J. Y.. and Gibbons, I. R. (1987). Photosensitized cleavage of dynein heavy chains. J. Biol. Chem. 263, 17728-17734. Tang, W. J. Y., Bell, C. W., Sale, W. S., and Gibbons, I. R. (1982). Structure of the dyneinI outer arm in sea urchin sperm flagella. J . B i d . Chem. 257, 508-515. Travis, S. M., and Nelson, D. L . (1988). Purification and properties of dyneins from Paramecium cilia. Biochim. Biophys. Acta 966, 73-83. Vale, R. D., Soll, D. R., and Gibbons, I. R. (1989). One-dimensional diffusionof microtubules bound to flagellar dynein. Cell (Cambridge, Mass.) 59, 915-925. Vallee. R. B., Wall. J. S. , Paschal, B. M., and Shpetner. H. S. (1988). Microtubule associated protein IC from brain is a two-headed cytosolic dynein. Nature (London) 332, 561-563. Vallee, R. B., Shpetner, H. S., and Paschal, B. M.(1989). The role of dynein in retrograde axonal transport. Trends NeuroSci. 12, 66-70. Walczak, C. E., Marchese-Ragona, S. P., and Nelson, D. L. (1993). Immunological comparison of 22s. 19s. and 12s dyneins from Paramecium cilia. Cell Motil. Cytoskel. 24, 17-28. Walker, J. E., Saraste. M., Runswick, M. J., and Gay, N. J. (1982). Distantly related sequences in the alpha and beta subunits of ATP synthase. myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J . 1, 945-951. Warner, F. D., and McIlvain, J. H. (1986). Kinetic properties of microtubule-activated 13s and 21s dynein ATPases. J. Cell Sci. 83, 251-267. Warner, F. D., Perreault, J. G.. and McIlvain. J. H. (1985). RebindingofTetrahymena 13s and 21s dynein ATPases to extracted doublet microtubules. J . Cell Sci. 77, 263-287. Warner, F. D., Satir. P.. and Gibbons. I. R., eds. (1989). "Cell Movement." Vol. I . Liss, New York. Warr, J. R., McVittie. A., Randall, J., and Hopkins, J. M. (1966). Genetic control of flagellar structure in Chlamydomonas reinhardii. Genet. Res. 7, 335-351. Wilkerson, C. G., King, S. M.. and Witman, G. B. (1994). Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer arm dynein. J. Cell Sci. 107, 497-506. Williams, B. D., Mitchell, D. R., and Rosenbaum. J. L. (1986). Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas. J . Cell Biol. 103, 1-11.

Williams, B. D.. Velleca. M. A., Curry, A. M.. and Rosenbaum, J. L. (1989). Molecular cloning and sequence analysis of the Chlamydomonas gene coding for radial spoke protein 3: Flagellar mutation of pf-14 is an ochre allele. J. Cell Biol. 109, 235-245. Witman, G. B., and Minervini, N. (1982). Dynein arm conformation and mechanochemical transduction in the eukaryotic flagellum. Prokaryotic Eukaryotic Flagella 35, 203-223. Witman, G. B., Plummer. J., and Sander, G. (1978). Chlamydomonas flagellar mutants lacking radial spokes and central tubules. J . Cell B i d . 76, 729-747. Witman, G. B.. Johnson, K. A., Pfister, K. K., and Wall, J. S. (1983). Fine structure and molecular weight of the outer arm dyneins of Chlamydomonus. J . Submicrosc. Cvtol. 15, 193- 197. Yano, Y., and Miki-Noumura, T. (1981). Recovery of sliding ability in arm-depleted flagellar axonemes after recombination with extracted dynein I. J . Cell Sci. 48, 223-239.

Morphological and Functional Reorganization of Human Carcinomas in Vitro Petra Kopf-Maier, Birgit Kolon, and Markus Bugenings Institut fur Anatomie, Freie Universitat Berlin. D-14195 Berlin-Dahlem, Germany

I. Introduction Despite the general ability of transformed cells to proliferate in uitro indefinitely, it is usually very difficult to establish primary cultures of human carcinomas obtained by surgery. This paradoxical situation reveals that human carcinoma cells are obviously more dependent upon environmental requirements than is usually assumed and that their growth depends upon factors that are present in the in uivo situation but commonly lacking in in v i m systems. Because of these difficulties, numerous methods and techniques, ranging from very simple approaches t o rather complicated and sophisticated arrangements, have been attempted to cultivate individual human carcinomas. In this chapter, we review currently available systems for growing human carcinomas and describe another approach to growing surgically removed carcinomas as primary cultures in the high-density culture system that was originally established for the cultivation of embryonic tissues (Moscona, 1952; Zimmermann, 1987; Zimmermann et ul., 1990, 1991).According to the techniques of this culture method, the tissues are dissociated into single-cell suspensions and dropped at high cell density on a membrane filter lying at the gas-medium interface. This arrangement preserves the cellular heterogeneity of the carcinoma tissues in uitro and allows the establishment of structural and functional intercellular contacts. This is made possible, especially, by the high density of the growing cells and the experimental arrangement, which intentionally avoids the submersion of the cultures in nutrient medium. This latter would deeply disturb the establishment of a cell-specific microenvironment and interfere with the

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action of autocrine and paracrine mechanisms which are known to be important for functional cell-cell interactions and control of growth and differentiation processes in mammalian tissues. It was an unexpected result of the investigations reported here that this culture arrangement allowed most human carcinomas that had been removed surgically and derived from different organs to reorganize and to differentiate histotypically in uitro to a much higher extent than is commonly seen by using other culture systems.

II. Cell Culture Systems for Growing Human Carcinomas

A. Monolayer Cultures One of the most established and best-known methods for cultivating mammalian cells, including carcinoma cells, is the monolayer culture in which isolated cells adhere to the glass or plastic bottom of the culture vessels and grow there as one cell layer that continuously becomes confluent. Cultivation of mesenchymal cells by this technique does not cause any problems, whereas the cultivation of epithelial cells is much more difficult (Merker et af., 1981). In the case of carcinoma cells which are also derived from epithelial tissues, only very few primary human carcinoma cells are actually able to adapt to the modest and spartan in uitro conditions of the conventional two-dimensional monolayer culture (Matsuoka el al., 1987; Scheithauer et af., 1986; Balconi et af., 1988; Kao and Collins, 1989; Fricker and Buckley, 1989; Smit et al., 1992). In most cases, fibroblasts rapidly overgrow the carcinoma cells during the early passages in uitro and, only sporadically, carcinoma cells with a very high proliferative activity and a correspondingly low level of differentiation are able to succeed in overgrowing the fibroblasts. This inevitably results in a pronounced cell selection and, at later passages, there is usually only a single type of cell growing in the monolayer culture. These cells increasingly dedifferentiate in uitro, but proliferate rapidly. This means that the monolayer culture is a very artificial system which selects highly proliferating cells and represents the original carcinoma tissues insufficiently because it does not allow heterogeneous types of cells, for example, carcinoma and connective tissue cells, to coexist in uitro, and impedes the cells from growing in three dimensions. There is no production of extracellular matrix material or formation of organized, differentiated structures in monolayer cultures as there are in the organism. These

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parameters, however, were shown to profoundly influence the drug sensitivity of human carcinomas (Miller et ul., 1985). In the rare cases when human carcinoma cells could be established successfully in a monolayer culture, they have been usually passaged continuously and developed into permanent tumor cell lines which have been used in many laboratories over the years, guaranteeing high reproducibility and comparability of the experimental results obtained with these lines, even in different laboratories. Because of the strong selection of highly proliferative cells, however, these monolayer cultures are only suited for determining general cytotoxic and antiproliferative properties of chemicals in vitro. They are inappropriate for predicting the outcome of clinical antitumor chemotherapy. The correlation rates between chemotherapeutical trials in monolayer cultures of human carcinomas and in clinical investigations are poor (Miller et ul., 1985; Kao and Collins, 1989; Smit et al., 1992). It is known from cytobiological experiments that the presence of extracellular matrix material, especially of a basal lamina, is important for the maintenance of functional and structural characteristics of epithelial cells and the induction of differentiation processes in epithelial tissues (Vracko, 1974, 1982; Banerjee et al., 1977; Ingber et al., 1986; Karst and Merker, 1988; Kopf-Maier and Kestenbach, 1989, 1990). Thus, recent approaches have been attempted to overcome the very low rate of successfully growing individual human carcinomas in the monolayer culture by plating them on supports coated with extracellular matrix, such as type I collagen, fibronectin, type IV collagen, laminin, or a reconstructed basal lamina. Under these conditions, the plating success rates were markedly enhanced and the differentiation level of the growing carcinoma cells was remarkably elevated (Vlodavsky et al., 1982; Miller et d., 1985; Bulbul et ul., 1986; Shea et al., 1989). Even human carcinomas such as testicular and prostatic tumors, which usually do not grow in common experimental in uivo and in vitro models, were cultivated with a success rate exceeding 50% when they were plated on extracellular matrix material (Bulbul et ul., 1986; Shea et al., 1989). However, three-dimensionally organized tissue nodules as they are characteristic for carcinoma tissues in vivo did not develop under the applied conditions in vitro. Nevertheless, the results demonstrate that the low success rate of plating individual human carcinomas as monolayers on uncoated surfaces is not an inherent property of human carcinomas, but that a greater number of carcinomas would grow if the in v i m conditions were brought closer to the in uivo situation. This means that environmental factors are very important for the survival, growth, and differentiation of human carcinoma cells in vitro.

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6. Stem Cell Cultures Another widely used system for growing human carcinoma cells in v i m is that of clonogenic colony cultures, the so-called stem cell cultures (Hamburger and Salmon, 1977; Salmon er al., 1978, 1980; Maurer and Ali-Osman, 1981; Neumeier et al., 1981; Salmon, 1984; Shoemaker et al., 1984). This culture method has been used to selectively grow human tumor stem cells which are responsible for tumor cell proliferation and for the indefinite growth of malignant human tumors. In this culture assay, human carcinomas are dissociated into single cell suspensions and plated in semisolid, agar-containing media where proliferating stem cells form monoclonal colonies by repeated cell divisions. These colonies consist of carcinoma cell aggregates lacking the typical histological features of the original carcinoma tissues (Salmon, 1980). As in monolayer cultures, the colony cultures are limited by diverse substantial shortcomings. For example, the rate of successful plating from fresh material is only 40 to 70% (Salmon, 1984; Fiebig et al., 1987) or even less (Shoemaker et al., 1984). The culture conditions are highly artificial and lead to rigorous cell selection. Only a minority of even highly proliferative cells of a human carcinoma are allowed to grow in the stem cell assay and to form colonies, whereas cells which do not cycle actively are excluded from consideration a priori. This is an intrinsic limitation since the majority of the cells present in original human carcinomas are not cycling actively, but are quiescent cells resting in the Go phase of the cell cycle (Bertoncello and Bradley, 1987). Moreover, as in monolayer cultures, the cells growing in the human tumor stem cell assay are deprived of contacts with the extracellular matrix and of interactions with other cell types which are part of cell growth and differentiation, and are critical in determining sensitivity to cytostatic drugs. As a result, the development of cellular and histological differentiation phenomena is largely resisted in colony cultures and, when cytostatic drugs are tested in colony cultures, it is just possible to determine their antiproliferative potency, but not to see their influence upon quiescent, noncycling or differentiated cells, which predominate in original human carcinomas and markedly influence the long-term success of a clinical chemotherapy. Thus, a correct prediction of a positive response to a drug is possible in only 61 to 64% (Salmon et al., 1980) or 71% (Salmon, 1984) of those carcinomas cloned successfully in human stem cell assays (-40 to 70%). This rate is too low to use the clonogenic colony culture method on a broad basis as “antioncogram,” that is, as an assay for screening individual human tumors for drug sensitivity before beginning clinical antitumor chemotherapy in a manner analogous to the techniques used for testing bacterial antibiotic sensitivity (antibiogram).

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In an opposite approach, some authors tried to establish organ cultures in which representative parts of original human carcinomas are explanted in culture, but can be maintained in uitro for only a few days (Volm et ul., 1970; Wolff and Wolff, 1975; Lawson et d . , 1986; Daidone et al., 1987). During this short period, the carcinoma tissue fragments that were explanted in uitro consist of various components of stroma and cells in a normal relationship and preserve the interactions between these components, without showing noticeable proliferative activity or differentiation processes. After a few days in uitro,the carcinoma pieces become unstable with respect to crucial functional and structural properties, and decompose. This relatively short-term integrity of human carcinoma tissues in the organ culture certainly enables one to observe, under in uitro conditions for a short period, the complex tissue structure that was grown in uiuo, but simultaneously results in a nonphysiological and labile experimental situation which is always threatened by the loss of viability of the tumors and the beginning of decomposition processes. These conditions have limited widespread use of the organ culture system for routine investigations, especially for testing cytostatic drugs. The organ culture method for human carcinomas was modified by Abaza et al. (1978) and, later on, by Hoffman and co-workers insofar as they explanted fragments of human carcinoma tissues on the top of a spongy material consisting of a hydrated collagen gel (Freeman and Hoffman, 1986; Vescio et al., 1987,1990; Hoffman et a[., 1989). In this gel-supported primary culture system, both the cellular heterogeneity and the threedimensional, native tissue architecture of individual human carcinomas were maintained for a few weeks. In addition, numerous carcinoma cells invaded the matrix, proliferated there vigorously for several weeks, and differentiated to a limited extent. They built up a cell framework that markedly incorporated [3H]thymidine and [3H]deoxyuridine as signs of an intense cellular proliferation. In some cases, the development of certain histological features was observed, such as melanogenesis in human melanoma cells o r the formation of simple adenoid structures in a human gastric adenocarcinoma. However, no detailed light-microscopic, immunohistochemical, or ultrastructural studies were reported by the authors that would allow one to clearly estimate the degrees of reconstitution, of cytological and histotypical differentiation, and of tumor-stromal interactions that were established in the cellular aggregates grown in the supporting collagen sponge. This in uitro system was proposed by Hoffman and co-workers as model for testing the anticancer activity of cytostatic drugs in uirro in individual human carcinomas by determining the incorporation rates of [3H]thymidine and [3H]deoxyuridine with autoradiographic techniques (Vescio e f ul., 1987; Hoffman er ul., 1989). As in the monolayer and colony culture

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assays, however, these parameters only measure the proliferation activity of the growing carcinoma cells without considering the behavior of less proliferative but more highly differentiated and often quiescent noncycling cells which are usually responsible for the later regrowth of treated carcinomas and the failure of chemotherapy in patients. N o studies have been published that make a broad-based comparison of the results of chemotherapeutic studies in gel-supported primary cultures and the response rates found in established experimental in vivo systems or in clinical trials. C. Spheroidal Aggregate Cultures

Another type of a three-dimensional culture arrangement is the spheroidal aggregate culture in which mammalian tissues that have been dissociated into single-cell suspensions reaggregate to multicellular spheroids when they are submerged in growth medium under certain culture conditions (Ono et al., 1979; Landry and Freyer, 1984; Landry et al., 1985; MuellerKlieser, 1987; Sutherland, 1988; Durand, 1990a). These spheroids are composed of several types of cells, that is, epithelial cells and some connective tissue cells, which rapidly establish structural cell-to-cell contacts and functional intercellular interactions in the spheroidal clusters. These intercellular contacts, which are known to regulate cellular growth and differentiation, obviously trigger the development of some differentiation processes which are manifested, for example, by the formation of bile duct-like structures and the production and deposition of extracellular matrix components in liver cell spheroids (Landry et al., 1983, the synthesis and release of insulin in pancreatic islet cell spheroids (Ono et al., 1979), the formation of some simple pseudoglandular structures and the expression of the carcinoembryonic antigen in spheroids of an experimental adenocarcinoma (Mueller-Klieser, 1987; Sutherland, 1988), or the aggregation of large cell clusters showing signs of melanogenesis in the spheroids of an experimental melanoma (Rofstad et al., 1985). It seems probable that the induction of these differentiation processes is effected by the presence of cellular heterogeneity, homologous and heterologous cell-cell contacts, and preserved tumor-stromal interactions in the multicellular spheroids, although no detailed histological, immunohistochemical, or electron-microscopic studies have been performed with carcinoma spheroids to illustrate and to define clearly the level of differentiation attained in comparison with the original tissues, and to estimate the degree of tumor-stromal interactions in the spheroidal carcinoma clusters. Moreover, all investigations with carcinoma tissue spheroids done so far used established tumor lines which had been grown before either as monolayer cell lines in uitro or as experimental tumors in animals.

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There are no clues in the literature on whether spheroid cultures can also be established using primary human carcinomas obtained directly from surgery . Spheroid cultures are mainly used and especially suited for physiological investigations in uitro, for example, to measure O2 consumption, glucose concentration, pH level, and the distribution of other metabolic gradients, since spheroids grown in uitro are usually characterized by an ideal, regularly geometrical configuration. With increasing size of the spheroids, they include a rising fraction of nonproliferating, quiescent cells, and cell death and necrosis occur in the centers of the spheroids in a manner similar to that seen in carcinoma nodules derived from human patients (Landry and Freyer, 1984; Mueller-Klieser, 1987; Sutherland, 1988; Durand, 1990a). On the other hand, only a few therapeutic trials were performed with spheroid cultures of some experimental tumors treated with hyperthermia, ionizing radiation, or chemotherapeutic agents (Durand and Sutherland, 1972, 1973; Rofstad et al., 1985; Durand, 1990a,b; Sasaki et al., 1993; Kubota et al., 1993). These investigations demonstrated that spheroids react to therapeutic manipulation with delayed growth, a reduction of the surviving cell fraction, and an increased number of cellular necroses, whereas, in general, the spheroids are less sensitive to chemotherapy and ionizing radiation than monolayer cultures, even under similar supply conditions (Durand and Sutherland, 1972, 1973). This may indicate that the response of cancer cells to treatment may be mimicked in spheroids more closely than in conventional monolayer cultures. However, broadbased comparative studies on the results of nude mouse experiments or clinical studies have not been done so far.

111. High-Density [Organoidl Culture

A. Methods The high-density culture method makes a compromise between ( I ) monolayer and colony culture techniques, both using isolated cells and growing them under highly artificial conditions far away from the in uiuo situation, and (2) organ cultures in which representative parts of mammalian organs are explanted and kept in uitro forjust a few days. The high-density culture method is based on the observation that discrete and viable embryonic cells grown in uitro at high cell density reaggregate into small, solid clumps within 1 to 2 days (Moscona, 1952). When mixed suspensions composed of cells from different types of tissue are used, the cells segregate during the constitution of the clusters, the cells of each type forming a different

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part of the same cluster (Moscona and Moscona, 1952; Moscona, 1957). This process, called “cell sorting-out,” was recognized to be an important step in morphogenesis and tissue differentiation (Steinberg, 1962, 1963). The high-density culture method has been applied in some variety to the in uitro cultivation of embryonic tissues. In dense cultures of limb blastema cells, the formation of blastema-like aggregates was observed within the first 3 days in uirro. Thereafter, hyaline cartilage nodules developed and the extracellular space contained type I1 collagen fibrils and cartilage-typical glycoprotein molecules (Zimmermann and Cristea, 1993; Schroter-Kermani et al., 1991). The processes of desmal and enchondral mineralization could be followed up in detail in dense cultures of osteoblasts or in cartilage cultures, respectively, under defined experimental conditions (Zimmermann er ul., 1990, 1991). In high-density cultures of the lung at different stages of fetal development, a clearly stage-dependent differentiation was observed. The cells differentiated into either a ciliated epithelium encircled by a basal lamina and a differentiated mesenchyme or, in lung cultures from older fetuses, into alveolar-like structures containing pneumocytes type I and I1 which were again separated from the surrounding connective tissue by a typical basal lamina (Merker er d., 1981 ; Zimmermann, 1987). In these lung cultures, epithelial differentiation culminated in the formation of lamellar bodies in pneumocytes I1 and the production and secretion of surfactant material. Analogous differentiation phenomena occurred in high-density cultures of liver and brain anlagen. Within several days in uitro, immature fetal liver cells differentiated into hepatocytes that included the characteristic organelles of liver cells, formed bile canaliculi that were surrounded by junctional complexes, and produced and secreted typical liver cell enzymes (Zimmermann, 1991). In cultures of isolated cells of the brain stem anlage, a neuroepithelium reorganized in uirro and differentiated into neurons with axons, dendrites, and synapses (Zimmermann, 1991). These results demonstrate that a high-density culture is obviously a useful tool for growing embryonic tissues in uirro and studying differentiation processes. Since embryonic and carcinoma tissues resemble one another insofar as they are not fully differentiated, we intended to see whether high-density (organoid) culture may also be an appropriate tool for growing human carcinoma tissues in v i m . In a pilot experiment, we cultivated five human carcinomas of different origins and various histopathologies that had been heterotransplanted to athymic mice earlier and passaged there for several years. All five carcinomas reorganized under the applied high-density culture conditions and redifferentiated into organoid structures which exhibited the typical histological characteristics of the original tissues (Kopf-Maier and Zimrnerman, 1991 ).

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It remained unclear whether this development was rendered possible by the cellular selection and tumor modification that occurred during the nude-mouse passages or whether it reflected a fundamental ability of human carcinomas to grow in an organoid, high-density culture. Therefore, we extended these studies to about 80 primary human carcinomas of diverse origin and varying histopathologies to examine whether primary human carcinomas would also reorganize and redifferentiate in the highdensity culture and, if they would do so, what would be the success rates in comparison with other in virro models. The experimental procedure summarized in Fig. 1 was used to grow primary human carcinomas in vitro. After surgical removal of the carcinomas, pieces of about 0.5 to 2 cm3 were immersed under aseptic conditions in 30 to 50 ml of a transportation medium consisting of Dulbecco's minimum essential medium (MEM) supplemented by 10% fetal calf serum and 10% penicillin/streptomycin solution (5000 U/ml; 5000 pg/ml) (all purchased from Gibco, Berlin). We endeavored to keep the time interval between the surgical removal of the carcinomas and the beginning of the experimental procedure as short as possible, that is, less than 2 hr. In a few cases, however, it was necessary to store the tumor pieces in the refrigerator overnight. This storage had no profoundly negative effect on the success of the following in v i m cultivation.

Removal of tumor material

t t

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Mechanical and enzymatic disaggregation into a single-cell suspension

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Concentration of the cells to 5 x lo4 cells/pl

4

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Incubation at 37"C/5% for several days

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Organoid growth and macroscopic enlargement of the cultures FIG. 1 Survey of the main experimental steps of the high-density culture method for growing human carcinomas in uifro.

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First, two small fragments were resected from the original carcinomas, one of them being embedded in Epon for further histological and electronmicroscopic investigations, the other one quick frozen in liquid nitrogen for immunohistochemical analysis. The remaining carcinoma pieces were then rinsed in Hanks’ balanced salt solution (HBSS), minced carefully with a small pair of scissors, and washed several times in HBSS. Thereafter, the tumor pieces were suspended in a 20-fold volume of a 1 : 1 mixture of dispase (2.4 U/ml; Boehringer) and collagenase D (250 U/mg; Boehringer, Mannheim, Germany), and incubated at 37°C in a water bath with gentle agitation for 50 to 120 min. The time of incubation depended on the consistency of the carcinomas. In case there was a high content of dense connective tissue in the original tumor specimens, the collagenase D in the enzyme solution has increased to exceed the dispase portion. At the end of the incubation period in the enzyme solution, the suspension was pipetted vigorously to further mince the enzymatically disaggregated tumor pieces. After centrifugation at 800 rpm, the supernatant was decanted and the disaggregated tumor tissue washed three times in growth medium. After 5 ml of the supplemented growth medium were added to the sediment, the suspension was again pipetted vigorously and filtered through a nylon mesh (pore diameter 20 pm) to obtain a single-cell suspension. After another centrifugation at 1000 rpm for 10 min, the supernatant was carefully sucked off and the sediment suspended homogeneously in the remaining fluid drop. Thus, a concentration of about 5 x lo4 cells/pl was obtained. Volumes of 20 pl of this dense cell suspension containing about lo6 cells were then dropped on filter pieces of cellulose nitrate (Figs. 1 and 2) which had been prepared as described later. Before seeding, viability in

FIG. 2 Schematic representation of the experimental arrangement for growing human carcinomas in a high-density culture. The surgically removed carcinoma tissues are minced mechanically and enzymatically, and dropped as dense cell suspensions onto a cellulose nitrate filter lying on the top of a bridge-like support at the gas-medium interface. There, the cell suspensions reorganize and differentiate into carcinoma tissue nodules.

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the single-cell suspension was determined by the trypan blue exclusion method (Durkin er id., 1979). For this purpose, 5 pl of the single-cell suspension were added to 495 pl of a 0.2% solution of trypan blue in saline. Under these conditions, viable cells appeared bright since they were able to exclude the dye, whereas dead cells were stained dark blue. During enzymatic incubation of the tumor pieces, bridge-like supports consisting of stainless steel grids with an upper area of 5 x 15 mm2 and a mesh size of about 1 x 1 mm’ were placed in petri dishes with a diameter of 35 mm and covered by a membrane filter consisting of cellulose nitrate with a pore diameter of 0.2 p m (Sartorius, Gottingen, Germany, SM 11307). The petri dishes were then filled with 2 ml of growth medium, the fluid surface just attaining the level of the support (Fig. 2). This arrangement was prepared at least 10 min before the cell suspensions were deposited on it. The growth medium usually consisted of Ham’s F 12 medium, 80 ml of which were supplemented by 20 ml of fetal calf serum, 1 ml L-glutamine (200 m M ) , 1 ml penicillin/streptomycin (5000 U/ml; 5000 pg/ml), 1 ml amphotericin B (250 pg/ml, Fungizone), 1 ascorbic acid (7.5 mg/ml), 1 ml nonessential amino acids (BME, loox), and 0.5 ml of a 40% (mlm) aqueous glucose solution. All ingredients were purchased from Gibco, Berlin. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO,. The growth medium was renewed every day in order to supply sufficient nutrients and to prevent infections with bacteria and fungi. For light-microscopic and ultrastructural investigations, small pieces of the original carcinomas and culture nodules taken at various days between days 1 and 30 after seeding and measuring about 1 X 1 mm2 were fixed in a solution containing I% glutaraldehyde (v/v) and 1% tannic acid (v/v) in 0.1 mol phosphate bufferlliter at pH 7.2 for 1 to 3 hr. After the carcinoma tissue pieces were rinsed three times in the phosphate buffer, they were postfixed in a 2% solution of OsO, in 0.1 mol aqueous phosphate bufferhiter for I to 2 hr, dehydrated in the alcohol series, and embedded in Epon. Semithick sections with a thickness of 0.5 to 0.6 p m were stained with a 0.2% aqueous solution of toluidine blue, viewed for orientation, and analyzed by light microscope. Ultrathin sections cut with an ultramicrotome were contrasted with an aqueous 1% uranyl acetate and a 2% lead citrate solution, and observed in a Zeiss EM 109 electron microscope. The specimens that had been quick frozen in liquid nitrogen were cut with a cryocut to a thickness of 5 to 8 pm; mounted on glass slides; rinsed in phosphate-buffered saline (PBS) which contained 0.5% human albumin; dried carefully; and incubated with the primary, monoclonal, or polyclonal antibodies against cell- and tissue-typical intracellular and extracellular molecules, certain tumor markers, and some oncogens. The following

192

PETRA KOPF-MAIER ET AL.

preparation of the specimens was done according to the alkaline-phosphatase antialkaline-phosphatase (APAAP) immunostaining techniques described in detail earlier (Kopf-Maier and Schroter-Kermani, 1993).

B. Success Rates of Carcinomas Grown in Vitro According to the techniques of the high-density culture method described in Section III,A, about 80 individual, surgically removed human carcinomas of different origin were grown in uitro as primary cultures. They were derived from the gastrointestinal tract (Table I), the oral cavity, pharynx, and esophagus (Table II), the lung (Table III), and the genitourinary tract, including the female breast (Tables IV and V ) . After surgical removal (day 0 ) , the carcinomas were minced and dropped as dense single-cell suspensions on the filter membrane at the gas-medium interface. During the following 3 to 10 days, the drops diminished in size and gave rise to several nodules of varying size growing on the upper side of the filter membrane (Figs. 3 and 4). These macroscopic alterations reflect profound changes in the inner organization of the cultures and are accompanied by histological signs of reorganization and histotypical redifferentiation in the culture nodules (cf. Sections III,C and D). When the growing carcinomas reorganized in uitro, all nodules of a given culture developed a similar state of reaggregation and differentiation, remaining stable for 1-4 weeks in uitro. Thereafter, the culture nodules became soft and viscous, and dissolved within a few days. Tables I-V document in detail the origin and histopathological classification of the carcinomas investigated and indicate the success rates of human carcinomas in the high-density (organoid) culture, classified according to the rates of reorganization and redifferentiation in vitro. Reorganization is defined as the revival and reaggregation of the minced and dissociated human carcinomas growing in uitro, and organoid redifferentiation as the development of essential morphological characteristics of cellular and histological differentiation in the culture nodules. Table VI summarizes the total numbers of carcinomas investigated and the success rates within the diverse tumors. In the case of gastrointestinal carcinomas, 28 carcinomas were grown in v i m . Five of them could not be evaluated since they either contained very few viable carcinoma cells or were contaminated with bacteria (Tables I and VI). Many of the remaining 23 carcinomas of the stomach, pancreas, bile duct, large bowel, and rectum were grown successfully in uitro, the rates of reorganization and redifferentiation amounting to 72% and 57%, respectively (Tables I and VI). Moderately differentiated carcinomas obviously grew more successfully than poorly differentiated ones;

IN VlTRO REORGANIZATION OF HUMAN CARCINOMAS

193

TABLE I Characteristicsand in Vitro Behavior of the Human Gastrointestinal Carcinomas Evaluateda

Organ origin Stomach' Pancreas

Bile duct

Large bowel'

Rectum'

Histopathology Adenocarcinoma Poorly differentiated Adenocarcinoma Moderately differentiated Adenocarcinoma Moderately differentiated Adenocarcinoma Poorly differentiated Moderately differentiated Carcinoid Poorly differentiated Adenocarcinoma Poorly differentiated Moderately differentiated

Total

Number of carcinomas evaluated

Reorganization in uirroh,(

Differentiation in uitro',d

3

313

2/3

1

-/I

-11

111

111

315

3/5

2i2

212

Ill

111

8

618

318

2

212

I 12

23

18/23

13/23

~

" All gastrointestinal carcinomas were kindly supplied by Prof. Dr. J. Konradt, Krankenhaus ZehlendorflBerlin. Reorganization was defined as the revival and reaggregation of human carcinomas in the organoid culture. ' The ratios are the numbers of reorganized or differentiated carcinomas, respectively, to the total number of carcinomas grown in uitro. Organoid differentiation was defined as the occurrence of morphological characteristics of cellular and histological differentiation under in uifro conditions. One stomach and one rectal carcinoma were not grown in uitro since there were fewer than lo4 viable tumor cells in the single cell suspensions before seeding. Three colon carcinomas were not evaluated since they developed bacterial infection in uitro within 48 hr.

rectal carcinomas generally showed a worse success rate than carcinomas derived from other parts of the gastrointestinal tract. The latter effect may be due to the comparably high content of connective tissue in human rectal tumors. Head and neck carcinomas, including esophageal malignancies, grew in vitro more successfully than gastrointestinal carcinomas and showed an average success rate of about 90%. Ninety-four percent of the head

194

PETRA KOPF-MAIER ET AL.

TABLE II Characteristics and in Vitro Behavior of Human Carcinomas Derived from the Head, Neck and Esophagusa ~~

Organ origin

Histopathology

Mouth floor Keratinizing and pharynx‘’ squamous-cell carcinoma Poorly differentiated Moderately differentiated Nonkeratinizing squamous-cell carcinoma Dedifferentiated Poorly differentiated Moderately differentiated Esophagus Keratinizing squamous-cell carcinoma Poorly differentiated Moderately differentiated Nonkeratinizing squamous-cell carcinoma Poor1y differentiated Large-cell anaplastic carcinoma Thyroid gland Follicular carcinoma Well differentiated Total

~

Number of carcinomas Reorganization Differentiation in vitro”‘ in vitrol.d evaluated

1

3

-/

I

41

313

113

1

111

111

4

414

414

3

313

313

I

1/1

111

1

111

111

1

111

111

I

1/1

111

I

Ill

Ill

17

16/17

14/17

The carcinomas derived from the mouth floor, the pharynx and the thyroid gland were kindly provided by Dr. Dr. R. Tausch-Treml. Universitatsklinikum Steglitz/Berlin and the esophageal carcinomas by Prof. Dr. J. Konradt, Krankenhaus Zehlendorf/Berlin. Reorganization was defined as the revival and reaggregation of human carcinomas in the organoid culture. The ratios are the numbers of reorganized or differentiated carcinomas, respectively, to the total number of carcinomas grown in uirro. Organoid differentiation was defined as the occurrence of morphological characteristics of cellular and histological differentiation under in virro conditions. ‘ One hypopharynx carcinoma was not grown in v i m since there were less than 20% viable cells in the single-cell suspension. (1

”

IN VlTRO REORGANIZATION OF HUMAN CARCINOMAS

195

TABLE 111 Characteristics and in Vitro Behavior of the Human Lung Carcinomas Evaluateda

Organ origin

Histopathology

Lung' Keratinizing (bronchial tract) squamous-cell carcinoma Moderately differentiated Well differentiated Nonkeratinizing squamous-cell carcinoma Moderately differentiated Well differentiated Adenocarcinoma Poorly differentiated Moderately differentiated Large-cell carcinoma Carcinoid Total

Number of carcinomas Reorganization Differentiation evaluated in vitroh,' in uitro',d

2

212

112

2

212

212

2

212

212

I

Ill

Ill

5

415

415

3

213

213

2

I 12

112

I

111

111

18

15/18

14/18

" All lung carcinomas were kindly supplied by Prof. Dr. D. Kaiser, Krankenhaus Zehlendorf-HeckeshornIBerlin. Reorganization was defined as the revival and reaggregation of human carcinomas in the organoid culture. ' The ratios are the numbers of reorganized or differentiated carcinomas. respectively, to the total number of carcinomas grown in uitro. Organoid differentiation was defined as the occurrence of morphological characteristics of cellular and histological differentiation under in vitro conditions. Two lung carcinomas were not grown in vitro since they contained less than 5% viable cells due to previous chemotherapy.

and neck carcinomas that were investigated reorganized in uitro; 82% developed signs of histotypical differentiation (Tables I1 and V1). Nonkeratinizing squamous cell carcinomas grew better in uitro than keratinizing ones, whereas there was no apparent dependence of the rates of reorganization and redifferentiation in uitro upon the degree of in uiuo differentiation.

196

PETRA KOPF-MAIER ET AL.

TABLE IV Characteristics and in Vitro Behavior of the Human Urological Carcinomas Evaluateda

Organ origin Prostate

Testis

Kidney

Bladder'

Total

Histopathology Adenocarcinoma Moderately differentiated Well differentiated Seminoma Embryonal carcinoma Teratoma Well differentiated Renal-cell carcinoma Moderately differentiated Well differentiated Transitional-cell carcinoma Poorly differentiated Moderately differentiated

Number of carcinomas evaluated

Reorganization in uitroh,'

Differentiation in vitroc,d

2

212

212

1

111

111

4

314 111

314 111

1

I

-/I

-/I

2 2

212 212

212 212

1

111

111

1

111

111

15

13/15

13/15

All urological carcinomas were kindly provided by Prof. Dr. L. Weissbach, Krankenhaus Am UrbanIBerlin. Reorganization was defined as the revival and reaggregation of human carcinomas in the organoid culture. The ratios are the numbers of reorganized or differentiated carcinomas, respectively, to the total number of carcinomas grown in vitro. Organoid differentiation was defined as the occurrence of morphological characteristics of cellular and histological differentiation under in vitro conditions. ' Two other bladder carcinomas were not considered; one of them lacked viable tumor cells in the original tumor removed by electrocoagulation, the other one developed bacterial infection in vitro within 48 hr.

Human lung carcinomas, which usually include quite different histopathological types of carcinomas, grew in uirro with an overall success rate of about 80% (Tables 111 and VI). Eighty-three percent of all bronchial carcinomas investigated reorganized in uirro; 78% redifferentiated histotypically in the organoid culture. Two out of 20 carcinomas could not be evaluated since they contained less than 5% viable cells as a consequence of previous chemotherapy. Although there were only small numbers per

197

IN vim0 REORGANIZATION OF HUMAN CARCINOMAS TABLE V Characteristics and in Vitro Behavior of the Human Gynecological Carcinomas Evaluateda

Organ origin Ovary

Cervix uteri

Breast'

Total

Histopathology Carcinoma (omental metastasis) Granulosa-cell carcinoma Pseudomucinous cystadenocarcinoma Squamous-cell carcinoma Poorly keratinizing Duct carcinoma Moderately differentiated (solid-adenoid)

Number of carcinomas evaluated

Reorganization

Differentiation

in uitroh,c

in uitroc,d

1

111

111

I

111

111

I

111

lil

1

111

-1 1

3

213

213

7

617

517

The omental metastasis of an ovary carcinoma and the granulosa-cell carcinoma were kindly provided by Prof. Dr. J. Konradt, Krankenhaus Zehlendorf/Berlin; all other gynecological carcinomas were provided by Dr. G. P. Breitbach, Stadtisches Krankenhaus Neunkirchen. Reorganization was defined as the revival and reaggregation of human carcinomas in the organoid culture. The ratios are the numbers of reorganized or differentiated carcinomas, respectively, to the total number of carcinomas grown in uitro. Organoid differentiation was defined as the occurrence of morphological characteristics of cellular and histological differentiation under in uifro conditions. ' Three breast carcinomas were not grown in uitro since they contained fewer than lo4 viable tumor cells in the single-cell suspension.

histological type, we continually found that squamous cell carcinomas of the lung obviously grew better in the organoid culture than other types of non-small-cell lung carcinomas. Small-cell carcinomas of the lung could not be investigated in this study since they are usually treated primarily by chemotherapeutic drugs without being removed surgically. Urological carcinomas derived from the kidney, bladder, and male genital tract represent another group of human carcinomas that grew successfully in the organoid culture, and reorganized and redifferentiated with an 87% success rate in uitro (Tables IV and VI). This group of carcinomas includes both prostatic and testicular carcinomas, which are known to grow only sporadically in common experiments in in uitro and in uiuo

198

PETRA KOPF-MAIER ET AL.

FIG. 3 A human lung carcinoma (moderately differentiated, keratinizing squamous-cell carcinoma) growing in high-density culture for 4 weeks. The figure documents time-dependent development in uitro, starting from a highly condensed cell suspension dropped on the filter at the gas-medium interface (day 1) and reorganizing into multiple nodules of carcinoma tissue within 12 days.

systems. Two carcinomas derived from the bladder could not be established in a high-density culture; one of them lacked any viable tumor cells after being removed by electrocoagulation and the other was contaminated with bacteria. The human gynecological carcinomas investigated in organoid culture included several ovarian and breast carcinomas, but only one uterine carcinoma. Whereas human ovarian carcinomas could be established in vitvo with a success rate of loo%, the success in growing human breast carcinomas was much lower and only amounted to 33%. This may be explained by the fact that 3 out of the 6 breast carcinomas investigated contained much less than lo4 viable carcinoma cells in the single-cell suspension after the mechanical and enzymatic disaggregation of the original carcinomas. Histological analysis of the corresponding specimens of the original breast carcinomas revealed that most of the carcinomas that did not grow in the high-density culture consisted mainly of fatty and dense collagen connective tissue and included only a few and small carcinoma cell clusters. Since the original specimens of the carcinomas that grew successfully in v i m contained many more carcinoma cell clusters

IN VlTRO REORGANIZATION OF HUMAN CARCINOMAS

199

FIG. 4 A human testicular carcinoma (seminoma) growing in high-density culture for 4 weeks. Starting from a homogeneous single-cell suspension that contained numerous erythrocytes and was seeded at day 0, a single and rather voluminous carcinoma tissue nodule surrounded by a few smaller tumor tissue nodules developed within 8-10 days.

and more than lo4 viable cells in the single-cell suspension that was dropped on the filter membrane, it can be concluded that the rather low success rate for human breast carcinomas in the high-density culture was obviously due to the low number of carcinoma cells in the specimens obtained. Table VI summarizes the total number of human carcinomas received, the portion that could be evaluated, and the rates of reorganization and organoid differentiation in the five classes of human carcinomas examined in our study. There was an overall success rate of 85 and 74%, respectively, with regard to in uitro reorganization and redifferentiation; the single values in the different carcinoma groups ranged from 72 to 94% and 57 to 87%, respectively (Table VI). In afurther attempt, we analyzed whether the success of in uitro cultivation depended upon some experimental factors such as the absolute number of viable cells or the viability in the single-cell suspension dropped on the filter membrane at the gas-medium interface (Figs. 5 and 6; Tables VII and VIII). It became obvious that growth success was highly uncertain when there were fewer than 2.5 x lo5viable cells per drop (20 pl) or less

200

PETRA KOPF-MAIER ET AL.

TABLE VI Summary of the Success Rates Classified According to the Rates of Reorganization and Organoid Differentiation of Human Carcinomas Grown in High-Density Culturea

Type of carcinoma

Reorganization in carcinomas evaluatedc,d

Organoid differentiation in carcinomas evaluated',d

82%

18/23 = 72%

13/23 = 57%

17/18 = 94%

16/17 = 94%

14/17

18/20 = 90%

15/18 = 83% 13/15 = 87%

14/18 = 78%

No. of carcinomas evaluated/No. of carcinomas obtainedb

Gastrointestinal carcinomas Carcinomas of the oral cavity, pharynx, and esophagus Lung carcinomas Urological carcinomas Gynecological carcinomas

23/28

13/15

=

87%

7/10

=

70%

617

=

86%

517

=

71%

Total

80193

=

86%

68/80

=

85%

59/80

=

74%

=

15/17 = 88%

=

82%

The values are summarized from Tables I-V. Some of the carcinomas obtained could not be evaluated since they either developed infection in vitro or did not contain enough viable cells (cf. Tables I-V). The ratios are the numbers of reorganized or differentiated carcinomas, respectively, to the total number of carcinomas grown in uitro. Reorganization was defined as the revival and reaggregation of human carcinoma in the organoid culture: organoid differentiation was defined as the occurrence of morphological characteristics of cellular and histological differentiation under in vitro conditions.

than 40% viable cells in the single-cell suspension dropped on the filter membrane (Figs. 5 and 6; Tables VII and VIII). At higher values of both the absolute number of viable cells per drop and the cell viability in the single-cell suspension dropped onto the membrane filter, there was no clear correlation between the success of in uitro growth and one of both parameters, whereby the cell viability, that is, the density of viable cells (Fig. 5; Table VII), seemed to be more important than the absolute number of viable cells dropped on the filter membrane (Fig. 6; Table VIII). Summarizing these results, it seems to be fruitless to grow human carcinomas in a high-density, organoid culture when there are fewer than 2.5 X los viable cells per drop (20 pl) or the viability in the single-cell suspension is lower than 40%. On the other hand, when there are higher values of both parameters, there is obviously a high probability of successfully growing human carcinomas derived from different origins as primary cultures in uitro. The success rates of human carcinomas in the highdensity culture markedly exceed those in common in uitro models such

IN VIJRO REORGANIZATION OF HUMAN CARCINOMAS

201

Viability in the single cell suspension (%)

80

60

** *** ***

** **

::

*:

**

8**3

** *. ***

%*** **

*:t*

**

***

***

*** t

40

20

0

9

I

I

I

No growth

Moderate

Good

Very good

Reorganization in vitro FIG. 5 Dependence of the success of growing 80 human carcinomas of different origin in high-density culture upon the cell viability in the single-cell suspension dropped on the filter membrane at the gas-medium interface (cf. Table VII). The parameter exploited was the degree of reorganization in uitro. estimated from the histological appearance of the culture nodules in semithick sections.

as monolayer and colony cultures (Von Hoff et al., 1980; Salmon, 1984; Bertoncello and Bradley, 1987). C. Histological Findings

All human carcinomas grown in uitro were examined histologically by preparing and evaluating semithick sections of Epon-embedded culture nodules taken on days 1 to 30 in uitro. They were evaluated with respect to reorganization phenomena occurring in uitro and compared with the specimens of the original, surgically removed human carcinomas in order to estimate the degree of redifferentiation under in uitro conditions. The following paragraphs describe these results qualitatively on the basis of those carcinomas that reorganized and redifferentiated in uitro.

5x10' -

.

. ..

. .. ..

8

t

0.

.. . . . . . .

0.

- ~ 5~10

0.

0. 0:.

0.

0.

0.

lo5 -

.

5x104 I

.I.

No

I

1

I

Moderate

Good

Very good

growth Reorganization in vitro FIG. 6 Dependence of the success of growing 80 human carcinomas of diverse origin in high-density culture upon the number of viable cells per growing culture nodule at the beginning of the cultivation period (cf. Table VIII). For further explanations, cf. legend to Fig. 5 . TABLE VII Dependence of Growth Success for Human Carcinomas in High-Density Culture upon Cell Viability in the Single-Cell Suspension Useda ~~

Cell viability

No growth

< 30%

3 (4%) 2 (2%)

2

30%

< 50% 2

50%

< 60% 1 60%

< 80% Total

1

Reorganization Moderate

Good

Very good

-

-

-

5 (6%)

3 (4%)

4 (5%)

3 (4%) 14 (17%)

5 (6%)

20 (25%)

16

3

Total

(1%)

(4%)

I1 (14%)

6 (8%)

3 (4%)

(22%)

(20%)

43 (54%)

12 (15%)

I1 (14%)

32 (40%)

25 (31%)

80 (100%)

18

" Cell viability given as a percentage in relation to the total cell number was determined by the trypan blue exclusion assay.

203

IN VITRO REORGANIZATION OF HUMAN CARCINOMAS TABLE Vlll Dependence of Growth Success for Human Carcinomas in High-Density Culture upon the Number of Viable Cells per Growing Nodule at the Beginning of the Cultivation Period Reorganization

Number of viable cells

No growth

< 2.5 x 10'

3 (4%) 1 (1%)

1 (1%)

6 (8%)

8 (10%)

2

2

2.5 x los < 5 x 10s 5 x 105

< I x 106 2

I x lo6

< 2.5 x 10' 2

2.5

X

lo6

< 5 x lo6 Total

Moderate

Good

Very good

-

-

-

6

2 (3%)

1

2

(7%) 16 (20%) 7

9 (11%) 10

Total

3 (4%) 10 ( I 2%) 39 (49%)

20 (25%)

(1%)

(3%)

(9%)

(12%)

1 (1%)

-

3 (4%)

(5%)

8 (10%)

12 (15%)

I1 (14%)

32 (40%)

25 (31%)

80 (100%)

4

1. Gastrointestinal Carcinomas Generally, about 70% of the gastrointestinal carcinomas that were investigated reorganized in uitro. This means that many of the segregated cells dropped on the filter membrane revived and reaggregated in uitro. They showed clear signs of cellular proliferation, manifested by the occurrence of mitotic figures in semithick sections and the remarkable enlargement of the islets of viable cells seen on days 2 to 4 in uitro to prominent nodules of densely packed carcinoma cells on later days. Within these culture nodules, signs of histological differentiation, that is, the appearance of adenoid structures resembling the main histological characteristics of the original adenocarcinomas, occurred in 57% of the gastrointestinal carcinomas between days 4 and 20 in uitro..This is clearly demonstrated for a human colon adenocarcinoma and a mucigenous adenocarcinoma of the bile duct in Figs. 7 and 8. In both cases, in the first days in uitro there was a clearly disorderly situation, the cultures consisting of numerous degenerating cells mixed with some viable and morphologically intact cells (Figs. 7b and 8b). Thereafter, the viable cells both proliferated and differentiated, giving rise to carcinoma cell spheroids organized as adenoid structures and composed of one or several central lumina and diverse layers of encircling, polarized, epithelial carcinoma cells (Figs. 7c,d and

204

PETRA KOPF-MAIER ET AL

IN VITRO REORGANIZATION OF HUMAN CARCINOMAS

205

8c,d). These findings are remarkably like those in the in uiuo situation within 19 days in culture. In the case of the mucin-producing bile duct adenocarcinoma, the carcinoma cells grown in uitro also developed the cytological features of mucoid secretion; the central lumina in the cultures filled with mucus and desquamated cells (Fig. 8d) as in the original carcinoma (Fig. 8a). On the bottom of both cultures, especially the bile duct carcinoma, fibroblasts and myoepithelial cells formed a separate layer of cells on the surface of the filter membrane (Fig. 8d). This phenomenon demonstrates that heterogeneous types of cells obviously coexist in a high-density culture in uitro.

2. Head and Neck Carcinomas The in uitro success rates for head and neck carcinomas, including esophagus malignancies, were clearly higher than in the case of gastrointestinal carcinomas and amounted to about 90% (Table VI). Histological analysis revealed that both moderately differentiated and dedifferentiated keratinizing and nonkeratinizing squamous-cell carcinomas reorganized and differentiated in uitro with 93 and 80% success, respectively. In the case of moderately differentiated squamous-cell carcinomas, the stratum basale and stratum spinosum of stratified squamous epithelia were reestablished in uitro; later, keratinizing carcinomas also developed the cytological features of keratinization and formed small globules of keratinized cells resembling horny pearls. On the other hand, dedifferentiated squamous-cell carcinomas of the head and neck, which consisted of large agglomerates of undifferentiated cells in uiuo (Fig. 9a), built up thick layers of undifferentiated carcinoma cells in vitro within 7 to 1 1 days (Fig. 9b-d) and exhibited clear signs of proliferative activity in uitro (Fig. 9d). Another example of a neck carcinoma that was grown successfully in uitro was a well-differentiated human follicular carcinoma of the thyroid gland. It was composed of numerous, densely arranged follicles that were FIG. 7 Moderately differentiated human colon adenocarcinoma. (a) Original carcinoma, (b) day 8. (c)day 19. (d)day 21 in high-density culture. The original carcinoma is characterized by numerous atypically organized adenoid structures (a).After the tumor tissue was minced to a single cell suspension and grown in uirro, numerous tumor cells revived and reorganized to islets of tumor tissue nodules (*) that were still surrounded by numerous degenerating cells during the first days in uitro ( + , b). On day 19. most degenerating cells had disappeared and the cultures mainly consisted of larger nodules of viable carcinoma cells. They built up adenoid structures with one or several central lumina that were encircled by a brush border and surrounded by one or several layers of carcinoma cells (c). Within the following days. the carcinoma tissue aggregates markedly enlarged and formed voluminous three-dimensional nodules which always included several adenoid lumina that often contained mucoid material (d). Semithick sections; a.b.d. x470: c. x600.

206

PETRA KOPF-MAIER ET AL.

IN VIJRO REORGANIZATION OF HUMAN CARCINOMAS

207

encircled by a cuboidal, sometimes columnar epithelium and included small central lumina filled with colloid (Fig. 10a). After this tumor was disaggregated to a single cell suspension and dropped on the filter membrane at the gas-medium interface, it consisted of mostly single cells on days I and 2 in uirro (Fig. lob). These reaggregated and redifferentiated to follicle-like structures until day 4 in uitro (Fig. 1Oc). Thereafter, the number of follicles markedly increased in the culture nodules until day 9 (Fig. 10d). They consisted of large follicular lumina which were encircled by a squamous epithelium that was obviously functionally inactive. Connective tissue cells and parafollicular cells were found between the follicles (Fig. 10d). There was a remarkable degree of similarity between the original carcinoma (Fig. 10a) and the organoid culture nodules of this tumor grown in uirro (Fig. lOc,d) with respect to both cellular composition and histological organization.

3. Lung Carcinomas Human lung carcinomas, which usually consist of quite different histological types of tumors, reorganized and redifferentiated in uitro with a success rate of about 80%. This was true for most keratinizing and nonkeratinizing squamous-cell carcinomas, adenocarcinomas, and large-cell carcinomas, as well as for one carcinoid tumor. They all redifferentiated in uitro to morphological structures that clearly resembled the characteristic features in the original, surgically removed carcinomas. Figure I 1 documents this process for a well-differentiated, keratinizing, epidermoid carcinoma of the lung (Fig. 1 la) grown in high-density culture in v i m (Fig. 1 lb-d). Whereas on day 1, there were both viable and necrotizing single cells that all lacked any sign of reorganization and differentiation (Fig. I I b), some viable cells reaggregated to clusters of densely packed, large and polygonal

FIG. 8 Moderately differentiated, mucin-producing human adenocarcinoma of the bile duct. (a) Original carcinoma, (b) day I , (c) day 4, (d) day 19 in the high-density culture. The original carcinoma contained numerous large adenoid structures surrounded by atypically arranged epithelial cells which included a great number of mucoid droplets in their cytoplasm (a). On day I in uitro, the culture nodules consisted of numerous degenerating cells, erythrocytes. some connective tissue cells and regenerating carcinoma cells that formed small cell clusters (b). Typical adenoid structures that included a small central lumen and showed first signs of a mucoid secretion developed until day 4 (c). During the following days, the adenoid structures markedly increased in size. They included large lumina that were filled with mucus and cellular debris and surrounded by one or two layers of mucin-producing epithelial cells (d). In addition, cells of heterogeneous appearance (carcinoma cells, myoepithelial cells, fibroblasts) covered the surface of the filter membrane (*) underneath the adenoid nodules. Semithick sections: a s . X600: b,d, x470.

IN vim0 REORGANIZATION OF HUMAN CARCINOMAS

209

cells (Fig. 1 Ic) which continuously differentiated and formed globules of corneous material surrounded by cells that exhibited the typical cytological signs of keratinization (Fig. I Id). In many lung carcinomas, two additional phenomena could be observed. A cell sorting-out phenomenon occurred in uitro which resulted in the formation of a two-compartment system consisting of ( I ) several layers of fibroblasts growing on the filter membrane and (2) histotypically organized carcinoma cell clusters lying above them. In one case of a moderately differentiated adenocarcinoma, the arrangement was reverse insofar as the carcinoma cells organized as adenoids were lying on the filter membrane and were superimposed by several layers of fibroblasts and another covering layer of carcinoma cells. Moreover, in many lung carcinomas of diverse histological types, collagen and elastic fibers appeared increasingly at later days in uitro and were deposited in sometimes large amounts in the extracellular space. This synthesis of extracellular matrix material under in uirro conditions is a remarkable indication of an active cell function that obviously revived within several days in the organoid culture. 4. Urological Carcinomas

In the case of human urological carcinomas, a total success rate of more than 80% was found over all investigated human prostatic, testicular, kidney, and bladder carcinomas. In most urological carcinomas, within 6 to 10 days in culture there was an evident reorganization and redifferentiation of the typical histological features characterizing the original carcinomas. Figure 12 shows this development for a moderately differentiated adenocarcinoma of the prostate and confirms both adenoid differentiation and mucoid secretion within 6 days in uitro (Fig. 12c). On day 14, large adenoid structures built up the culture nodules. They consisted of peripherally arranged, epithelial carcinoma cells and voluminous central lumina containing some mucus and desquamated cells (Fig. 12d). Large amounts of necrotizing cells and extracellular matrix material were detected all around.

FIG. 9 Dedifferentiated human carcinoma of the tongue. (a) Original carcinoma, (b) day 1, (c) day 7, (d) day I 1 in high-density culture. The original carcinoma was composed of large aggregates of round and spindle-shaped carcinoma cells devoid of the signs of special cytological and histotypical differentiation (a). One day after seeding. the culture plaques mainly consisted of separated. degenerating and viable carcinoma cells. a few erythrocytes, and fibroblasts (b). Some of the carcinoma cells were just passing the mitotic phase (+, b). During the following days, the carcinoma cells reorganized into solid tissue aggregates resembling the features of the original dedifferentiated carcinoma tissue and lacking particularly differentiated structures (c.d). Semithick sections; a-c. ~ 4 5 0 d. ; ~800.

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Other urological carcinomas investigated were some classical seminomas and nonseminomas of the testis, renal-cell carcinomas of the kidneys, and transitional-cell carcinomas of the bladder and the urethra. Three of the four seminomas that were analyzed reaggregated in culture into large agglomerates of spermatic cells at different stages of differentiation. They contained, in addition, some connective tissue cells that covered the filter membrane at the gas-medium interface. Another testicular tumor, a highly differentiated teratocarcinoma, neither reorganized nor redifferentiated in vitro, probably because of its high level of in uiuo differentiation. All four representatives of renal-cell carcinomas reorganized in uitro and developed typical signs of organoid differentiation within the remarkably short in uitro period of 1 to 3 days. During this time, the single-cell suspension that was dropped onto the filter membrane on day 0 reorganized to an organoid tissue and built up numerous canalicular and spherical structures. They contained a central lumen, which was encircled by cuboidal, partially brush-bordered epithelial cells. and widely resembled the renal tubular structures of the original carcinomas. These features remained stable for 3 to 4 weeks in vitro. Transitional-cell carcinomas of the bladder and the urethra were found to be rather problematic for in vitro growth because they are often removed by electrocoagulation methods which thermically damage the carcinomas so profoundly that there are not enough viable cells left for successful cultivation in vitro. On the other hand, when bladder carcinomas are resected by surgical cystectomy , they reorganize in vitro and redifferentiate in an organoid, histotypical manner within 9 to 1 1 days.

5. Gynecological Carcinomas The last group of human carcinomas that was investigated in the organoid culture consisted of human gynecological carcinomas, mainly represented by ovarian and breast malignancies. All ovarian carcinomas recovered in vitro within 4 to 8 days and built up large tissue nodules that were comFIG. 10 Human follicular carcinoma of the thyroid gland. (a) Original carcinoma, (b) day 2. (c) day 4. Id) day 9 in high-density culture. The original carcinoma was composed of numerous follicles which were encircled by a cuboidal to columnar epithelium and filled with small amounts of colloid (a). Whereas on day 2 the culture nodules contained both degenerating and some viable cells as well as clusters of undifferentiated cells (b). there were clear signs of histotypical differentiation during the following days. resulting in the formation of follicular structures with large central lumina encircled by a simple cuboidal to squamous epithelium (c.d). On day 9 (d). the differentiation of the carcinoma tissue nodules had clearly progressed from that of day 4 (c). On the top of the filter membrane (*I. an additional thin layer of fibroblastic cells appeared on day 9 and later (d). Semithick ; ~450. sections: a.d ~ 2 3 0 b.c

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posed of histotypically organized carcinoma cell clusters, some connective tissue cells, and extracellular matrix material. In the case of a pseudomucinous cystadenocarcinoma of the ovary (Fig. 13a), a remarkable and extraordinary approximation to the in uiuo situation was manifested within 13 days in uitro by the formation of adenoid structures that were encircled by proliferating, cuboidal epithelial cells and filled with mucus and desquamated cells, and by the presence of some fibroblasts and large amounts of extracellular matrix material around the adenoid structures (Fig. 13b-d). On the other hand, no reorganization and redifferentiation of human breast carcinomas occurred in uitvo when the structural components of fatty and collagen connective tissues predominated in the original carcinomas. When there were larger carcinoma cell clusters present in the original tissues, the breast carcinomas reorganized in uitro within 3 to 6 days to solid, spheroid nodules consisting of densely arranged carcinoma cells, some peripherally located fibroblasts, and a central area filled with cellular debris. These results obtained with human gastrointestinal, head and neck, lung, urological, and gynecological malignant tumors demonstrate that most human carcinomas were grown successfully in a high-density culture and redifferentiated in an organoid manner that is not seen in other in uitro culture systems. It seems probable that the spatial separation of the growing cells and the culture medium; the high cell density, which favors the establishment of structural contacts and functional interactions between the seeded cells; and the presence of heterogeneous cell types within the culture nodules are important factors leading to the exceptional success in growing human carcinomas in this in v i m arrangement. D. Ultrastructure

Additional electron-microscopic investigations were performed with selected human carcinomas of different origins and various histopathologies, for which a good level of reorganization had been confirmed by histological FIG. 11 Well-differentiated keratinizing epidermoid carcinoma of the human lung. (a)Original carcinoma, (b) day 1. (c) day 6, (d) day 8 in high-density culture. The original carcinoma consisted of globules of an atypically arranged, stratified squamous and keratinizing epithelium which was composed of a stratum basale ( I ) , a stratum spinosum (2), and centrally located corneous material ( 3 ) (a). On day I , the cell suspension seeded in uirro consisted of separated viable cells, degenerating cells. and extracellular matrix material (b). Within the following days, the viable carcinoma cells quickly reorganized and built up clusters of keratinizing squamous cells with a peripheral stratum basale ( 1 ) and centrally located corneous material (3) (c,d). Among the nonorganized viable and degenerating cells around the stratified squamous cell clusters, numerous elastic fibers were detectable on days 6 and 8 in vitro (+, c,d). Semithick sections: a. x275: b.c.x450; d. x600.

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examination. The ultrastructural studies were aimed at a more detailed description of the pattern of cellular and histological redifferentiation of human carcinomas in the organoid culture. 1. Gastrointestinal Carcinomas All investigated gastrointestinal carcinomas were moderately or poorly differentiated adenocarcinomas derived from the stomach, the bile duct, and the colon, including the rectum. They all consisted of large cell aggregates which formed adenoid structures with a central lumen in the case of the better differentiated carcinomas. Around the lumina, single and sometimes aggregated microvilli, organized as a brush border, were found at the apical carcinoma cell surface. The cell clusters were encircled by a basal lamina which was only rarely continuous, but mostly fragmentary. It separated the carcinoma cell clusters from the surrounding connective tissue. The carcinoma cells around the central adenoid lumina tightly contacted one another and formed lateral interdigitations. desmosomes, and other specialized cell contacts. Most carcinoma cells of the colon and bile duct tumors included numerous mucigen droplets in their cytoplasm and showed signs of mucous secretion into the adenoid lumina. When these carcinomas were dissociated into single-cell suspensions and grown in uitro, within 3 to 6 days they reorganized into globular cell aggregates consisting of densely arranged, clear and structurally intact cells (Fig. 14a). Around these clusters, some fibroblasts and extracellular matrix material was seen in most culture nodules after day 8 in uitro. Fragments of a basal lamina and, sometimes, a continuous basal lamina of irregular thickness (Fig. 14h) underlay the basal carcinoma cell membrane, marking the borderline with the surrounding connective tissue. The carcinoma cells were always densely arranged and connected by numerous desmosomes and lateral invaginations. In addition to these signs of reorganization and cytological redifferentiation, the better-differentiated gastrointestinal carcinomas formed adenoid structures within 2 to 6 days in FIG. 12 Moderately differentiated adenocarcinoma of the human prostate. (a) Original carcinoma, (b) day 1. (c) day 6. (d) day 14 in high-density culture. The original carcinoma is characterized by numerous adenoid structures consisting of atypically arranged epithelia and voluminous lumina filled with some mucoid material (a) and desquamated. degenerating cells (a). When this tumor was dissociated into a single-cell suspension and seeded at the gas-medium interface. it consisted of cytologically and histologically undifferentiated aggregates of separated viable and degenerating cells and of some decomposing material of the extracellular matrix during the first days in uitro (b). Thereafter, the viable cells reorganized and differentiated into histotypically organized adenoid structures that increased in size and in degree of differentiation during the following days (c,d). Semithick sections; a. ~ 2 3 0 b-d. ; ~600.

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v i m (Fig. 14b). These consisted of carcinoma cells that were arranged circularly around a central lumen and projected microvillus-like cellular extensions into the lumen (Fig. 14a-c). In the case of the moderately differentiated bile duct and some colon carcinomas, the microvilli were very frequent, braced by a central core of straight filaments, organized as a striated border and covered by a thick glycocalyx (Fig. 14e). Near the central lumen of the adenoid structures, the cell membranes of neighboring cells were in close contact via junctional complexes consisting of tight junctions, zonulae adhaerentes, and desmosomes (Fig. 14b,c). In the more abluminal parts of the lateral cell membranes, the cells were also connected by desmosomes and lateral interdigitations (Fig. 14c,f). A further sign of cellular differentiation developed in the bile duct tumor and most of the colon carcinomas within 4 to 8 days in uitro. During this period, some mucigen droplets appeared in the cytoplasm of numerous tumor cells. Their contents were obviously discharged into the lumen of the adenoid structures, appearing there as a fine granular material on day 8 and later. Beginning on day 28, a lot of lipid droplets were seen in the cytoplasm of many carcinoma cells, the striated border disaggregated, and an increasing number of necrotic cells were scattered among the clusters of viable cells. These phenomena hinted at a beginning degeneration of the high-density cultures at the end of the fourth week in uitro. 2. Head and Neck Carcinomas The head and neck carcinomas that were investigated ultrastructurally were some poorly and moderately differentiated, nonkeratinizing squamous-cell carcinomas which consisted mainly of polyhedral cells equipped with large bundles of tonofilaments, thin and spine-like cytoplasmic extensions, and a lot of desmosomes located usually at the top of the spiny cytoplasmic processes. A continuous or defective basal lamina

FIG. 13 Moderately differentiated pseudomuciiioua cystadenocarcinoma of the human ovary. (a) Original carcinoma, (b) day 5 . (c,d) day 13 in high-density culture. The original carcinoma was composed of a reticular network of epithelial cell strands encircling globular and polygonal lumina of different sizes. These lumina contained some mucoid material and a few desquamated cells (a). After the original carcinoma tissue was dissociated into a singlecell suspension and seeded onto a membrane filter at the gas-medium interface. the viable cells again proliferated within 2 to 4 days and reorganized to solid nodules of carcinoma tissue within 3 to 5 days (b). The arrows indicate some mitotic figures (+, b). Until day 13, the carcinoma tissue nodules markedly enlarged and differentiated into histotypically organized, mucin-producing cystadenoid structures (c.d) which remarkably resembled the histological features of the original carcinoma (a). There were still numerous mitotic figures on day 13 (+. d). Semithick sections; a. X175; b. X370; c. X135; d. X900 (detail of the boxed area in c).

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FIG.14 Moderately differentiated adenocarcinomas of the gastrointestinal tract and lung in high-density culture. (a-d) Stomach adenocarcinorna of the intestinal type. (a) Day 3; (b-d) day 4 in uifro; (e) colon adenocarcinoma, day 24: (f,g) lung adenocarcinoma. days 21 and 28; (h) bile duct carcinoma, day 9 in uitro. Most adenocarcinomas grown in the high-density culture reorganized and differentiated into adenoid structures within a few days in v i m (a-d). These consisted of a central, spherical or notched lumen and one or two layers of surrounding epithelial carcinoma cells (a-c) which projected microvilli into the lumen and contacted their neighboring cells by specialized cellular contacts such as desmosomes (+, a-d), tight junctions (jb,c), , and some more or less pronounced lateral interdigitations (c,f). In numerous colon carcinomas, a striated border consisting of straight and equally sized cellular extensions. covered by a thick glycocalyx. developed at the luminal surface

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in uitro (e). The formation of a more or less continuous basal lamina (+)

encircling the adenoid carcinoma cell clusters was observed both in human lung (g) and gastrointestinal (h) carcinomas. Sometimes, cell sorting-out processes occurring frequently in the high-

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density culture resulted in the segregation of epithelial ( I ) and connective tissue ( 2 ) compartments. both being separated by a basal lamina of irregular thickness (+, g). Electron micrographs: a,g. ~ 5 6 0 0 b, ; X 12.200; c , e , x20.650: d. X65.800; b,h, X 11.250.

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FIG. 15 Moderately differentiated squamous-cell carcinoma of the hypopharynx. The segregated carcinoma tissue cells grown in the high-density culture differentiated into aggregates of stratified squamous epithelium-like tissue within 7 days in uitro (a-d). These aggregates consisted of polyhedral cells with an irregular cell shape and short cytoplasmic processes interdigitating with neighboring cells and contacting them by typical desmosomes (a.b). Numerous intracytoplasmic bundles of tonofilaments originated from and terminated at these desmosomes (b,c). Sometimes the desmosomes were found on the top of thin. elongated,

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spiny cytoplasmic processes traversing the enlarged intracellular space (*, c). In other epidermoid carcinoma cells grown in uirro. irregularly shaped keratohyalin granules (A, d) occurred within a few days in uirro and closely associated with cytoplasmic tonofilament bundles (+, d), Electron micrographs: a, ~ 5 2 5 0 b. : X23.000: c . X46.500: d. x33,600.

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surrounded the carcinoma cell aggregates and was sometimes linked by some half-desmosomes to the cell membrane of adjacent carcinoma cells. After having been dissociated into single-cell suspensions, the head and neck carcinomas reorganized in uitro to solid aggregates of carcinoma tissues within 5 to 7 days (Fig. 15a). They built up multiple cell layers with an often enlarged intercellular space that was traversed by a varying number of thin cytoplasmic extensions. The spiny processes of neighboring cells usually contacted each other and where cytoplasmic bundles of tonofilaments terminated or originated, they were attached to one another by typical desmosomes (Fig. 15b,c). Some carcinoma cells also contacted their neighboring cells by lateral interdigitations. Cytoplasmic tonofilament bundles were commonly present in the cytoplasm of most cultivated head and neck carcinoma cells. In one epidermoid carcinoma grown in uirro, typical and irregularly shaped keratohyalin granules appeared in the cytoplasm on day 7 and later, and closely attached the tonofilament bundles (Fig. 15d), as is seen in the stratum granulosum of normal keratinizing squamous epithelia. Granular extracellular matrix material was deposited in the extracellular space on day 10 in uirro and later, sometimes assembling into short fragments of a basal lamina. Collagen fibrils and fibroblasts were seen only rarely in high-density cultures of human epidermoid carcinomas of the head and neck. A well-differentiated follicular carcinoma of the thyroid gland was another type of neck carcinoma investigated in our study. The original carcinoma was composed of numerous thyroid follicles consisting of an irregular cuboidal epithelium equipped with numerous cytoplasmic bundles of tonofilaments, single secretory granules, and some microvilli projecting into the central cavity, which was filled with colloid. The follicles were surrounded by a basal lamina and some other extracellular matrix material. When this tumor was dissociated into a single-cell suspension and grown in a high-density culture, the carcinoma cells reorganized and redifferentiated into follicular structures within 9 days in uitro. They consisted of circularly arranged cuboidal epithelial cells which projected a few small microvilli into the central lumen of the follicles and contained some secretory granules in their cytoplasm. Some mitotic activity was seen during this period that resulted in the enlargement of the follicles within the following 3 to 5 days. On days 14, 18, and 28, the culture nodules of the follicular carcinoma included large follicles which were encircled by squamous and sometimes cuboidal epithelial cells. These contained a few secretory granules and bundles of tonofilaments, projected some microvilli covered by a glycocalyx into the follicular lumen, and contacted their neighboring cells by desmosomes, zonulae adhaerentes, and tight junctions, the latter being located adjacent to the central cavity of the follicles. Around the follicles, some fibroblasts, collagen fibrils, and other extracel-

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lular matrix material were found, the latter sometimes assembling into short fragments of a basal lamina. Beginning on day 28, degeneration phenomena occurred and became vast during the following 2 weeks.

3. Lung Carcinomas Lung carcinomas were represented by different nonsmall-cell carcinomas, that is, some bronchial adenocarcinomas and some bronchial epidermoid carcinomas. The original adenocarcinoma tissues were all characterized by adenoid structures built up by circularly arranged carcinoma cells. These surrounded a central lumen; contained some secretory granules; contacted the neighboring cells by desmosomes and lateral interdigitations; and were encircled by a basal lamina, collagen fibrils, and some fibroblasts. Within 4 days in vitro, the carcinoma tissue cells seeded as single-cell suspensions onto the filter membrane at the gas-medium interface and reaggregated into large clusters of clear cells which included some lipid droplets and a few secretory granules. They contacted the neighboring cells by lateral interdigitations, and began to redifferentiate into adenoid structures with a central lumen, some microvilli projecting into the lumen, and specialized cell contacts like desmosomes and tight junctions between apposed carcinoma cell membranes, preferably adjacent to the glandular lumen (cf. Fig. 14a-c). There were also some fibroblasts in the growing tissue nodules as well as collagen and elastic fibers. During the following 10 to 20 days, the adenoid structures in the culture nodules remarkably increased in size and number; the degree of differentiation that was attained in vitro clearly depended upon the differentiation in the original carcinoma tissue. There was an increasing number of fibroblasts, collagen, and elastic fibers, and some granular extracellular matrix material deposited in the culture nodules between days 6 and 28. These elements of the connective tissue were found around the carcinoma cell clusters, being separated from them by small or larger fragments of a basal lamina that parallelled the course of the basal carcinoma cell membrane, the latter being stabilized by a sublemmal filamentous network. This sorting-out process for epithelial carcinoma and connective tissue cells culminated in a two-compartment system in the carcinoma tissue nodules between days 21 and 28 (Fig. 14g). The original epidermoid carcinomas of the lung were composed of large aggregates of cells resembling the morphological features of the stratum basale and the stratum spinosum of multilayered squamous epithelia. The main histological characteristics of this type of tissue were reconstituted in vifro within 8 to 15 days after previous dissociation into single-cell suspensions. Then, the cultures consisted of large aggregates of polyhedral cells with thin and spine-like cytoplasmic processes crossing the enlarged

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intercellular space and making contact with the neighboring cells by typical desmosomes. Bundles of tonofilaments and a few lipid droplets were seen in the cytoplasm of the carcinoma cells. Some fibroblasts, collagen, and elastic fibers were also present in the culture nodules. Short segments of a basal lamina incompletely separated the carcinoma cells from the surrounding elements of the connective tissue.

4. Urological Carcinomas The urological carcinomas derived from the prostate, the testis, and the kidneys differed from one another profoundly with respect to histological type and thus differentiated in uitro into quite different histological features. The adenocarcinoma of the prostate, which was examined ultrastructurally, redifferentiated quickly into adenoid structures within 3 days in uitro. As in the original carcinoma, the culture nodules of this tumor mainly consisted of circularly arranged carcinoma cells with short apical microvilli covered by a glycocalyx and encircling a central lumen. They contained some secretory granules, contacted the neighboring cells by desmosomes and numerous tight junctions, and were surrounded by some collagen fibrils and granular extracellular matrix material which obviously did not assemble into basal lamina-like structures in this carcinoma grown in uitro. An increasing number of necrotic cells occurred in the culture nodules on day 21 and later. The original testicular seminoma, which was analyzed electron microscopically, was composed of aggregates of large and clear cells resembling type A and type B spermatogonia and of surrounding strands of connective tissue, both being separated by a mostly continuous basal lamina. When this tumor was dissociated into a single-cell suspension and grown in uitro, the carcinoma cells and fibroblasts sorted out in uitro within 7 days. From this day forward, the spermatogonia-like carcinoma cells were aggregated into large clusters and separated from the surrounding connective tissue by a monolayered basal lamina that was interrupted by numerous gaps. On day 13 and later, there was an increasing amount of collagen fibrils and granular extracellular matrix material in the connective tissue compartment of the seminoma grown in uitro. Both renal-cell carcinomas that were investigated ultrastructurally were composed of tubular structures with a central lumen and surrounding clear cells which contained a varying number of cytoplasmic organelles and projected either a few and small, or numerous and tall microvilli into the central tubular lumen. Both carcinomas reorganized quickly in the highdensity culture and redifferentiated into organ-typical features within 2 to 4 days (Fig. 16a). These remained stable for at least 28 days in uitro. During this period, the culture nodules contained numerous tubular and

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spherical structures with a central lumen that was surrounded by one or two layers of clear cuboidal cells (Fig. 16a). They were connected by desmosomes, tight junctions, and lateral invaginations; and equipped with microvilli of varying size and number which projected into the adjacent central lumen (Fig. 16b). The carcinoma cells included numerous cytoplasmic organelles and were surrounded by fragments of a basal lamina and some excessive, nonassembled granular extracellular matrix material (Fig. 16c).

5. Gynecological Carcinomas In the group of gynecological carcinomas, one ovarian and one breast carcinoma were examined ultrastructurally. The ovarian carcinoma was a pseudomucinous cystadenocarcinoma which was composed of large glandular aggregates of mucin-producing cells. These surrounded big lumina filled with mucus and cellular debris, and showed the cytological features of mucoid synthesis and secretion. They projected numerous stump-like microvilli with a thick central core of microfilaments into the lumen and rested on a continuous basal lamina that separated the carcinoma cell clusters from the surrounding connective tissue. After this carcinoma tissue was disaggregated into single cells and grown in uitro, reorganization and redifferentiation into large adenoid structures occurred within a few days. As in the in vivo situation, the culture nodules consisted of large cells which contained a well-developed rough endoplasmic reticulum, a Golgi zone, and numerous cytoplasmic mucoid droplets. There were numerous bundles of filaments within the cytoplasm of the carcinoma cells grown in uitro; these passed over into the central core of numerous short microvilli at the luminal cell surface. Most carcinoma cells were connected to their neighboring cells by desmosomes, tight junctions, and lateral interdigitations. The lumen of the adenoid structures was filled with mucus and degenerating cells. The breast carcinoma investigated ultrastructurally was a typical adenocarcinoma consisting of voluminous, irregularly formed globules of epithelial cell clusters scattered between a rich network of connective tissue stroma and fat cell islets. In vitro, typical adenoid structures reorganized within 3 to 6 days and developed the morphological features known from the in uiuo situation. Densely packed, large, and clear cells encircled the central glandular lumina, formed microvilli at the luminal surface, and contacted the neighboring cells by desmosomes and lateral interdigitations. These cell clusters were encircled by segments of a basal lamina of varying length which separated the epithelial carcinoma cells from some fibroblasts and bundles of collagen fibrils surrounding the carcinoma cell clusters.

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FIG. 16 Moderately differentiated renal-cell carcinoma. (a) Day 2, (b) day 14, (c) day 25 in uifro. Within 2 days in the high-density culture, the segregated carcinoma tissue cells reorganized and differentiated into tubular structures (a). These were composed of one or two layers of a cuboidal epithelium surrounding a central lumen (L; a,b). The tubular cells

projected numerous microvilli into the lumen (a,b) and were connected by desmosomes (+, a,b). tight junctions (j, b), and some lateral invaginations (a). The abluminal membrane of the tubular carcinoma cells rested on a basal lamina of irregular thickness (j c). ,Electron micrographs; a, X5600; b, X35,500; c, X 14,400.

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These electron-microscopic results obtained with diverse carcinomas of the gastrointestinal tract, the lung, the head and neck, and the male and female urogenital tract, confirm that there is a remarkable cytological and histotypical redifferentiation of human carcinoma tissue cells in a high-density culture. This was not only documented in light-microscopic studies, but also verified at the ultrastructural level. In many carcinomas, cell sorting-out phenomena occurred in uirro which are known to be an important factor for the induction of differentiation processes. The ultrastructural findings moreover revealed that there was often a restoration of active cell functions in uitro, such as the synthesis and excretion of mucus and other secretions, the establishment of specialized cell contacts, the production of extracellular matrix material, and the formation of a more-or-less continuous basal lamina. This means that human carcinomas reestablish in a high-density culture with respect to both structural and functional parameters, and that this in uitro system obviously comes close to the in uiuo situation as far as certain differentiation parameters are concerned. E. lmmunohistochemical Findings

In a further trial, the expression and presence of certain cell-specific molecules and secretion products, some tumor markers, and oncoproteins were analyzed in the organoid culture nodules of human carcinomas by using immunohistochemical techniques. The results were compared with the findings in the original carcinomas in order to define the cellular composition of the culture nodules in more detail and to estimate the level of cytological and functional differentiation in uitro in a more quantitative manner. Table IX summarizes the data concerning the monoclonal and polyclonal nature of the antibodies used, their animal origin, and the dilution applied. With the exception of the antibody against type IV collagenase, which was kindly supplied by PD Dr. N. Ulbrich, Berlin, all antibodies were purchased from commercial firms. They were all applied in concentrations which failed to stain the control specimens in which the primary antibodies against the investigated cellular and extracellular antigens were omitted, but which were handled identically in all other respects. Different groups of antigens were analyzed to determine the structural and functional stability of the human carcinomas grown in the high-density culture. The cell markers cytokeratin, vimentin, desmin, and myoglobin, which characterize epithelial, mesenchymal, and muscle cells, respectively (Moll, 1987; Altmannsberger et al., 1987), were used to determine whether the cell heterogeneity of the original carcinomas was preserved

TABLE IX Cellular and Extracellular Antigens Identified by lmmunohistochemical Techniques (APAAP method) in Human Carcinomas Grown in High-Density Culture

Cell markers Cytokeratin' Vimentin Desmin Myoglobin Receptor molecules Laminin receptor EGF' receptor Secretion products Type IV collagenase Thyroglobulin Tumor markers CEA' AFP' ACTH' P-HCG' Calcitonin NSE' Serotonin CA 125' CA 19-9' Oncoproteins fos mYc ras Extracellular matrix components Type IV collagen Laminin Fibronectin

Nature of the antibody

Origin of the antibody

Dilution of the antibody

Pol yclonal Pol yclonal Pol yclonal Pol yclonal

Rabbit' Goat' Rabbit' Rabbitd

1 : 2,500 1 : 2,500 1 : 1,500 1 : 2,000

Monoclonal Polyclonal

Ratr Rabbitr

1 : 3,000 I :50

Polyclonal Polyclonal

Rabbitg Rabbitd

1 : 15,000 1 : 2,500

Polyclonal Pol yclonal Pol yclonal Pol yclonal Pol yclonal Polyclonal Monoclonal Monoclonal Monoclonal

Rabbitd Rabbitr Rabbitd Rabbitd Rabbitr Rabbitr Moused Mouseh Mouseh

1 : 2,000 1:5

Pol yclonal Monoclonal Pol yclonal

Sheep' Mouse' Sheep'

1 : loo 1 : 500

Monoclonal Pol yclonal Polyclonal

Mouser Rabbit' Rabbitd

I : 750 1 : 1.000 I : 20,000

1:3,00 1 :4,000 1 : 10 1:15 1 : 50 1 : 100 1 : 250

I : 200

The cytokeratin antibody used was a broad-spectrum antibody reacting with both acidic and basic (class I and 11) cytokeratins. The antibody was purchased from Paesel & Lorei. Frankfurt, Germany. The antibody was purchased from Sigma, Deisenhofen, Germany. The antibody was purchased from Dako, Hamburg, Germany. ' Abbreviations: EGF, epidermal growth factor; CEA, carcinoembryonic antigen; AFP, a-I-fetoprotein; ACTH, adrenocorticotropin; P-HCG, beta human chorionic gonadotropin; NSE, neuron-specific enolase; CA 125, cancer antigen 125; CA 19-9, carboanhydrate antigen 19-9 The antibody was purchased from Dianova, Hamburg, Germany. The antibody was kindly supplied by PD Dr. N. Ulbrich, Institut fur Biochemie, Freie Universitat Berlin, Germany. The antibody was purchased from Biogenesis/Quartett, Berlin, Germany. The antibody was purchased from Serva, Heidelberg, Germany. j The antibody was purchased from ICN, Meckenheim, Germany.

'

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in the high-density culture. The laminin and EGF receptor molecules at the surface of carcinoma cells were investigated since the expression and presence of both molecules at the cell surface is known to be an active function of epithelial cells found usually in most (laminin receptor) or only a few (EGF receptor) carcinoma tissues derived from human patients. The enzyme type IV collagenase is assumed to be secreted by actively invading and metastasizing carcinoma cells (Liotta et af., 1982; Liotta, 1984), and thyroglobin is known to be secreted from differentiated follicular thyroid carcinomas at an elevated level (Heitz, 1987; Lamerz et af., 1988). The group of tumor markers investigated included quite different types of molecules which are correlated with malignant transformation and the growth of malignant tumors. These molecules comprise the oncofetal antigens carcinoembryonic antigen (CEA) and a-fetoprotein (AFP); the tumor-associated carboanhydrate antigens CA 125 and CA 19-9; as well as adrenocorticotropic hormone (ACTH), human chorionic gonadotropin (p-HCG), calcitonin, serotonin, and the neuron-specific enolase (NSE) (Staab, 1987; Kloppel, 1987; Heitz, 1987; Lamerz et af., 1988; Spona and Kuzmits, 1992). Moreover, the expression of the oncoproteins fos, myc, and ras in carcinoma cells and the local distribution of the extracellular matrix components-type IV collagen, laminin, and fibronectin-were analyzed in uiuo and in uitro with type IV collagen and laminin representing main and intrinsic constituents of the basal lamina which usually separates epithelia from the surrounding connective tissue and simultaneously supports essential functions and properties of epithelial cells. 1. Gastrointestinal Carcinomas

Eight human gastrointestinal carcinomas derived from the stomach, the colon, and the rectum were investigated immunohistochemically for the presence and distribution of the cellular and extracellular antigens described, both in the original tissue and the high-density culture nodules. The results, which are summarized in Table X, confirm that when human gastrointestinal carcinomas were grown in the high-density culture, a high stability of cytological and functional parameters was found in comparison with the in uiuo situation. In all cases, heterogeneous cell types of epithelial and mesenchymal origin, which usually compose carcinoma tissues in patients, coexisted in uitro. When human carcinoma cells grown in uitro coexpressed epithelial and mesenchymal cell markers, as was seen in most of the eight gastrointestinal carcinomas, this pattern of coexpression was also maintained in uitro in a similar and comparable manner. The expression or nonexpression of the laminin and EGF receptor molecules

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in uitro also corresponded to the features found in uiuo,and the type IV collagenase produced by most gastrointestinal carcinomas investigated was sometimes found in uitro at a higher concentration than in human patients. All gastrointestinal carcinomas expressed CEA both in uiuo and in uitro. Some of them showed an additional single-cell reactivity for ACTH; three carcinomas reacted with the antibody against the CA 19-9 antigen; and one carcinoma of the colon was characterized by a faint reactivity for calcitonin and NSE. All these tumor markers were found both in uiuo and in uitro in a similar amount and pattern of distribution. By analyzing the oncoproteins fos, myc, and ras, a remarkable stability with respect to the expression or nonexpression of these molecules was again confirmed in most carcinomas that were investigated. Another important factor for determining the functional stability of human carcinomas is known to be their ability to reconstitute a basal lamina. The analysis of the intrinsic basal lamina components-type IV collagen and laminin-revealed that ( 1) many carcinoma cells exhibited an intracytoplasmic reactivity for both components in uiuo and in uitro, indicating that the synthesis of intrinsic basal lamina components is continued in uitro under conditions of high-density culture, and that (2) basal lamina segments of varying length assembled in uitro in most carcinomas both at the border between carcinoma and connective tissue cells and, in contrast to normal tissues, also in enlarged extracellular spaces between carcinoma cells apart from the contact zone to the connective tissue. The latter finding reflects the loss of polarity in most human carcinomas, which is also known from investigations of human carcinomas grown in uiuo (Ingber et af.,1981;Van Den Hooff, 1989; Kopf-Maier and Merker, 1991).

2. Head and Neck Carcinomas Analysis of the immunohistochemical parameters in three squamous-cell carcinomas of the head and the neck and one follicular thyroid carcinoma (Table XI) similarly revealed a remarkable stability of the main cytological and functional features under conditions of high-density culture. This became true for the intracellular epithelial and mesenchymal cell markers cytokeratin, vimentin, desmin, and myoglobin; for the laminin and EGF receptor molecules which are incorporated into the cell membrane; and for the enzyme type IV collagenase. Thyroglobulin was present at a high concentration in the original follicular carcinoma of the thyroid gland, but was found in a weak concentration in the high-density culture nodules of this tumor. Although malignant thyroid tissue is often functionally autonomous, the remarkably decreased synthesizing activity of thyroglob-

TABLE X Comparison of the lmmunohistochemical Detection of Cellular and Extracellular Antigens in the Original Tissue and in High-Density Culture Nodules of Eight Human Gastrointestinal Carcinomas

Cell markers Cytokeratin Vimentin Desmin Myoglobin Receptor molecules Laminin receptor EGF receptor Secretion product Type IV collagenase Tumor markers CEA AFP ACTH P-HCG Calcitonin NSE Serotonin CA 19-9

Poorly differentiated adenocarcinoma of the stomach Originallculture"

Poorly differentiated adenocarcinoma of the colon Originallculture"

+I+

+I+ +I+

-1+I+ ++I+ -1+'I+' -1-1-1-1-1-

Poorly differentiated adenocarcinoma of the colon Originallculture"

+I+ +I(+)

-I+

+I+

+I+ +I+

(+)I+ +I+ -1+ ll( + ) C -I-1-1-I(+)

Mod. differentiated adenocarcinoma of the colon Originallculture"

+I+ -1+ ' I ( +)' -1-1-I-1-1-

+I+ -1+I+ -1( + )I( + ) +I(+) -1+I+

Oncoproteins fos mYc ras Extracellular matrix components Type IV collagen Laminin Fibronectin

+I+ -1-

(+)I-/-

-1-

-I-

Receptor molecules Laminin receptor EGF receptor Secretion product Type IV collagenase Tumor markers CEA AFP

(+)I-

+I+ +I(+)

-1(

+ )/( + )

++I+ (+)I+I+ f

+/+ -1-

+I+ + +I+ + dl-

-I+ +/+ +I+

+I+ +I+ +I+

Mod. differentiated adenocarcinoma of the stomach Originallculture" Cell markers Cytokeratin Vimentin Desmin Myoglobin

-1-1-

Poorly differentiated carcinoid of the colon Originallculture" +/+ + +I+ ( +)I(

+)

-1-

Poorly differentiated adenocarcinoma of the colon Originallculture"

+ +I+ (

+ )I( + )

(+)I-

+ +I+ -1-

+/+ -1-

- 1-

- 1-

++I+ -1-

-b

-1-

+

++I+ -1-

Mod. differentiated adenocarcinoma of the colon Originallculture" ++I(+)

+I+

+

+) h

( (+

+) h

+I+

+I(+)

+

++I+

+

-1(confiniced)

TABLE X (continued)

ACTH P-HCG Calcitonin NSE Serotonin CA 19-9 Oncoproteins fos mYc ras Extracellular matrix components Type IV collagen Laminin Fibronectin

Mod. differentiated adenocarcinoma of the stomach Originallculture"

Poorly differentiated carcinoid of the colon Originallculture"

Poorly differentiated adenocarcinoma of the colon Originallculture"

Mod. differentiated adenocarcinoma of the colon Originallculture"

++I+'

(+)I+ -1-

-1-1-1-1-1+I+

+/-1-1-

-/-

-I(+)I-/-

-/-1-1-1-

+CI+C

+I-I-I++I+

-/-

-I-I-

+

+I+

++d/+

+d

-I-1-1( + )/( + )

-1-1-I-

-I-/-

+/+ +I+ +dl-

a Comparison between the antigen expression in the original carcinoma tissue and the high-density culture nodules during the period of maximum reorganization and differentiationin uitro. The intensity of antigen expression was estimated semiquantitatively from immunohistochemicalpreparations (APAAP method). - , no expression; (+), very weak detection of the antigen; + , moderate detection of the antigen; + , strong detection of the antigen. Only single cells show a cytoplasmic immunoreactivity. Single aggregated carcinoma cells show a coarse-grained, cytoplasmic immunoreactivity. Immunoreactivity only in the connective tissue components.

+

IN vim0 REORGANIZATIONOF HUMAN CARCINOMAS

237

ulin in the follicular carcinoma under in vitro conditions can be explained only by a lack of hormonal stimulation in vitro. With respect to the other tumor markers investigated, CEA was the only marker that was expressed by all four head and neck carcinomas with an intensity comparable in uiuo and in uitro. On the other hand, the dedifferentiated carcinoma of the tongue produced traceable intracytoplasmic amounts of the ras oncoprotein, the thyroid carcinoma of the fos oncoprotein. Again, both oncoproteins were found in a similar concentration in the carcinoma cells of the original tissues and the culture nodules. As in the gastrointestinal carcinomas mentioned earlier, there was both an intracellular and extracellular reactivity with the antibodies against the intrinsic basal lamina components, type IV collagen and laminin, in all head and neck carcinomas investigated. At later days in uitro, these components assembled into distinct segments of a basal lamina that remained more discontinuous and irregular in the high-density culture nodules than in the original carcinomas.

3. Lung Carcinomas Another group of human carcinomas that were investigated immunohistochemically were four nonsmall-cell lung carcinomas (Table XII). They were again characterized by a high in vitro stability with respect to the expression or nonexpression of the cell markers cytokeratin, vimentin, desmin, and myoglobin, and the receptor molecules for laminin and EGF. These carcinomas similarly produced type IV collagenase and the tumor marker CEA both in uiuo and in uitro, but failed to express the analyzed oncoproteins fos, myc, and ras under both conditions. All four lung carcinomas produced extracellular matrix components in uiuo and in vitro, resulting in the formation of fragments of a basal lamina in the high-density culture nodules.

4. Urogenital Carcinomas The fourth group, human urogenital carcinomas, consisted of quite heterogeneous carcinomas, that is, a renal-cell carcinoma, a bladder carcinoma, an ovary carcinoma, and a granulosa-cell tumor (Table XIII). As h the carcinoma types mentioned earlier, when they grew for several days in the high-density culture in vitro, the tumors of the urogenital tract developed the typical cytological and functional features characterizing the original carcinomas. In all tumors, epithelial cells which are identifiable

TABLE XI Comparison of the lmmunohistochernical Detection of Cellular and Extracellular Antigens in the Original Tissue and in High-Density Culture Nodules of Four Human Head and Neck Carcinoma

Mod. differentiated keratinizing squamous-cell carcinoma of the hypopharynx Originallculture" Cell markers Cytokeratin Vimentin Desmin Myoglobin Receptor molecules Laminin receptor EGF receptor Secretion products Type IV collagenase Thyroglobulin

Mod. differentiated nonkeratinizing squamous-cell carcinoma of the hypopharynx Originallculture"

Dedifferentiated squamous-cell carcinoma of the tongue Originallculture"

+ +I+ + +I+ -1-

+I+ + +'I+' -1(+)I(+)

+I+ -1-

++I+ -1-

++I+

+I+ -1-

Well-differentiated follicular carcinoma of the thyroid gland Originallculture"

+ cl(+ y -1-

(+)I+ n.d.dln.d.d

+'/+ ++I(+)

Tumor markers CEA AFP P-HCG Calcitonin Oncoproteins fos myc ras Extracellular matrix components Type IV collagen Laminin Fibronectin

2

+bl+b

+ +/+

+I+ -I-

+/+

-/-

-I-

-I-I-

-/-

-/-

-l-

-I-

-I-

-I-

-I-I-

-I-

-I-I-

+/+

-/-

-/-

-I-

+/+

-I-

+/+ +/+ -/-

+/+I(+)

-/-

(+

-I-

+ +/+ ++I+ +/+

+)

Comparison between the antigen expression in the original carcinoma tissue and the high-density culture nodules during the period of maximum reorganization and differentiation in uitro. The intensity of antigen expression was estimated semiquantitatively from immunohistochemical preparations (APAAP method). - , no expression; (+), very weak detection of the antigen; + , moderate detection of the antigen; + , strong detection of the antigen. Immunoreactivity only in single cells. Immunoreactivity only in the connective tissue strands. n.d. = not determined.

+

TABLE XI1 Comparison of the lmmunohistochemical Detection of Cellular and Extracellular Antigens in the Original Tissue and in High-Density Culture Nodules of Four Human Lung Carcinomas 0

Poorly differentiated Moderately differentiated Moderately differentiated Well differentiated adenocarcinoma adenocarcinoma keratinizing squamous-cell nonkeratinizing squamous-cell of the lung of the lung carcinoma of the lung carcinoma of the lung Originallculture" Originallculture" Originallculture" Originallculture" Cell markers Cytokeratin Vimentin Desmin Myoglobin Receptor molecules Laminin receptor EGF receptor Secretion product Type IV collagenase

+I+ +I(+) -1(+)b/-

+I+ -I-

(+)/+

+I+ -1+b/-

+I+ (+)I-

+/+ -1-

+I+

+I+

Tumor markers CEA AFP ACTH 0-HCG Calcitonin NSE Oncoproteins fos mYc ras Extracellular matrix components Type IV collagen Laminin Fibronectin

+I+ -I-I-

(+)/(+)

-I-

-I-I-I-I-

-/-

-/-

-/-

+I+ -/-

+ +/+

-/-I-I-/-

-I-/-

-/-/-I-

-I-/(+)I(+)

-I-/-I-

+I+ +I+ ( +)
+I+ +I+ +/-

+I+ +I+ (+)I+

-I-

(+)I(+)/-

-/-

-I-/+/4

+I+ +‘I+<

h)

P

Comparison between the antigen expression in the original carcinoma tissue and the high-density culture nodules during the period of maximum reorganization and differentiation in uirro. The intensity of antigen expression was estimated semiquantitatively from immunohistochemical preparations (APAAP method). - , no expression; (+), very weak detection of the antigen; + , moderate detection of the antigen; + + , strong detection of the antigen. Immunoreactivity only in single cells. Immunoreactivity only in the connective tissue components.

TABLE Xlll Comparison of the lmmunohistochemical Detection of Cellular and Extracellular Antigens in the Original Tissue and in High-Density Culture Nodules of Four Human Urogenital Carcinomas

Moderately differentiated renal-cell carcinoma Original/culture"

h)

P

h)

Cell markers Cytokeratin Vimentin Desmin Myoglobin Receptor molecules Laminin receptor EGF receptor Secretion product Type IV collagenase Tumor markers CEA AFP

+I+ +/+ -/+I(+)

Mod. differentiated transitionalcell carcinoma of the bladder Original/culture"

Carcinoma (omental metastasis) of the ovary Originallculture" ++I++ (+)I(+) +/+ +/+

-/-

+/+ +/+ (+)I-1++I+ -1-

+I+ -1-

+=/+c

Granulosa-cell tumor of the ovary Originallculture"

+/+ -I-

-1-I-

r~

f

-1-

-I-

-/-1-

-/-

ACTH P-HCG Calcitonin NSE CA 125

n.d.d/n.d.d

Oncoproteins fos myc ras

+I+ -1-1-

Extracellular matrix components Type IV collagen Laminin Fibronectin

+I+ +I+ +I(+)

(+)/-

-I-/n.d.d/n.d.d

(+)/(+)

-1-

-/-1+ +/+ +

-I-/-

-I-/-I-

-1-1-

+I+ -1-

+/(+) -1-

-1-

+/(+I

-1-

+I(+) +I+

+I+ +I+ +I+

( + ) I (+ ) +I+ ( + ) I (+ )

+CI+'

Comparison between the antigen expression in the original carcinoma tissue and the high-density culture nodules during the period of maximum reorganization and differentiation in uitro. The intensity of antigen expression was estimated semiquantitatively from immunohistochemical preparations (APAAP method). - , no expression; (+), very weak detection of the antigen; + , moderate detection of the antigen; + + , strong detection of the antigen. Immunoreactivity only in the connective tissue strands. ' Immunoreactivity only in single cells. n.d. = not determined.

244

PETRA KOPF-MAIER ET AL.

by the intracytoplasmic presence of cytokeratin coexisted in uitro with cells of mesenchymal origin expressing vimentin, desmin, and/or myoglobin. In the renal-cell, ovary, and granulosa-cell tumors, numerous carcinoma cells coexpressed epithelial and mesenchymal cell markers in the original carcinoma tissue and continued this behavior in uitro after being grown in the high-density culture for a few days. Many urogenital carcinomas expressed the laminin and EGF receptor molecules at their cell surface and produced type IV collagenase both in uiuo and in uitro. The tumor marker CEA was expressed in the kidney, the bladder, and the ovary carcinomas in uivo and in uitro in an analogous strength, and the CA 125antigen was found in a similarly high concentration in the original ovary carcinoma and the culture nodules following the first days of in uitro culture. The expression and nonexpression of the oncoproteins fos, myc, and ras remained stable in all cases under the applied in uitro conditions, and the synthesis of the extracellular matrix components-type IV collagen, laminin, and fibronectin-revived after a few days in vitro, leading to the assembly of a fragmentary basal lamina at the borderline between epithelial and connective tissue cells in the organoid culture nodules. These immunohistochemical studies broaden the qualitative histological and electron-microscopic investigations performed with different human carcinomas in a high-density culture and reveal that most carcinomas that were investigated preserved their cellular heterogeneity in the high-density culture as well as the pattern of coexpression of epithelial and mesenchyma1 cell markers in the carcinoma cells grown in uitro. About half of the carcinomas showed only faint to moderate reactivity with the EGF receptor antibody both in uiuo and in uitro, whereas most of them expressed the laminin receptor being incorporated, other than in normal tissues, circumferentially in the cell membrane of most carcinoma cells. This was seen not only in the original carcinoma specimens, but also in the highdensity culture nodules. A similar correspondence was observed with respect to the detection of the oncoproteins fos, myc, and ras in uiuo and in vitro. These results confirm a pronounced cytological stability for most human carcinomas analyzed under the in vitro conditions of the organoid, highdensity culture that was not similarly observed in common and established culture systems. Further immunohistochemical investigations which aimed at the determination of the functional stability of human carcinomas in a high-density culture revealed that all investigated indicators of important carcinoma cell functions, for example, the secretion of the basal lamina-degrading enzyme, type IV collagenase; the synthesis of relevant tumor markers such as CEA, AFP, ACTH, CA 19-9, or CA 125; and the production and secretion of the extracellular matrix components, type IV

IN VlTRO REORGANIZATION OF HUMAN CARCINOMAS

245

collagen and laminin, revived after some days in culture and attained a strength of expression equal to that seen in the original human carcinoma specimens. In addition to the earlier-mentioned histological and ultrastructural studies, these immunohistochemical results clearly confirm a high cytological and functional stability for most of the individual human carcinomas of quite different types and origin investigated in the high-density culture and thus approximate the in uiuo situation to a much higher extent than has been observed in competing in uitro models such as monolayer, suspension, colony, spheroid, or gel-supported culture systems.

IV. Concluding Remarks The results documented in the preceding sections reveal that most surgically removed, primary human carcinomas that were dissociated into single-cell suspensions and seeded in high cell densities at a gas-medium interface, grew and redifferentiated histotypically under the applied culture conditions. The success rates estimated from the reorganization phenomena occurring in uitro ranged from 83 to 94%, with an overall average of 85%. Insofar as the success rates were assessed on the basis of the histotypical pattern of differentiation developing in uitro, they amounted to 57 to 87%, with an overall average of 74%. By use of electron microscopy it was illustrated that heterogeneous types of cells coexisted in uitro and separated from one another by cell sorting-out phenomena which are known to be important for the induction of differentiation processes (Moscona and Moscona, 1952; Steinberg, 1963; Zimmermann, 1987). Moderately differentiated features such as adenoid, pseudoglandular, and renal tubular structures, stratified squarnous epithelium-like formations, specialized cell contacts and brush border-like arrangements, the production of extracellular matrix material, and the assembly of a fragmentary basal lamina were the morphological signs of cytological and tissue-typical differentiation in uitro. Irnmunohistochemical investigations moreover revealed that the expression of oncoproteins and typical cell markers such as cytokeratin, virnentin, and desrnin as well as coexpression patterns of epithelial and rnesenchymal cell markers were reestablished in uitro; cell activities such as the synthesis of tumor markers and extracellular matrix components revived in the high-density culture within several days. These results confirm that the high-density culture comes close to the in uiuo situation as far as phenomena of histological reorganization and

246

PETRA KOPF-MAIER ET AL.

differentiation, and the recovery of important cell functions are concerned. A similarly high level of cytological and functional redifferentiation in uitro was not attained after growing human carcinoma cells or tissues in monolayer and colony cultures (Bulbul et al., 1986; Balconi et al., 1988; Shea et al., 1989; Hamburger and Salmon, 1977; Salmon, 1980) or in the three-dimensional gel-supported and multicellular spheroid culture systems (Abaza et al., 1978; Freeman and Hoffman, 1986; Hoffman et al., 1989; Rofstad et al., 1985; Mueller-Klieser, 1987; Sutherland, 1988). In a comparison of high-density culture with these established in uitro models for growing human carcinomas, the high-density culture is distinguished from the outset by the presence of multiple cell types in the culture nodules and the separation of the growing cells from the nutrient medium by a permeable membrane filter. This type of culture setup, which intentionally avoids the submersion of the seeded cells in nutrient medium and the dilution of molecules secreted by the growing cells in a fluid surrounding them, should allow the establishment of an appropriate cell- and tissue-typical microenvironment around the cells and the functioning of autocrine and paracrine mechanisms working within the culture nodules. These mechanisms, which are the molecular basis of intercellular communication and permit the establishment of functional cell-to-cell contacts, together with the high cell density which facilitates the formation of close structural contacts between neighboring cells, are obviously important factors for the induction of differentiation processes in human carcinoma cells in uitro, as was observed earlier with embryonic tissues (Merker et al., 1984; Zimmermann, 1987). The cellular heterogeneity in the culture nodules and the cell sorting-out phenomena that lead to the segregation of epithelial and mesenchymal cells, which are often separated by a fragmentary basal lamina, should also stimulate cellular differentiation processes (Steinberg, 1962, 1963), the basal lamina maintaining the polarity, organization, and functional control of the growing epithelial cells (Vracko, 1974, 1982; Karst and Merker, 1988). Other experimental possibilities for growing human carcinomas outside human patients are some animal systems, such as the nude mouse model (Povlsen and Rygaard, 1971, 1972; Fiebig and Lohr, 1984) and the subrenal capsule assay (Bogden et al., 1981; Bogden and Von Hoff, 1984). In both models, surgically removed individual human carcinomas were heterotransplanted with a rather high overall success rate of 75 and 89%, respectively (Fiebig and Lohr, 1984; Fiebig et al., 1984; Bogden and Von Hoff, 1984). In the nude mouse model, extensive and detailed studies were performed with different types of human carcinomas. It was shown that initial growth in the first passage occurred in the case of 83% of smallcell and 82% of nonsmall-cell lung tumors, 78% of colorectal and 68% of stomach carcinomas, 59% of renal-cell carcinomas, but only with 36% of

IN VITRO REORGANIZATION OF HUMAN CARCINOMAS

247

testicular and 26% of head and neck malignancies (Fiebig e f al., 1984; Braakhuis et al., 1984). Serial passages could be established successfully in 50 to 60% of those carcinomas that had grown in the first passage in athymic nude mice. By comparing these data with the success rates of human carcinomas in the high-density culture, it becomes obvious that the rates are similar in both models in the case of lung and gastrointestinal carcinomas, but much higher in the high-density culture when urological malignancies and head and neck carcinomas are considered. Both the nude mouse model and the subrenal capsule assay were tested as experimental systems for screening antitumor drug activity and drug resistance in individual human carcinomas before clinical chemotherapy. Only a few investigations were done using the subrenal capsule assay (Bogden et al., 1981; Bogden and Von Hoff, 1984) since the model was found to be burdened by severe practical and methodical limitations, such as the need to surgically implant the human tumor pieces under the renal capsules of mice, the occurrence of degeneration phenomena in the transplants, and lymphocytic infiltration into the xenografts, both factors that influence the size of the transplants, which is a crucial parameter for determining the effect of chemotherapy. On the other hand, the nude mouse model is at present the best predictive and most relevant experimental system for preclinical screening of newly developed antitumor agents and for predicting the outcome of clinical chemotherapy for individual cancer patients (Povlsen and Jacobsen, 1975; Bellet et al., 1979; Fiebig el al., 1984; Braakhuis ef af., 1984). It was shown that xenografts in nude mice gave a correct prediction for drug resistance in 97% and for tumor response in 92% (Fiebig et al., 1984) of the cases. This model, then, clearly has a higher correlation with the results of clinical chemotherapy than, for example, the stem cell assay as the main in uifro model for antitumor drug testing (Salmon et al., 1980; Salmon, 1984; Shoemaker e f al., 1984). In spite of excellent correlation rates, the nude mouse could not be developed as a model for realizing the antioncogram, that is, as a pretherapeutic screening system for determining the drug sensitivity or resistance of individual human carcinomas. The need to use living animals, the low testing rate of only 20 to 50% and especially, the long duration of the testing procedure of surgically removed, individual tumor material in the nude mouse model, which usually requires 30 to 60 weeks to obtaining results, are the main limitations and practical handicaps of this model. Here we have described an organoid, high-density culture for growing primary human carcinomas in uifro with an overall success rate of 75 to 85%, which clearly exceeds the growth rates of primary human carcinomas in other culture systems. Since, in contrast to the nude mouse model, high-density culture does not require living animals o r take several months,

248

PETRA KOPF-MAIER ET AL.

but needs only 6 to 20 days to grow surgically removed human carcinomas in an organoid, histotypical manner which comes close to the in uiuo situation, we made great efforts to develop an “antioncogram” on the basis of an organoid, high-density culture, that is, an in vitro system for testing the response of individual human carcinomas to cytostatic drugs within a short time (Kopf-Maier, 1992; Kopf-Maier and Kolon, 1992). It is easy to treat the organoid culture nodules growing on a membrane filter at the gas-medium interface with cytostatic drugs by adding the drugs to the nutrient medium beneath the filter. As in the in uiuo situation, the cytostatic drugs do not flow around the carcinoma cells and do not contact them directly in this setup, but have to diffuse through the filter sheet before they reach the carcinoma tissue nodules. In numerous experiments we then evaluated the modalities of testing cytostatic drugs in a high-density culture and of determining the cytostatic effects achieved (Kopf-Maier and Kolon, 1992; Kopf-Maier, unpublished results), and found the following procedure to be convenient and appropriate for predicting the sensitivity or resistance of human carcinomas to cytostatic drugs (Fig. 17). After human carcinomas were grown in a high-density culture for 5 to 15 days according to the techniques described in Section 111, the cytostatic drugs were added to the nutrient medium below the membrane filter for

of human carcinomas at the gas-medium interface

+

t bT&&%d

Addition of cytostatic drugs to the growth medium (e.g., for two days)

+

t

@ @ Is/\

rrjr

Detachment of the nodules and bisecting them

A i B

C i D

I

I

A, C: determination of the viable cell fraction by the neutral red method B, D: determination of the total cell mass by measuring the total cell protein (e.g., via the Lowry or the BCA method)

t

FIG. 17 Survey of the main experimental steps of the organoid culture assay, proposed as a method for realizing the pretherapeutic “antioncogram.” that is, as an assay that may allow one to predict the outcome of clinical chemotherapy in individual cancer patients.

IN VlTRO REORGANIZATION OF HUMAN CARCINOMAS

249

4 or 24 hr. Then the drug-containing medium was removed, replaced by a growth medium containing 50 pg neutral red/ml, and the cultures incubated for another 24 hr. [The neutral-red method is a colorimetric assay for the assessment of cytotoxicity in v i m (Borenfreund and Puerner, 1984; Borg ef a!., 1984). It is based on the fact that the relatively nontoxic, supravital dye, neutral red, is taken up selectively by live, viable cells and incorporated into their cytoplasmic lysosomes.] After this, the culture nodules were detached and bisected. In one half, the neutral red taken up by the cells was extracted by the addition of 2 ml of an acetic acid-ethanol mixture and the amounts of the dye extracted were measured spectrophotometrically by determining their absorbance at 540 nm. In the other half, the total mass of both viable and dead cells was estimated by determining total protein either by BCA assay (Smith er al., 1985; Slocum and Deupree, 1991 ;Bicinchoninic acid protein assay kit, Sigma, Deisenhofen, Germany) or the Lowry method (Lowry et al., 1951; Oyama and Eagle, 1956). The quotient of neutral red absorbance divided by total protein in treated cultures, related as a percentage of the control value, indicated the fraction of viable cells and gave a measure of the cytotoxic injury caused by the cytotoxic drug. In a pilot experiment, we investigated whether this organoid culture assay (OCA, Fig. 17) is suitable for detecting differences in the drug sensitivity and resistance of human carcinomas in vitro. We used this method with three strains of a human, moderately differentiated, squamous-cell carcinoma of the hypopharynx that had been xenografted into athymic, nude mice and distinguished there by different levels of experimentally induced resistance to the inorganic cytostatic drug cisplatin ( Jackel and Kopf-Maier, 1991). These differences in resistance were clearly retained in uirro and mirrored by increasing EC,, values after treatment with cisplatin in the OCA (Fig. 18). The EC,, values amounted to 0.9 x mol cisplatin/liter in the original, highly sensitive strain, which regressed completely in uiuo after administration of optimum doses of cisplatin. In the partially resistant strain, which was accustomed to mollliter. weekly doses of 6.5 mg cisplatidkg, the ED,, has 5.5 X In the highly resistant strain, which had been treated in mice at least twice per week with 6.0 mg cisplatinlkg, it was 0.9 x lo-’ molhter. These results confirm that the different levels of resistance which had been induced experimentally in uiuo can be detected in uitro by statistically significant ( p < 0.01) differences in the response levels, which vary by factors from 5 to 10, and that the organoid culture assay may be suitable for revealing differences in the sensitivity and resistance of human carcinomas to cytostatic drugs in a rapid and feasible manner. Nevertheless, it is at present too early to decide whether the OCA can be developed as a new approach to establish the antioncogram, that is, as

PETRA KOPF-MAIER ET AL.

250 Oh

of control

Original sensitive strain

20 -

t 80 60 40

20 0 C

5x107

5x10-6

+ Concentration of

5x105

5~10.4

cisplatin (mol/l)

FIG. 18 Testing of three strains of a human hypopharynx carcinoma against increasing concentrations of cisplatin (abscissa) in the organoid culture assay. The strains differ in their level of resistance to cisplatin. The parameter evaluated is cell viability given as a percentage of the control value (ordinate). It was determined by either trypan blue exclusion assay (0 - 0 ) (Durkin er a / . , 1979) or neutral red uptake (Borenfreund and Puerner, 1984) in relation to the total cell mass: the latter was measured by the BCA method ( O ~ ~ I I I I I O ) . The ECSovalues (concentration effecting a loss of viability in 50% of the cells compared with the controls) are indicated by downward arrows and amount to 0.9 x (original sensitive strain), 5.5 x (partially resistant strain), and 0.9 x 10-5 mol cisplatin/liter (highly resistant strain), respectively.

an approach to predict the outcome of clinical chemotherapy in individual cancer patients. For this decision, numerous additional in uitro data based on other carcinomas and cytostatic drugs are required. For the present, a broad-based study has been performed in our laboratory with the scope to determine the correlation rate between the treatment results for human carcinomas of different origin and varying histopathology in the OCA and in nude mouse experiments, which are known to reflect quite well the clinical situation of cancer chemotherapy (Bellet et al., 1979; Shorthouse

251

IN VlTRO REORGANIZATION OF HUMAN CARCINOMAS

et al., 1980; Fiebig et al., 1984; Braakhuis et al., 1984). Moreover, some initial experiments have been done in the OCA in parallel with clinical chemotherapy trials. One example of an ovary carcinoma is illustrated in Fig. 19 (P. Kopf-Maier, D. Miihlen, G. P. Breitbach, unpublished results). This carcinoma had relapsed to a clinical first-line chemotherapy with cyclophosphamide and carboplatin, and actually showed resistance in the OCA to both mafosfamide (generously supplied by Dr. J. Pohl, ASTA company, Bielefeld, Germany), which represents a stabilized cytostatic metabolite of cyclophosphamide (Klein et al., 1984; Niemeyer et al., Viability (%)

I

lZO 100

Mafosfamide

Viabiliw (%)

Treosulfan

Plasma peak concentration

80

60 40

20 0 Viability

C

(YO)

4x104 4 ~ 1 0 - ~ r n o l / l

Viability (%)

Carboplatin

I

Cisplatin

lZo

100

80

Ioo1 80

60 40

6o

20

20 40 C

1 . 3 ~ 1 0 ~1 . 3 ~ 1 0 - ~ Concentration in vitro

0

1

1i

c

2x10-5 2x104 Concentration in vitro

FIG. 19 Testing of a human ovary carcinoma that relapsed under clinical first-line therapy with cyclophosphamide and carboplating in the OCA (P. Kopf-Maier. D. Miihlen, and G. P. Breitbach, unpublished results). The carcinoma was tested in vitro against mafosfamide, which represents a stabilized cytostatic metabolite of cyclophosphamide; treosulfan, which is another alkylating agent; carboplatin; and cisplatin. All four cytostatics were applied for 24 hr in plasma peak and 10-fold higher concentrations. The cell viability appointed in the treated cultures was related as percentage to the values found in untreated control cultures (C, 100%). Mafosfamide was generously supplied by Dr. J. Pohl, ASTA, Bielefeld, Germany, and treosulfan by G. Sass, medac. Hamburg, Germany.

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1989), and to carboplatin; however, it was sensitive to other cytostatics such as treosulfan (generously supplied by Mrs. G. Sass, medac company, Hamburg, Germany) and cisplatin. Although this example suggests that the OCA may be suitable for reflecting the clinical situation of drug responsiveness and resistance of human carcinomas, it is necessary to perform numerous other comparative, experimental and clinical trials with different human carcinomas and cytostatic drugs in order to judge the actual value of the OCA as an “antioncogram.” In any case, irrespective of the results of these experiments, which have to be done in the future, high-density culture is a valuable method for growing individual human carcinomas as primary cultures, for performing experimental studies with human carcinomas in uitro, and for investigating differentiation processes in human carcinoma cells and tissues under in uitro conditions in a manner that is not possible in common culture systems. Thus, the high-density, organoid culture is obviously an important addition to the arsenal of experimental methods for growing human carcinomas outside human patients without using living laboratory animals. Acknowledgments The authors thank Dr. G. P. Breitbach, Frauenklinik des Stadtischen Krankenhauses Neunkirchen, Prof. Dr. D. Kaiser, Abteilung fur Thoraxchirurgie des Krankenhauses Zehlendorf-Heckeshorn/Berlin, Prof. Dr. J. Konradt, Chirurgische Abteilung des Krankenhauses Zehlendorf/Berlin, Prof. Dr. H. Scherer and Dr. Dr. R. Tausch-Treml, HalsNasen-Ohren-Klinik des Klinikum Steglitz/Berlin. and Prof. Dr. L. Weissbach, Urologische Klinik des Krankenhauses Am UrbadBerlin for their cooperation and for having provided the surgically resected specimens of human carcinomas, and PD Dr. N. Ulbrich, Institut fur Biochemie der Freien Universitat Berlin, for having kindly supplied the antibody to the type-lV collagenase. They thankfully acknowledge the expert technical assistance of Mrs. C. Schreiner, and thank Mrs. M. Risch for typing the manuscript and for preparing a computer-driven typeset version. The work was supported by a grant from the Trude Goerke Foundation.

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Index

A Acetabularia, cytoplasmic streaming analysis using tonoplast-free cells, 112 microtubule system-supported, 109-1 10 Acidic fibroblast growth factors, migration induction by, 70-71 Actin, cytoplasmic streaming mechanism affected by calcium sensitivity, 123-124 identification in plants, 108 Actin-myosin system, motive force generation, 100-108 actin identification in plants, I08 Characeae, 100-103 myosin identification in plants, 103-108, 130 Action potential, cytoplasmic streaming affected by, 120-125, 130 Amino acids hormonal character. 23 sequence homology with gonadotropic hormone receptors. 27 Amoeba, hormone effects, 2 Antigens, in immunohistochemical identification of carcinomas gastrointestinal carcinomas, 233-236 gynecological carcinomas, 242-244 lung carcinomas, 240-241 technique, 230-232, 245 urological carcinomas, 242-243 ATP cytoplasmic streaming role concentration as determining factor, 112-11s temperature effects, 127

dynein outer arm changes caused by, 159-160, 169 ATPase, related activity of dynein. 146- I47 vanadate-dependent, 151 Autocrine motility factor, migration induction by, 79-80

Basic fibroblast growth factors, migration induction by, 71-73 Bladder carcinomas characteristics, 196 high-density cultures, 21 1 Boyden chamber assay, as migration analysis method, 53, 55-57 Breast carcinomas characteristics, 197 high-density cultures, 198-199, 21 1, 213 ultrastructure, 227 Bryospsis, cytoplasmic streaming supported by microtubule system, 109

C Calcium, cytoplasmic streaming affected by action potential role, 120-125 gravity role, 128-129 inhibition in Characeae, 118-120, 130 temperature role, 126-127

259

260 Carcinomas, human, cultures cell culture systems monolayer, 182-183 spheroidal aggregate, 186- 187 stem cell, 184-186 cytostatic drug experiments, 248-250 high-densit y gastrointestinal carcinomas, 100. 192-193 gynecological carcinomas, 197-200 head and neck carcinomas, 193-195, 200 histological findings, 201-214. 216 lung carcinomas, 195-196, 198. 200 methods overview. 187-192 success rates, 192-201 urological carcinomas, 196-200 histological findings, 201-214, 216 gastrointestinal carcinomas, 203-206 gynecological carcinomas, 21 1, 213, 216 head and neck carcinomas, 205. 207-2 10 lung carcinomas, 207, 209, 212-213 urological carcinomas, 209, 21 I . 214 immunohistochemical findings gastrointestinal carcinomas. 232-236 gynecological carcinomas, 237, 242-245 head and neck carcinomas, 233, 237-239 lung carcinomas, 237. 240-241 methods overviews, 230-232 urological carcinomas. 237, 242-245 keratinizing carcinomas, 205 methods, 181-182 mouse models, 247 organoid culture assay, 248-252 ultrastructure, 213, 215, 217-229 gastrointestinal carcinomas, 215, 217-221 gynecological carcinomas, 227 head and neck carcinomas, 217. 222-225 lung carcinomas, 218-221, 225-226 urological carcinomas, 226-229 Caulerpa, cytoplasmic streaming supported by microtubule system, 109 Cell migration, see Migration Cell motility, definition, 50

INDEX Chara, cytoplasmic streaming in

action potential as factor, 121 calcium-regulated inhibition, 118 light as factor, 125 microtubule system-supported, 109 reconstruction experiments, 116 Characeae, cytoplasmic streaming in actin-myosin system role, 100-103 demembered cell models calcium-regulated inhibition, 1 18- 120, 130 energy requirements, 112-1 14 longitudinally cut model, 112-1 13 magnesium requirement, 114-1 15 pH dependence. I15 plasma membrane permeabilized cells, 110- I I I preparation, 110-1 13 reconstruction experiments, 116-1 17 tonoplast-free cells, 111-112, 115 gravity role, 127-129 mechanism, 129-130 Chemotaxis, in migration analysis, 53. 55-57 Chlamydomonas, flagellar dyneins characteristics, 141 cross-bridge cycle, 169 flagellar motility in uivo, 166-167 gene modification and expression, 173 heavy chains, 150, 152-154 inner row in siru localization, 164 intermediate chains, 148-151 light chains, 147-148 microtubule translocation in uirro, 167-168 outer arm, 155-160 outer row composition, 147 structure-function relationships, 161- I62 structure, 146 Chlamydomonas reinhardtii, dynein assembly, 146 Coating assays, as migration analysis method, 60 Colon carcinomas, ultrastructure, 215 Cultures human carcinoma, see Carcinomas, human, cultures system analysis, 52-53

261

INDEX

Cyclic AMP receptors, in ameba, 2, 8 Cytoplasmic streaming Characeae. actin-myosin system role. 100-103 Characeae, demembered cell models calcium-regulated inhibition, I 18-120, I30 energy requirements, 112-1 14 longitudinally cut model, 112-1 13 magnesium requirement, 114-1 15 pH dependence, I15 plasma membrane-permeabilization, 110-111

preparation, 110-1 13 reconstruction experiments, 116-1 17 tonoplast-free cells. 1 11-1 12, 115 classification, 97-98 extracellular factors affecting action potential, 120-125, 130 gravity. 127-129 light, 125 low temperature, 125-127 mechanism, 97-98. 129-130 motive force generation mechanism actin identification in plants, 108 actin-myosin system involvement, 100-108 Characeae, 100- 103 microtubules, 109-1 10 myosin identification in plants, 103-108, 130 site, 98-100 sliding theory, 99-100 Cytostatic drugs. carcinoma culture experiments with, 248-250

D Dictyostelium cyclic AMP receptors, 8 dynein. outer row heavy chains, 153 Drugs. cytostatic. carcinoma culture experiments, 248-250 Dynein regulatory complex definition, 170 mutations. I7 1-173 Dyneins. flagellar characteristics, 141-142. 173-174 cross-bridge cycle, 168-169

in flagellar motility in uiuo, 166-167 inner row. 162-166 purification. 162-164 in sitrr localization. 164-166 subunit compostiion, 162- 164 in microtubule motility doublet sliding, 142-145 sliding converted to bending, 145-146 translocation in uitro, 167-168 outer row heavy chains, 150-155 intermediate chains, 148-150 light chains, 147-148 outer arm structure. 155-161 purification, 146-155 structure-function relationships, 161- I62 subunit composition, 146-155 regulation, 170-173 mutations, 171- I73 radial spokes, 170-171 sliding velocity, 170-171 structure, 142-145

E Endothelial cell growth factor, migration induction by, 80 Energy. cytoplasmic streaming requirements. 112-1 14 Epidermal growth factor, migration induction by, 67-70 Esophageal carcinomas. high-density cultures. 193- I94 Evolution, hormonal imprinting studies, 37 Extracellular matrix cell migration coating assay as analytical tool, 60 composition effects, 51-52, 55, 57 cellular interactions, 52 Extracellular matrix-track assay, migration analysis. 58-60

F Fibroblast growth factors, migration induction by, 70-73 acidic fibroblast growth factors, FGF-I, 70-7 I

262

INDEX

basic fibroblast growth factors, FGF-2, 7 1-73 Flagella doublet sliding, 142-145 dyneins, see Dyneins, flagellar sliding converted to bending, 145-146 structure. 142-145 Follicle-stimulating hormone receptor, homology with related receptors, 27

G Gastrointestinal carcinomas high-density cultures, 192-193, 200 histological findings, 203-206 immunohistochemical findings, 232-236 ultrastructure, 2 15, 2 17-22 I Genes, ontogenic open system, 36 Gonadotropic hormone receptors, homology of related receptors, 27 Granulocyte-colony stimulating factor, migration modulation by, 80 Granulocyte macrophage-colony stimulating factor, migration modulation by, 80 Gravity, cytoplasmic streaming affected by, 127-129 Growth factors, in migration, see Migration Gynecological carcinomas high-density cultures, 197-200 histological findings, 211, 213, 216 immunohistochemical findings, 237, 242-245 ultrastructure. 227

H Head and neck carcinomas high-density cultures, 193-195, 200 histological findings, 205, 207-210 immunohistochemical findings, 233, 237-239 ultrastructure, 2 17, 222-225 Hepatocyte growth factor/scattering factor, migration induction by, 63-64, 78-79

Hormone receptors, 1-38 characteristics, 37-38 multicellular organisms, 3 ontogeny conclusions, 35-37 imprinting cell-to-cell transmission, 34-35 in developing adult cells, 33-35 optimal time for, 29-30 pathological, 30-33 perinatal period, 24-26 steroid receptors, 32-33 maturation, 24-26 perinatal period imprinting, 24-26 specificity, 27-29 phylogeny, lowest level, 1-3 unicellular organisms imprinting mechanism, 19-24, 36 microbes, 3-7 protozoa, 3-7 second messengers, 8- I 1 signal molecules in receptor development, 11-18 specificity of hormone and receptor, 7-8 Human carcinoma cultures, see Carcinomas, human, cultures Hydrocarbons, aromatic, imprinting of steroid receptors induced by, 32-33

I Imprinting, hormone receptors cell-to-cell transmission, 34-35 developing adult cells, 33-35 developmental phases as determining factor. 36-37 evolutionary studies, 37 induced by aromatic hydrocarbons, 32-33 learning process induction, 34 maturation affected by, 25 optimal time for, 29-30 pathology, 30-33 during perinatal period, 27-29 steroids, 32-33 unicellular organisms, 19-24, 36

263

INDEX

Ins u I in binding capacity, 11, 13-15, 17, 26 imprinting in Tetrahymena. 9-10 Insulin-like growth factors, migration induction by, 77-78 Interferon, migration modulation, 81 Invasion assay, as migration analysis method. 63-66

K Keratinizing carcinomas, human, cultures, histological findings, 205

L Light, cytoplasmic streaming affected by, 125 Lung carcinomas high-density cultures, 195-196, 198, 200 histological findings, 207, 209, 2 12-213 immunohistochemical findings, 237, 240-24 1 ultrastructure, 218-221, 225-226 Luteinizing hormone receptor, homology with related receptors, 27

M Magnesium, cytoplasmic streaming requirements, 114-1 15 Microbes, hormone receptors, 3-7 Microcarrier assay, as migration analysis method, 63 Microtubules cytoplasmic streaming and, 109-1 10 motive force generation mechanism, 109-1 10 translocation in virro by flagellar dyneins, 167-168 Migration, 49-81 Boyden chamber assays, 53, 55-57 cell culture system analysis, 52-53 cell-extracellular matrix interactions, 52 chemotaxis, 53, 55-57 coating assays, 60 definitions, 50

direct observation, 53-54 extracellular matrix-track assay, 58-60 growth factor-induced acidic fibroblast growth factors, 70-71 autocrine motility factor, 79-80 basic fibroblast growth factors, 71-73 capacity, 52 characterization, 50 endothelial cell growth factor, 80 epidermal growth factor, 67-70 fibroblast growth factors, 70-73 hepatocyte growth factor/scattering factor, 63-64, 78-79 insulin-like growth factors, 77-78 migration-stimulating factor, 79-80 nerve growth factor, 80 platelet-derived growth factor, 75-77 transforming growth factor-a, 68-69 transforming growth factor-p, 74-75 inhibition, 51 initiation, 51 invasion assays, 63-66 microcarrier assays, 63 migration track assays, 58-60 modulating factors granulocyte-colony stimulating factor, 80 granulocyte macrophage-colony stimulating factor, 80 interferon, 81 tumor necrosis factor-a, 80 phagokinetic track assay, 58 phases, 51 reaction specificity, 51 scatter assay, 63-64 spheroid assays, 62 studies, 49-50, 66, 81 time-lapse recording, 53 translocation, 51 "under agarose" assay, 57 in vitro wound assay, 61-62 Migration-stimulating factor, characterization, 79-80 Motility, definition, SO Mutations, flagellar dyneins, 171-173 Myosin, see also Actin-myosin system cytoplasmic streaming mechanism affected by calcium sensitivity, 123-124 identification in plants, 103-108, 130 phosphorylation, 123

264

INDEX

N Neck carcinomas, see Head and neck carcinomas Nerve growth factor discovery, 49 migration induction by, 80 Neurospora, hormone receptors, second messengers, 9 Nitella, cytoplasmic streaming action potential as factor, 121 reconstruction experiments, I16

R Radial spokes, flagellar dynein regulation, 170-171 Reconstruction, in demembered cell models of Characeae, 116-1 17 Rectal carcinomas characteristics, 193 high-density cultures, 193 ultrastructure, 2 I5 Renal-cell carcinomas high-density cultures, 21 1 ultrastructure, 226

0 Ontogenesis, open system during, and hormonal imprinting, 36 Organoid culture assay, human carcinoma experiments, 248-252 Ovarian carcinoma characteristics, 197 high-density culture methods, 198 histological findings, 21 I , 213 organoid culture assay, 251 ultrastructure, 227

P Paramecium, dynein, outer row composition, 147 Perinatal period, hormonal imprinting during, 24-29 pH, cytoplasmic streaming requirements, 115 Phagokinetic track assay, as migration analysis method, 58 Physarum. cytoplasmic streaming affected by action potential, 121 Plants actin identification in, 108 myosin identification in, 103-108, 130 Platelet-derived growth factor, migration induction by, 75-77 Protozoa hormone receptors characteristics, 3-7 imprinting, 2, 21 membrane poll, 12 offspring generations, 17 plasma membrane structure, 22

S Scatter assay, as migration analysis method, 63 Second messengers, and hormone receptors, 8-1 1 Sliding velocity, flagellar dynein regulation, 170-171 Spheroid assay, as migration analysis method, 62 Steroid receptors imprinting by aromatic hydrocarbons, 32-33 pathological, 31 inducibility, 16 Streaming, cytoplasmic, see Cytoplasmic streaming

T Temperature. cytoplasmic streaming affected by, 125-127 Testicular carcinomas characteristics, 196 high-density cultures, 21 1 ultrastructure, 216 Tefruhymena dynein cross-bridge cycle, 168-169 inner row in situ localization, 164 microtubule translocation, 168 outer row composition, 147 identification, 146 structure of outer arms, 157-160

INDEX

hormone receptor imprinting liver cells of higher mammals compared to. 18 mechanism, 19-21 hormone receptors antibody treatment, 1 I downregulation, 12 FITC-insulin binding in offspring generation, 17 of plasma membranes, 13-14 histamine-induced phagocytosis. 3 insulin imprinting. 9- 10 insulin treatment of nuclei. 15 mating receptor role in development, 4 model for evolutionary studies, 5 recognition of hormones, 6-7 second messengers, 9 specificity, 7-8 Thyroid gland carcinomas high-density cultures, 194 ultrastructure, 224-225 Thyroid-stimulating hormone receptor, homology with related receptors, 27 Transforming growth factor-a, migration induction by, 68-69 Transforming growth factor-p. migration induction by, 74-75 Transforming growth factors, discovery, 49

265 Tumor cells. as migration modulators, 49, 55 Tumor necrosis factor-a, migration modulation by, 80 Tumors spheroidal aggregate cultures, 186 stem cell cultures. 184

U Urological carcinomas high-density cultures, 196-20 histological findings, 209, 21 1, 214 immunohisrochemical findings, 237. 242-245 ultrastructure, 226-229

v Vanadate. dynein activity affected by, 150- 151

w Wound assay, in uitro, as migration analysis method. 61-62