- Email: [email protected]
Cytoskeleton structure and function
TIBS - April 1984
192
l0 11 12 13
Veeger, C. (1982) Eur. J. Biochem. 121, 483-491 Braaksma, A., Haaker, H. and Veeger, C. (1983) Eur. J. Biochem. 133, 71-76 Cordewener, J., Haaker, H. and Veeger, C. (1983) Eur. J. Biochem. 132, 47-54 Robson, R., Kennedy, C. and Postgate, J. R. (1983) Can. J. Microbiol. 29, 954-967 Dixon, R. A., Alvarez-Morales, A., Oements, J., Drummond, M., Merrick, M. and Postgate,
J. R. (1984) in Advances in Nitrogen Fixation Research (Veeger, C. and Newton, W., eds), pp. 635~o42, Nijhoff/Junk 14 Bergersen, F. J. (1984) inAdvances in Nitrogen Fixation Research (Veeger, C. and Newton, W. E., eds), pp. 171-180, Niihoff/Junk 15 Haaker, H., Laane, C. and Veeger, C. (1980) in Nitrogen Fixation (Stewart, W. D. P. and Gallon, J. R., eds), pp. 113-138, Academic Press
Cytoskeleton structure and function Walter Birchmeier It is reasonable to assume that in 1976 around 95% of experimental biologists were not aware of the facts that living cells have cytoskeletons and that such networks are somehow involved in cell motility/In 1984, however, 95% seem to know, and many of them now even consider the cytoskeleton to play a fundamental role in quite a wide variety of biological phenomena both in prokaryotes and eukaryotes. Thus, the first I00 issues of TIBS largely covered the period in which 'cytoskeletology' became fashionable and in which also much progress in this field was made. What was known about the cytoskeleton and its involvement in cell motility in 1976? The experts who met that year at a Cold Spring Harbor conference I had realized that a series of characteristic proteins - some of which were already known from the study of muscle tissues - form a complex cytoplasmic network in all cells. Visually striking immunofluorescence and electron micrographs had been published showing two different filament systems, the microfilaments2 and the microtubules3. We still had to wait for another year for the immunofluorescent localization of the third network, the intermediate filaments4 (although they were known from electron microscopic studies). It was also realized that these cytoskeletal elements interact with the plasma membrane, and that they are somehow responsible for intracellular positioning of organelles. Furthermore, an impressive amount of biochemical information on cell motility had already been collected, in particular on filament formation and contraction involving actin, myosin and tubulin (see Ref. 1 for reviews).
formation were characterized in vitro and in vivo (gelation factors, bundling and fragmenting proteins); proteins which might connect cytoskeletal elements to membranes were discovered (e.g. ankyrin, vinculin); calmodulin was established to be involved in many cytoskeletal functions; intermediate filament components were largely unknown in 1976 but now constitute a well characterized family of proteins (see Ref. 5 and below). Through the use of monoclonal antibodies further cytoskeletal proteins have been discovered, and it is expected that this technique will lead to the detection of many new components. Interestingly, a new set of microfilament-regulating proteins has recently been described; these turn out to be modified (e.g. phosphorylated) forms of actin itself9. This is a good example of how economically ceils have modified their genes and thus to fine-tune cytoskeletal action. It might be worth closely inspecting the intermediate filament and the microtubule systems for similar solutions to cytoskeleton regulation.
A flood of new proteins In the mid-seventies a search for new cytoskeleton-associated proteins began which has been quite successful. Table I lists a few examples and gives the network to which they are associated. These new proteins can be grouped according to their function: for example, proteins which promote or disturb actin filament
Cytoskelelal dynamics A variety of observations on live cells have indicated that cytoskeletal structures are not static but in a process of continuous assembly and disassembly. Research directed only toward the isolation of the molecular components and toward their microscopic localization in fixed cells would not take this fully into account. Some of these limitations have been overcome by the recent technique of microinjection into living cells of
Walter Birchmeier is at the Friedrich-MiescherLaboratorium der Max-Planek-Gesellscha]~, Spemannstr. 37-39, D-7400 Tiibingen, FRG.
t~ 1984.ElsevierSciencePublishersB.V., Amsterdam 0376- 5067184?$02.00
16 Rowell, P., Reed, R. H., Hawkesford, M. J., Ernst, A., Diez, J. and Stewart, W. D. P. (1981) in Current Perspectives in Nitrogen Fixation (Gibson, A. H. and Newton, W. E., eds), pp. 186-189, Elsevier/North-Holland 17 Haaker, H., Laane, C., Hellingerweff, K., Houwer, B., Konings, W. N. and Veeger, C. (1982) Eur. J. Biochem. 127,639-645 18 Scherings, G. S. (1983) PhD. Thesis, Pudoc, Wageningen
fluorescently labeled cytoskeletal proteins, of antibodies against them, and of cytoskeleton-specific mRNA. It has turned out that the microinjected proteins (e.g. actin, et-actinin, tubulin) faithfully integrate into the native cellular structures (see Fig. 1 and Ref. 10 for a review). In combination with image intensification (to protect the live cells from too much fluorescent light) and fluorescence quenching measurements the turnover of cytoskeletal elements in various parts of the cells (e.g. ruffles, microfilament bundles) has been monitored. In addition, it could be shown that single stress fiber sarcomeres of tissue culture cells are contractile 1°. After microinjection of antibodies against keratin filaments, this particular network collapses ll. Similarly, microinjected Factin fragmenting protein leads to the decay of microfilament bundles 12. Microinjected keratin-specific mRNA produced a new cytoskeletal network in otherwise non-permissive cells (keratin filaments in fibroblasts, see Ref. 13). It is expected that the microinjection technique in combination with innovative physicochemical measurements (fluorescence quenching, microspectrofluorimetry), and by using the full complement of cytoskeletal proteins, related antibodies, genes and RNA, will soon further our understanding of cytoskeletal dynamics. Lights in the intermediate filament jungle During the first eight years of TIBS the number of components which make up intermediate-sized filaments steadily increased, but this now seems to be coming to an end. There are five major classes of intermediate filament proteins presently recognized (see Ref. 14 and Table I): vimentin filaments in mesenchymal tissue (one protein of mol. wt 55 000), glial filaments in glial ceils (one protein of 53 000), desmin filaments in muscle (one protein of 52 000), neurofilaments in neurons (three proteins of 70 000, 150 000 and 200 000), and keratin filaments (some 20 proteins of 40-70 000). These filaments share a common ultrastructure, with the differ-
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T I B S - April 1984
C Fig. 1. Microinjected fluorescent actin is integrated into microfilament bundles of living human fibroblasts. Actin was rhodamine-labeled and microinjected into a single fibroblast through a microcapillary. The same cell was also examined by interference reflection microscopy which pictures the cell's focal contacts. (a) Micrograph showing fluorescent acting incorporated into bundles, (c) corresponding interference reflection micrograph indicating in dark the triangular focal contacts, (b) the superimposed pattern (red fluorescence becomes yellow on the green background) illustrating stress fiber~ overlapping with focal contacts' (see arrows, taken from Re]~ 26).
ent components being able to form mixed (tumors derived from muscle) are des- non-erythroid cells (fodrin, TW 260/240), filaments. Sequence analysis (on the min-positive, gliomas stain for glial fila- and furthermore, erythrocyte ankyrin level of the proteins and now also on the ments, and neuroblastomas are often was found to be related to a microtubulegenes, see Ref. 15) has revealed exten- positive for neurofilaments. Even in associated protein, MAP-1 (Refs 18, intermediate filaments 19). These findings have exposed a sive homologies, whereby the proteins metastases of one tissue type in different species are characteristic of the tissue of origin have series of questions which still await an more similar than those of different been observed (Ref. 14). answer. For instance, is this new nonfilament classes in one species H. erythroid cytoskeletal system truly Despite the facts that intermediate Cytoskeleton and membranes membrane-bound and if so, how is it It has been realized for quite some anchored to which integral membrane filaments form such impressive networks in cells and that their biochemical and time that cytoskeletal elements are protein; what will be the role of calstructural analysis has progressed so far, responsible for supporting the cell's modulin and of phosphorylation in the their function is entirely unknown. This plasma membranes and that they must fodrin-TW 260/240 system; and whether is reflected by the finding that even also interact with membranes of internal microtubules might also interplay with the though intermediate filament organiza- organelles. For instance, if red blood actin-related membrane meshwork. tion is disrupted after microinjection of cell membranes are treated with nonMembrane-attached cytoskeletal elespecific antibodies, the cells' gross shape ionic detergents their gross size and ments have gained even more attention and motility are not measurably altered shape is maintained (Triton shells). since they seem to be involved in the nor is cell division disturbed n~'~. Never- However, if the membrane-associated phenomenon of 'transmembrane sigtheless, there are a few clues as to where cytoskeleton (the spectfin-actin network) naling' through the plasma membrane z°. one should look for a possible function. is released at low ionic strength, the Bidirectional transmembrane flow of For instance, the network breaks down cells break down into small vesicles. information must occur continuously in many cell types during mitosis 16. During the time span of the first 100 during cell spreading and during coordiFurthermore, the expression of inter- issues of T I B S an intense effort has nated cell locomotion. It also seems to be mediate filament proteins faithfully been applied to the overall structure of essential for even more complex propermarks the different cell types in tissues this model membrane, and its cytoskel- ties of cells such as contact inhibition of during normal and malignant develop- eton has now been analysed to the growth and the integration of single cells ment ~4. Thus, intermediate filaments satisfaction of most researchers ~7. into whole tissues. A model organelle might be involved, perhaps in a way not According to this scheme the spectrin- where transmembrane signaling should yet examined in vitro, in a tissue- and actin complex is connected through the be manifested is the fibroblast focal concell-cycle-specific interaction between protein ankyrin to a major integral tact (see Ref. 21 for a review). The protein vinculin27 (an actin-binding prothe cells' environment and its genetic membrane component (Band 3). For a long time no similar components tein enriched at these sites), which is apparatus. Intermediate filaments have already in other cells could be detected by phosphorylated by the Rous sarcoma probeen quite valuable, however, for human immunological methods, although the tein kinase (pp60Src), was suggested to be tumor diagnosis. By immunofluorescence general feeling existed that nature should a mediator of such signaling. However, of tumor sections it can be seen that have developed membrane-associated there exist other likely candidates 22. carcinomas express keratins but not skeletons for other cells as well. This has vimentin, and that the converse is true now changed. Many laboratories have Cytoskeleton and organelles The plasma membrane of living cells in most sarcomas. Rhabdomyosarcomas discovered spectrin-like molecules in For technical reasons we are unable to reproduce this figure in colourin this edition- see the April issue of Trends in Biochemical Sciences for full colour illustration.
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T I B S - A p r i l 1984
encloses a variety of organdies, some of which undergo directed locomotion as in saltatory movement. One topic of current research is the degree to which the cytoskeletal network participates in the positioning and movement of the different organeUes. For instance, lipid granules of HeLa cells or pigment granules of fish chromatophores exhibit radial motion, which is inhibited by colchicine and vinblastine (both known to dissociate microtubules). Vesicles containing neurotransmitters are axonally transported, and both colchichine and microinjected D N a s e 1 (which destroys microfilaments) interfere with this m o v e m e n t . F u r t h e r m o r e , microscopic investigations
suggest a direct association between organelles and cytoskeletal structures; e.g. mitochondria with microtubules, cell nuclei with microtubules and intermediate filaments, synaptic vesicles with microtubules, and coated vesicles with microfilaments and microtubules (reviewed in Ref. 8). Recent experiments have focused on the association b e t w e e n coated vesicles (which are involved in receptor-mediated endocytosis and possibly also in exocytosis) and microtubules. It has become apparent that clathrin and also intact coated vesicles copurify with microtubules during isolation 8 and, vice versa, that tubulins are true c o m p o n e n t s of the
Table L Some new cytoskeletal proteins
Protein
Properties
Actin-associated
Actinogelin(1978) Acumentin (1982) Ankyrin (1978) Brevin (1978) Caldesmon (1981) Calmodulin (1973) Filamin (1975) Fibrin (1980) Fodrin (1981)
Fragmin(1980) Gelsolin (1979)
Profilin (1977) Spectrin (1971) Vfllin (1979) Vinculin(1979)
Ca2+ sensitivegelation factor Caps the pointed ends of microfilaments6 Connects the erythrocyte cytoskeletonwith the membrane, related to MAP-I Shortens actin filaments, present in serum, cf. gelsolin Binds to actin in the presence of calmodulinand Ca-,+ (Ref. 28) Ca2+-dependent activator of myosinkinase, associatedwith cytoskeletonof microvilli,decorates microfilamentsin interphase and binds to the spindle in mitosis CrosslinksF-actin filaments, cf. also actin-bindingprotein and HMW protein Actin bundling protein, present in the core of microvilliand in other membrane extensions Non-erythroid analogue of spectrin, forms a membrane-associated cytoskeletonin various cells, cf. TW 260/240 From Physarum, fragments F-actin filaments In macrophages, platelets and serum, shortens F-actin filaments. Gelsolin, villin, brevin, and fragminare Ca2+-dependentactin severingproteins, react with the barbed ends of microfilaments (cf. with acumentin) G-actin stabilizingprotein, acts similarto DNAse 1 Main cytoskeletalprotein of erythrocyte membranes, related to fodrin and TW 260/240 Shortens actin filaments in the presence of Ca2+, bundles actin filaments in the absence of Ca> Present in focal contacts of fibroblasts,bundles actin filaments
Intermediate-filament-associated
Cytokeratins (1972)
Main structural components of intermediate (tono) filamentsof epithelial cells Desmin (1976) Desmin filaments are found in muscle cells Filaggrin (1977) Suggestedto aggregate all intermediate filamenttypes into macrofibdls5 Glial filament protein (1971) Forms intermediate filamentsin glialcells Neurofilamentproteins (1975) Complex of proteins (mol. wt 70 003, 150 000 and 200 000) whichforms neurofilaments Plecfin (1980) High mol. wt complexthat copurifieswith vimentin filaments Synemin (1980) Associatedwith desmin filaments Myofibrilscaffoldprotein7 Titin (1979) Vimentin (1978) Forms the intermediate filamentsof mesenchymalcells (also termed decamin) Tubulin-associated
Clathrin (1976) Dynein (1973) MAPs (1974) Tan factors (1975)
Main component of coated vesicles,these can reversiblyassociatewith microtubules8 High mol. wt proteins with ATPase activitywhich form the arms of microtubules,involvedin microtubulessliding High tool. wt proteins associatedwith microtubules,MAP-1 is related to ankyrin, MAP-2 is phosphorylatedand possiblybinds to organelles Lower tool. wt tubulin-bindingproteins
The years in parentheses indicate the approximate first descriptionof the protein in the literature (see subject index in Refs I and 5). Actin, myosin, a-actinin, tubulin, troponin, and tropomyosin(whichhad been discovered earlier) are not included in this list.
clathrin lattice of coated vesicles23'24. Progress has also been made in the construction o f models of cytoskeletonorganelle complexes. Myosin ( H M M ) coated beads were placed on to actin filaments present at the inner surface of the giant algae Nitella. D e p e n d e n t on the presence of A T P , the artificial beads show large scale m o v e m e n t in one direction 29. Tips for the future Within the limited space provided I could not describe in detail (but see Ref. 15) the ever expanding field of molecular analysis of cytoskeletal genes. Let m e mention one case, however, which I find fascinating at the m o m e n t . Certain t u m o r viruses have acquired host cell genes which seem to be important for normal growth regulation, but which they have modified to become potent oncogenes. It is striking that one of them, the G a r d n e r - R a s h e e d feline sarcoma virus, has captured part of the yactin gene in tandem with an oncogene 25. Cytoskeletal structures are modified during malignant transformation of cells (another area of cytoskeletal research not described herein, but see Refs 1, 5, 21); it is therefore possible that during transformation such an oncogene--actin fusion protein directly interferes with proper cytoskeletal architecture, or that the new actin-containing oncogene (a tyrosine kinase) might be targeted to particular cytoskeletal structures.
Acknowledgements I w o u l d like to t h a n k Drs T. D o e t s c h m a n and S. G o o d m a n (Tiibingen) for critically reading the manuscript, and R. B r o d b e c k for secretarial assistance. The microinjection experim e n t shown in Fig. 1 was performed by D r T. Kreis (Heidelberg, see Ref. 26).
References 1 Goldman, R., Pollard, T. and Rosenbaum, J., eds (1976) Cell Motility, pp. 1-1376, Cold Spring Harbor Laboratory Press 2 Lazarides, E. and Weber, K. (1974) Proc. Natl Acad. Sci. USA 71, 2268-2273 3 Fuller, G. M., Brinkley, B. R. and Boughter, J. M. (1975) Science 187, 948-951 40sborn, M., Franke, W. W. and Weber, K. (1977) Proc. Natl Acad. Sci. USA 74, 2490-2494 5 Organization of the Cytoplasm (1976) Cold Spring Harbor Symposia on Quantitative Biology Vol. 46, pp. 1-1043, Cold Spring
Harbor Laboratory Press 6 Southwick, F. S. and Hartwig, J. (1982) Nature 297, 303-307 7 Wang, K., McCluire, J. and Tu, A. (1979) Proc. Natl Acad. Sci. USA 76, 3698-3702 8 Imhof, B. A., Marti, U., Boller, K., Frank, H. and Birchmeier, W. (1983) Exp. Cell Res. 145, 199-207
195
TIBS - April 1984 9 Maruta, H. and lsenberg, G. (1983) J. Biol. Chem. 258, 10151-10158 10 Kreis, T. E. and Birchmeier, W. (1982) in International Review of Cytology 75, 209227 11 Klymkowsky, M. W. (1981) Nature 291, 249-251 12 Fiichtbauer, A., Jockusch, B. M., Maruta, H., Kilimann. M. W. and Isenberg, G. (1983) Nature 304, 361-364 13 Kreis, Y. E., Geiger, B., Schmid, E., Jorcano, J. L. and Franke. W. W. (1983) Cell 32,
1125-1137 14 Osborn, M. and Weber, K. 11982) Cell 31, 303-306 15 Quax, W., Egberts, W. V., Hendfiks, W., Quax-Jeuken, Y. and Bloemendahl, H. (1983)
Cell 35,215-223 16 Lane, E. B., Goodman, S. L. and Trejdosiewiez, L. K. (1982) EMBO J. 11, 1365-1372 17 Branton, D., Cohen, C. M. and Tyler, J. (1981) Cell 24, 24-32 18 Repasky, E. A., Granger, B. L. and Lazarides, E. (1982) Cell 29, 821-833 19 Bennett, V. and Davis, J. (1981) Proc. Natl Acad. Sci. USA 78, 7550-7554 20 Singer, S. J., Ash, J. F., Bourguignon, L. Y. W., Heggenness, M. H. and Louvard, D. (1978) J. Supramol. Struct. 9, 373-389 21 Birchmeier, W. (1981) Trends Biochem. Sci. 6, 234-237 22 Oesch, B. and Birchmeier, W. 11982) Cell 31, 671~79
Mechanisms of mitosis J. Richard Mclntosh Research on the mechanisms o f mitosis can be divided into two categories: descriptions o f the mitotic" process and characterizations o f components in the mitotic machinery. In the first category, the major efforts over the last eight years have focused both on the time-dependent changes in the arrangements o f spindle microtubules and on the character o f mitotic forces. In the second category, there has been notable progress in developing extracts o f mitotic cells to define the factors which regulate spindle .formation and action, and in identifying new spindle proteins.
23 Pfeffer, S. R., Drubin, D. G. and Kelly, R. B. (1983) J. Cell BioL 97, 40-47 24 Kelly, W. G., Passaniti, A., Woods, J. W., Daiss, J. L. and Roth, T. F. 11983) J. Cell Biol. 97, 1191-1199 25 Naharro, G., Robbins, K. C. and Reddy, E. P. (1983) Science 223.6.3~6 26 Birchmeier, C., Kreis, T. E., Eppenberger, H. M., Winterhalter, K. H. and Birchmeier, W. (1980) Proc. Natl Acad. Sci. USA 77. 41084112 27 Geiger, B. (1979) Cell 18, 193-205 28 Sobue, K., Muramoto, Y., Fujita, M. and Kakiuchi, S. (1981) Proc. Natl Acad. Sci. USA 78, 5652-5655 29 Sheetz, M. P. and Spudich, J. A. 11983) Nature 303, 31 37
kinetochores are long enough to interdigitate with MTs from the opposite pole (Fig. la). During anaphase the short MTs, including those attached to the c h r o m o s o m e s , get shorter while the long, interdigitating MTs lengthen as the poles move apart (Fig. lb) 1'2. In some cells, the elongating MTs change their extent of interdigitation, giving the appearance of M T sliding (Fig. lc) a. In other cells the extent of interdigitation of the two M T families remains constant as they elongate ~.
Descriptions of the mitotic process Microtubule arrangements and rearrangements during mitosis T h e classic image of the metaphase spindle was a structure shaped like an A m e r i c a n or R u g b y football with c h r o m o s o m e s aligned at the equator. It was said to be built from two classes of fibers: those connecting the chromosomes to the poles and those running from pole to pole. Early electron microscopy showed that spindle fibers were bundles of microtubules (MTs), but in the early 1970s the end-points and the orientations of individual spindle MTs were largely unknown. During recent years, the spindle MTs o f several organisms have been tracked through serial sections to yield 3-dimensional structures at each of several times during mitosis (e.g. Refs 1,2). M e t h o d s have also been developed to determine the polar orientation of the spindle MTs. Most of the MTs in each half of the spindle, including those which attach to the chromos o m e s at their k i n e t o c h o r e s (centromeres), are oriented with their 'plus' (fast-growing) ends distal to the near-by pole 3. M T length is heterodisperse, and some of the MTs which do not end at J. Richard Mclntosh is at the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
LENGTHENING OFLONGMTs
:
~
SHORTENING l OFSHORTMTs ¢
WHATARETHESITESOF SUBUNIT ADDITIONANDLOSS?
Anaphase
~
SLIDING OFMTs?
(c) Telophase
CHROMATINCONTRACTION PRIORTOREFORFBTION WHEREARETHEFORCES OFNUCLEARENVELOPE FORSLIDINGGENERATED? Fig. l. Rearrangement ofspind~MTsdunng anaphase.
~) 1984.ElsevierSciencePublishersBV..Amsterdam 0376 5(~7/84/$(r2.011
192
l0 11 12 13
Veeger, C. (1982) Eur. J. Biochem. 121, 483-491 Braaksma, A., Haaker, H. and Veeger, C. (1983) Eur. J. Biochem. 133, 71-76 Cordewener, J., Haaker, H. and Veeger, C. (1983) Eur. J. Biochem. 132, 47-54 Robson, R., Kennedy, C. and Postgate, J. R. (1983) Can. J. Microbiol. 29, 954-967 Dixon, R. A., Alvarez-Morales, A., Oements, J., Drummond, M., Merrick, M. and Postgate,
J. R. (1984) in Advances in Nitrogen Fixation Research (Veeger, C. and Newton, W., eds), pp. 635~o42, Nijhoff/Junk 14 Bergersen, F. J. (1984) inAdvances in Nitrogen Fixation Research (Veeger, C. and Newton, W. E., eds), pp. 171-180, Niihoff/Junk 15 Haaker, H., Laane, C. and Veeger, C. (1980) in Nitrogen Fixation (Stewart, W. D. P. and Gallon, J. R., eds), pp. 113-138, Academic Press
Cytoskeleton structure and function Walter Birchmeier It is reasonable to assume that in 1976 around 95% of experimental biologists were not aware of the facts that living cells have cytoskeletons and that such networks are somehow involved in cell motility/In 1984, however, 95% seem to know, and many of them now even consider the cytoskeleton to play a fundamental role in quite a wide variety of biological phenomena both in prokaryotes and eukaryotes. Thus, the first I00 issues of TIBS largely covered the period in which 'cytoskeletology' became fashionable and in which also much progress in this field was made. What was known about the cytoskeleton and its involvement in cell motility in 1976? The experts who met that year at a Cold Spring Harbor conference I had realized that a series of characteristic proteins - some of which were already known from the study of muscle tissues - form a complex cytoplasmic network in all cells. Visually striking immunofluorescence and electron micrographs had been published showing two different filament systems, the microfilaments2 and the microtubules3. We still had to wait for another year for the immunofluorescent localization of the third network, the intermediate filaments4 (although they were known from electron microscopic studies). It was also realized that these cytoskeletal elements interact with the plasma membrane, and that they are somehow responsible for intracellular positioning of organelles. Furthermore, an impressive amount of biochemical information on cell motility had already been collected, in particular on filament formation and contraction involving actin, myosin and tubulin (see Ref. 1 for reviews).
formation were characterized in vitro and in vivo (gelation factors, bundling and fragmenting proteins); proteins which might connect cytoskeletal elements to membranes were discovered (e.g. ankyrin, vinculin); calmodulin was established to be involved in many cytoskeletal functions; intermediate filament components were largely unknown in 1976 but now constitute a well characterized family of proteins (see Ref. 5 and below). Through the use of monoclonal antibodies further cytoskeletal proteins have been discovered, and it is expected that this technique will lead to the detection of many new components. Interestingly, a new set of microfilament-regulating proteins has recently been described; these turn out to be modified (e.g. phosphorylated) forms of actin itself9. This is a good example of how economically ceils have modified their genes and thus to fine-tune cytoskeletal action. It might be worth closely inspecting the intermediate filament and the microtubule systems for similar solutions to cytoskeleton regulation.
A flood of new proteins In the mid-seventies a search for new cytoskeleton-associated proteins began which has been quite successful. Table I lists a few examples and gives the network to which they are associated. These new proteins can be grouped according to their function: for example, proteins which promote or disturb actin filament
Cytoskelelal dynamics A variety of observations on live cells have indicated that cytoskeletal structures are not static but in a process of continuous assembly and disassembly. Research directed only toward the isolation of the molecular components and toward their microscopic localization in fixed cells would not take this fully into account. Some of these limitations have been overcome by the recent technique of microinjection into living cells of
Walter Birchmeier is at the Friedrich-MiescherLaboratorium der Max-Planek-Gesellscha]~, Spemannstr. 37-39, D-7400 Tiibingen, FRG.
t~ 1984.ElsevierSciencePublishersB.V., Amsterdam 0376- 5067184?$02.00
16 Rowell, P., Reed, R. H., Hawkesford, M. J., Ernst, A., Diez, J. and Stewart, W. D. P. (1981) in Current Perspectives in Nitrogen Fixation (Gibson, A. H. and Newton, W. E., eds), pp. 186-189, Elsevier/North-Holland 17 Haaker, H., Laane, C., Hellingerweff, K., Houwer, B., Konings, W. N. and Veeger, C. (1982) Eur. J. Biochem. 127,639-645 18 Scherings, G. S. (1983) PhD. Thesis, Pudoc, Wageningen
fluorescently labeled cytoskeletal proteins, of antibodies against them, and of cytoskeleton-specific mRNA. It has turned out that the microinjected proteins (e.g. actin, et-actinin, tubulin) faithfully integrate into the native cellular structures (see Fig. 1 and Ref. 10 for a review). In combination with image intensification (to protect the live cells from too much fluorescent light) and fluorescence quenching measurements the turnover of cytoskeletal elements in various parts of the cells (e.g. ruffles, microfilament bundles) has been monitored. In addition, it could be shown that single stress fiber sarcomeres of tissue culture cells are contractile 1°. After microinjection of antibodies against keratin filaments, this particular network collapses ll. Similarly, microinjected Factin fragmenting protein leads to the decay of microfilament bundles 12. Microinjected keratin-specific mRNA produced a new cytoskeletal network in otherwise non-permissive cells (keratin filaments in fibroblasts, see Ref. 13). It is expected that the microinjection technique in combination with innovative physicochemical measurements (fluorescence quenching, microspectrofluorimetry), and by using the full complement of cytoskeletal proteins, related antibodies, genes and RNA, will soon further our understanding of cytoskeletal dynamics. Lights in the intermediate filament jungle During the first eight years of TIBS the number of components which make up intermediate-sized filaments steadily increased, but this now seems to be coming to an end. There are five major classes of intermediate filament proteins presently recognized (see Ref. 14 and Table I): vimentin filaments in mesenchymal tissue (one protein of mol. wt 55 000), glial filaments in glial ceils (one protein of 53 000), desmin filaments in muscle (one protein of 52 000), neurofilaments in neurons (three proteins of 70 000, 150 000 and 200 000), and keratin filaments (some 20 proteins of 40-70 000). These filaments share a common ultrastructure, with the differ-
193
T I B S - April 1984
C Fig. 1. Microinjected fluorescent actin is integrated into microfilament bundles of living human fibroblasts. Actin was rhodamine-labeled and microinjected into a single fibroblast through a microcapillary. The same cell was also examined by interference reflection microscopy which pictures the cell's focal contacts. (a) Micrograph showing fluorescent acting incorporated into bundles, (c) corresponding interference reflection micrograph indicating in dark the triangular focal contacts, (b) the superimposed pattern (red fluorescence becomes yellow on the green background) illustrating stress fiber~ overlapping with focal contacts' (see arrows, taken from Re]~ 26).
ent components being able to form mixed (tumors derived from muscle) are des- non-erythroid cells (fodrin, TW 260/240), filaments. Sequence analysis (on the min-positive, gliomas stain for glial fila- and furthermore, erythrocyte ankyrin level of the proteins and now also on the ments, and neuroblastomas are often was found to be related to a microtubulegenes, see Ref. 15) has revealed exten- positive for neurofilaments. Even in associated protein, MAP-1 (Refs 18, intermediate filaments 19). These findings have exposed a sive homologies, whereby the proteins metastases of one tissue type in different species are characteristic of the tissue of origin have series of questions which still await an more similar than those of different been observed (Ref. 14). answer. For instance, is this new nonfilament classes in one species H. erythroid cytoskeletal system truly Despite the facts that intermediate Cytoskeleton and membranes membrane-bound and if so, how is it It has been realized for quite some anchored to which integral membrane filaments form such impressive networks in cells and that their biochemical and time that cytoskeletal elements are protein; what will be the role of calstructural analysis has progressed so far, responsible for supporting the cell's modulin and of phosphorylation in the their function is entirely unknown. This plasma membranes and that they must fodrin-TW 260/240 system; and whether is reflected by the finding that even also interact with membranes of internal microtubules might also interplay with the though intermediate filament organiza- organelles. For instance, if red blood actin-related membrane meshwork. tion is disrupted after microinjection of cell membranes are treated with nonMembrane-attached cytoskeletal elespecific antibodies, the cells' gross shape ionic detergents their gross size and ments have gained even more attention and motility are not measurably altered shape is maintained (Triton shells). since they seem to be involved in the nor is cell division disturbed n~'~. Never- However, if the membrane-associated phenomenon of 'transmembrane sigtheless, there are a few clues as to where cytoskeleton (the spectfin-actin network) naling' through the plasma membrane z°. one should look for a possible function. is released at low ionic strength, the Bidirectional transmembrane flow of For instance, the network breaks down cells break down into small vesicles. information must occur continuously in many cell types during mitosis 16. During the time span of the first 100 during cell spreading and during coordiFurthermore, the expression of inter- issues of T I B S an intense effort has nated cell locomotion. It also seems to be mediate filament proteins faithfully been applied to the overall structure of essential for even more complex propermarks the different cell types in tissues this model membrane, and its cytoskel- ties of cells such as contact inhibition of during normal and malignant develop- eton has now been analysed to the growth and the integration of single cells ment ~4. Thus, intermediate filaments satisfaction of most researchers ~7. into whole tissues. A model organelle might be involved, perhaps in a way not According to this scheme the spectrin- where transmembrane signaling should yet examined in vitro, in a tissue- and actin complex is connected through the be manifested is the fibroblast focal concell-cycle-specific interaction between protein ankyrin to a major integral tact (see Ref. 21 for a review). The protein vinculin27 (an actin-binding prothe cells' environment and its genetic membrane component (Band 3). For a long time no similar components tein enriched at these sites), which is apparatus. Intermediate filaments have already in other cells could be detected by phosphorylated by the Rous sarcoma probeen quite valuable, however, for human immunological methods, although the tein kinase (pp60Src), was suggested to be tumor diagnosis. By immunofluorescence general feeling existed that nature should a mediator of such signaling. However, of tumor sections it can be seen that have developed membrane-associated there exist other likely candidates 22. carcinomas express keratins but not skeletons for other cells as well. This has vimentin, and that the converse is true now changed. Many laboratories have Cytoskeleton and organelles The plasma membrane of living cells in most sarcomas. Rhabdomyosarcomas discovered spectrin-like molecules in For technical reasons we are unable to reproduce this figure in colourin this edition- see the April issue of Trends in Biochemical Sciences for full colour illustration.
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T I B S - A p r i l 1984
encloses a variety of organdies, some of which undergo directed locomotion as in saltatory movement. One topic of current research is the degree to which the cytoskeletal network participates in the positioning and movement of the different organeUes. For instance, lipid granules of HeLa cells or pigment granules of fish chromatophores exhibit radial motion, which is inhibited by colchicine and vinblastine (both known to dissociate microtubules). Vesicles containing neurotransmitters are axonally transported, and both colchichine and microinjected D N a s e 1 (which destroys microfilaments) interfere with this m o v e m e n t . F u r t h e r m o r e , microscopic investigations
suggest a direct association between organelles and cytoskeletal structures; e.g. mitochondria with microtubules, cell nuclei with microtubules and intermediate filaments, synaptic vesicles with microtubules, and coated vesicles with microfilaments and microtubules (reviewed in Ref. 8). Recent experiments have focused on the association b e t w e e n coated vesicles (which are involved in receptor-mediated endocytosis and possibly also in exocytosis) and microtubules. It has become apparent that clathrin and also intact coated vesicles copurify with microtubules during isolation 8 and, vice versa, that tubulins are true c o m p o n e n t s of the
Table L Some new cytoskeletal proteins
Protein
Properties
Actin-associated
Actinogelin(1978) Acumentin (1982) Ankyrin (1978) Brevin (1978) Caldesmon (1981) Calmodulin (1973) Filamin (1975) Fibrin (1980) Fodrin (1981)
Fragmin(1980) Gelsolin (1979)
Profilin (1977) Spectrin (1971) Vfllin (1979) Vinculin(1979)
Ca2+ sensitivegelation factor Caps the pointed ends of microfilaments6 Connects the erythrocyte cytoskeletonwith the membrane, related to MAP-I Shortens actin filaments, present in serum, cf. gelsolin Binds to actin in the presence of calmodulinand Ca-,+ (Ref. 28) Ca2+-dependent activator of myosinkinase, associatedwith cytoskeletonof microvilli,decorates microfilamentsin interphase and binds to the spindle in mitosis CrosslinksF-actin filaments, cf. also actin-bindingprotein and HMW protein Actin bundling protein, present in the core of microvilliand in other membrane extensions Non-erythroid analogue of spectrin, forms a membrane-associated cytoskeletonin various cells, cf. TW 260/240 From Physarum, fragments F-actin filaments In macrophages, platelets and serum, shortens F-actin filaments. Gelsolin, villin, brevin, and fragminare Ca2+-dependentactin severingproteins, react with the barbed ends of microfilaments (cf. with acumentin) G-actin stabilizingprotein, acts similarto DNAse 1 Main cytoskeletalprotein of erythrocyte membranes, related to fodrin and TW 260/240 Shortens actin filaments in the presence of Ca2+, bundles actin filaments in the absence of Ca> Present in focal contacts of fibroblasts,bundles actin filaments
Intermediate-filament-associated
Cytokeratins (1972)
Main structural components of intermediate (tono) filamentsof epithelial cells Desmin (1976) Desmin filaments are found in muscle cells Filaggrin (1977) Suggestedto aggregate all intermediate filamenttypes into macrofibdls5 Glial filament protein (1971) Forms intermediate filamentsin glialcells Neurofilamentproteins (1975) Complex of proteins (mol. wt 70 003, 150 000 and 200 000) whichforms neurofilaments Plecfin (1980) High mol. wt complexthat copurifieswith vimentin filaments Synemin (1980) Associatedwith desmin filaments Myofibrilscaffoldprotein7 Titin (1979) Vimentin (1978) Forms the intermediate filamentsof mesenchymalcells (also termed decamin) Tubulin-associated
Clathrin (1976) Dynein (1973) MAPs (1974) Tan factors (1975)
Main component of coated vesicles,these can reversiblyassociatewith microtubules8 High mol. wt proteins with ATPase activitywhich form the arms of microtubules,involvedin microtubulessliding High tool. wt proteins associatedwith microtubules,MAP-1 is related to ankyrin, MAP-2 is phosphorylatedand possiblybinds to organelles Lower tool. wt tubulin-bindingproteins
The years in parentheses indicate the approximate first descriptionof the protein in the literature (see subject index in Refs I and 5). Actin, myosin, a-actinin, tubulin, troponin, and tropomyosin(whichhad been discovered earlier) are not included in this list.
clathrin lattice of coated vesicles23'24. Progress has also been made in the construction o f models of cytoskeletonorganelle complexes. Myosin ( H M M ) coated beads were placed on to actin filaments present at the inner surface of the giant algae Nitella. D e p e n d e n t on the presence of A T P , the artificial beads show large scale m o v e m e n t in one direction 29. Tips for the future Within the limited space provided I could not describe in detail (but see Ref. 15) the ever expanding field of molecular analysis of cytoskeletal genes. Let m e mention one case, however, which I find fascinating at the m o m e n t . Certain t u m o r viruses have acquired host cell genes which seem to be important for normal growth regulation, but which they have modified to become potent oncogenes. It is striking that one of them, the G a r d n e r - R a s h e e d feline sarcoma virus, has captured part of the yactin gene in tandem with an oncogene 25. Cytoskeletal structures are modified during malignant transformation of cells (another area of cytoskeletal research not described herein, but see Refs 1, 5, 21); it is therefore possible that during transformation such an oncogene--actin fusion protein directly interferes with proper cytoskeletal architecture, or that the new actin-containing oncogene (a tyrosine kinase) might be targeted to particular cytoskeletal structures.
Acknowledgements I w o u l d like to t h a n k Drs T. D o e t s c h m a n and S. G o o d m a n (Tiibingen) for critically reading the manuscript, and R. B r o d b e c k for secretarial assistance. The microinjection experim e n t shown in Fig. 1 was performed by D r T. Kreis (Heidelberg, see Ref. 26).
References 1 Goldman, R., Pollard, T. and Rosenbaum, J., eds (1976) Cell Motility, pp. 1-1376, Cold Spring Harbor Laboratory Press 2 Lazarides, E. and Weber, K. (1974) Proc. Natl Acad. Sci. USA 71, 2268-2273 3 Fuller, G. M., Brinkley, B. R. and Boughter, J. M. (1975) Science 187, 948-951 40sborn, M., Franke, W. W. and Weber, K. (1977) Proc. Natl Acad. Sci. USA 74, 2490-2494 5 Organization of the Cytoplasm (1976) Cold Spring Harbor Symposia on Quantitative Biology Vol. 46, pp. 1-1043, Cold Spring
Harbor Laboratory Press 6 Southwick, F. S. and Hartwig, J. (1982) Nature 297, 303-307 7 Wang, K., McCluire, J. and Tu, A. (1979) Proc. Natl Acad. Sci. USA 76, 3698-3702 8 Imhof, B. A., Marti, U., Boller, K., Frank, H. and Birchmeier, W. (1983) Exp. Cell Res. 145, 199-207
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TIBS - April 1984 9 Maruta, H. and lsenberg, G. (1983) J. Biol. Chem. 258, 10151-10158 10 Kreis, T. E. and Birchmeier, W. (1982) in International Review of Cytology 75, 209227 11 Klymkowsky, M. W. (1981) Nature 291, 249-251 12 Fiichtbauer, A., Jockusch, B. M., Maruta, H., Kilimann. M. W. and Isenberg, G. (1983) Nature 304, 361-364 13 Kreis, Y. E., Geiger, B., Schmid, E., Jorcano, J. L. and Franke. W. W. (1983) Cell 32,
1125-1137 14 Osborn, M. and Weber, K. 11982) Cell 31, 303-306 15 Quax, W., Egberts, W. V., Hendfiks, W., Quax-Jeuken, Y. and Bloemendahl, H. (1983)
Cell 35,215-223 16 Lane, E. B., Goodman, S. L. and Trejdosiewiez, L. K. (1982) EMBO J. 11, 1365-1372 17 Branton, D., Cohen, C. M. and Tyler, J. (1981) Cell 24, 24-32 18 Repasky, E. A., Granger, B. L. and Lazarides, E. (1982) Cell 29, 821-833 19 Bennett, V. and Davis, J. (1981) Proc. Natl Acad. Sci. USA 78, 7550-7554 20 Singer, S. J., Ash, J. F., Bourguignon, L. Y. W., Heggenness, M. H. and Louvard, D. (1978) J. Supramol. Struct. 9, 373-389 21 Birchmeier, W. (1981) Trends Biochem. Sci. 6, 234-237 22 Oesch, B. and Birchmeier, W. 11982) Cell 31, 671~79
Mechanisms of mitosis J. Richard Mclntosh Research on the mechanisms o f mitosis can be divided into two categories: descriptions o f the mitotic" process and characterizations o f components in the mitotic machinery. In the first category, the major efforts over the last eight years have focused both on the time-dependent changes in the arrangements o f spindle microtubules and on the character o f mitotic forces. In the second category, there has been notable progress in developing extracts o f mitotic cells to define the factors which regulate spindle .formation and action, and in identifying new spindle proteins.
23 Pfeffer, S. R., Drubin, D. G. and Kelly, R. B. (1983) J. Cell BioL 97, 40-47 24 Kelly, W. G., Passaniti, A., Woods, J. W., Daiss, J. L. and Roth, T. F. 11983) J. Cell Biol. 97, 1191-1199 25 Naharro, G., Robbins, K. C. and Reddy, E. P. (1983) Science 223.6.3~6 26 Birchmeier, C., Kreis, T. E., Eppenberger, H. M., Winterhalter, K. H. and Birchmeier, W. (1980) Proc. Natl Acad. Sci. USA 77. 41084112 27 Geiger, B. (1979) Cell 18, 193-205 28 Sobue, K., Muramoto, Y., Fujita, M. and Kakiuchi, S. (1981) Proc. Natl Acad. Sci. USA 78, 5652-5655 29 Sheetz, M. P. and Spudich, J. A. 11983) Nature 303, 31 37
kinetochores are long enough to interdigitate with MTs from the opposite pole (Fig. la). During anaphase the short MTs, including those attached to the c h r o m o s o m e s , get shorter while the long, interdigitating MTs lengthen as the poles move apart (Fig. lb) 1'2. In some cells, the elongating MTs change their extent of interdigitation, giving the appearance of M T sliding (Fig. lc) a. In other cells the extent of interdigitation of the two M T families remains constant as they elongate ~.
Descriptions of the mitotic process Microtubule arrangements and rearrangements during mitosis T h e classic image of the metaphase spindle was a structure shaped like an A m e r i c a n or R u g b y football with c h r o m o s o m e s aligned at the equator. It was said to be built from two classes of fibers: those connecting the chromosomes to the poles and those running from pole to pole. Early electron microscopy showed that spindle fibers were bundles of microtubules (MTs), but in the early 1970s the end-points and the orientations of individual spindle MTs were largely unknown. During recent years, the spindle MTs o f several organisms have been tracked through serial sections to yield 3-dimensional structures at each of several times during mitosis (e.g. Refs 1,2). M e t h o d s have also been developed to determine the polar orientation of the spindle MTs. Most of the MTs in each half of the spindle, including those which attach to the chromos o m e s at their k i n e t o c h o r e s (centromeres), are oriented with their 'plus' (fast-growing) ends distal to the near-by pole 3. M T length is heterodisperse, and some of the MTs which do not end at J. Richard Mclntosh is at the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
LENGTHENING OFLONGMTs
:
~
SHORTENING l OFSHORTMTs ¢
WHATARETHESITESOF SUBUNIT ADDITIONANDLOSS?
Anaphase
~
SLIDING OFMTs?
(c) Telophase
CHROMATINCONTRACTION PRIORTOREFORFBTION WHEREARETHEFORCES OFNUCLEARENVELOPE FORSLIDINGGENERATED? Fig. l. Rearrangement ofspind~MTsdunng anaphase.
~) 1984.ElsevierSciencePublishersBV..Amsterdam 0376 5(~7/84/$(r2.011