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Chapter 1 The Cytoskeleton: A Perspective
Chapter 1
The Cytoskeleton: A Perspective B. R. BRINKLEY Department of Cell Biology Baylor College of Medicine Houston, Texas
I. Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Microfilaments . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ill. Intermediate Filaments . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 4 6 8
This timely two-part volume on the cytoskeleton appears at the end of a productive decade in cell biology, accentuated by enormous progress in understanding the structural order and functional complexities of cytoplasm. The old concept of cytoplasm as an amorphous colloidal mass containing a suspension of organelles and inclusions must now be abandoned in favor of a more dynamic view in which organized arrays of fibrous elements interact to form a highly integrated structural network called the cytoskeleton. The capacity of cytoplasmic proteins such as tubulin, actin, and intermediate filament proteins to form polymers, to depolymerize, to intertwine and interact, and to elongate and contract in conjunction with subtle cell movements and shape changes gives cytoplasm its unique properties of life. Although the term cytoskeleton has become widely accepted, it is not totally descriptive. The expression applies to both muscle and nonmuscle cells and encompasses all of the fibrous elements of cytoplasm, including filamentous actin, myosin, intermediate filaments, microtubules, and a myriad of other proteins that anchor, cross-link, bind, or otherwise regulate the fibrous network in the cytoplasm. As the chapters in this volume will attest, however, the cytoskeleton provides a much more extensive function in cells than merely maintaining skeletal support and cytoplasmic consistency. Indeed, it appears to be actively involved in both force production and force transduction in various forms of cell motility, including cytoplasmic 1 METHODS IN CELL BIOLOGY, VOLUME 24
Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-564124-9
2
8 . R . BRINKLEY
streaming, organelle movement, cytokinesis , phagocytosis , secretion, axonal transport, and cell surface modulation. The suggested linkage of cytoskeletal components to transmembrane protein receptors implies a role in peptide hormone action. Components of the cytoskeleton may also be involved in mitogenesis and control of cell proliferation, but here the evidence is too new and incomplete to be fully convincing.
I.
Microtubules
Like other developments in the field of cell biology, advances in knowledge of the cytoskeleton were largely made possible by a series of technical achievements dating back over two decades. Although a fibrous network was detected in silver-stained neurons by Cajal in the nineteenth century, we generally credit early electron microscopists with the discovery of microtubules and microfilaments. The vast improvements in fixation afforded by glutaraldehyde (Sabatini et al., 1963) led to the widespread recognition of microtubules in most all eukaryotic cells. Prior to the glutaraldehyde era, discrete “filaments” were recognized in the mitotic spindles of amoebas (Roth and Daniels, 1962), and even earlier, fibrous elements were faintly seen to form a 9 2 pattern in dismembered and sectioned cilia (Manton and Clark, 1952; Fawcett and Porter, 1954). A significant contribution to our knowledge of microtubule substructures came from EM studies in meristematic cells of juniper. As a result of the natural electron opacity afforded by the cell wall material of this plant, Ledbetter and Porter (1964) identified 13 globular subunits in the walls of microtubules, a finding that in recent years has been widely confirmed in many diverse species using tannic acid as a stain (Tilney et al., 1973). Much of our current understanding of how subunits are arranged in the microtubule surface lattice has come from optical diffraction studies of negatively stained flagellar microtubules using optical filtering techniques and computer methods of image analysis (Amos and Klug, 1974; Erickson, 1974; Chasey, 1972). In addition, X-ray diffraction studies of unfixed, hydrated material has largely confirmed early electron microscopic studies (Mandelkow et al., 1977). In retrospect and considering all of the elegant electron microscopic studies, it is significant to point out that evidence for microtubules in living cells came a full decade before the EM era. By observing dividing marine oocytes through a polarizing microscope equipped with rectified, strain-free optics, Inoue detected weak form birefringence in the mitotic apparatus that was later shown to be due to highly oriented microtubules. Through a series of experimental manipulations Inoue and co-workers (1967, 1975) proposed that soluble subunits were in a dynamic equilibrium with their polymeric forms contained in spindle fibers. Moreover, Inoue proposed that the assembly was entropy driven and that the
+
1.
THE CYTOSKELETON: A PERSPECTIVE
3
subunits were maintained in the polymer by weak hydrophobic bonds. This important study has now been confirmed by three decades of research, including in vitro assembly experiments using purified microtubule protein. Currently, renewed interest is being placed on studies of the cytoskeleton in living cells using microinjection techniques and videomicroscopy. Thus Inoue ’s pioneering experiments were well in advance of their time. Although it is inappropriate to dwell excessively on microtubules in an overview of the cytoskeleton, I would be remiss in ignoring the exciting era of microtubule biochemistry that followed closely, and to some extent paralleled, the morphological studies of the 1960s and 1970s and continues unabated today. I need not elaborate the details here, since much of it will be covered in the chapters that follow. However, it should be pointed out that through the use of [3H]colchicine as a probe and the specificity of binding of this drug to microtubule protein, E. W . Taylor and his students (Shelanski and Taylor, 1967; Weisenberg ef al., 1968; Borisy and Taylor, 1%7a,b) and Wilson and Friedkin (1967) were able to isolate and characterize a single protein with a sedimentation coefficient of 6 S and a molecular weight of 110,000-120,000. Later it was shown that denaturation of the protein with guanidine hydrochloride produced two similar 55,000 M , a and /3 subunits. It was concluded that in aqueous solutions the larger colchicine-binding molecules existed as a dimer that came to be known as tubulin (Mohri, 1968). It was also discovered that a- and P-tubulin subunits existed in a constant I:] molar ratio in microtubules forming an (YP heterodimer (Bryan and Wilson, 1971). The 1970s saw the rise of tubulin biochemistry as a bona fide discipline with worldwide participation. One of the more significant developments came when Weisenberg (1972) succeeded in attaining microtubule assembly in vifro. He demonstrated that microtubules would form spontaneously from supernatants of brain homogenates when the solution was warmed to 37°C in the presence of GTP and magnesium. The key to his success was the addition of the calcium chelating agent EGTA to the reassembly mixture. He concluded that free calcium concentrations as low as 6 p M could inhibit the in vifro assembly of microtubules, an observation that may have physiological relevance concerning cellular control of microtubule assembly. For assembly to occur, a “critical concentration” of tubulin (0.2 mg/ml) was essential. This concentration is the lowest level at which cooperative association of subunits occurs forming structures that nucleate the assembly of microtubules. Thus assembly appears to be a two-step condensation-polymerization process in which nuclei (seeds) form and then elongate. At steady state polymerized tubulin is in equilibrium with a critical concentration of soluble tubulin. The in vitro assembly procedure of Weisenberg as utilized and modified by many other laboratories has contributed significantly toward an understanding of the biology and biochemistry of microtubules in eukaryotic cells. It has also provided a convenient means of purifying tubulin from a variety of tissues.
4
B . R. BRINKLEY
The availability of highly purified cytoskeletal proteins has enabled numerous investigators to produce monospecific antibodies to these proteins and to use them as immunofluorescent and immunoelectron microscopic probes in cells. The procedure of indirect immunofluorescence originally introduced by Coons and co-workers (1941) has had a popular rebirth in cytoskeleton research. As will be discussed in the chapters by Lazarides and by Osborn and Weber, this method has had a major impact on our understanding of the holistic organization of cytoskeletal elements in cells. For example, through the use of tubulin antibodies and indirect immunofluorescence, two microtubule arrays have been identified in proliferating cells: the mitotic spindle and a delicate lacework of tubules in interphase cells called the cytoplasmic microtubule complex or CMTC (Brinkley et al., 1975). The microtubules of the spindle and CMTC are morphologically and immunocytochemically similar and are both organized around discrete microtubule organizing centers. The two arrays differ, however, in overall morphology, numbers and length of microtubules, response to drugs and physical agents, time of appearance in the cell cycle, and, of course, function. These facts raise a number of new questions concerning regulation. Are there multiple populations of tubulin and microtubules in cells? How is the length, number, and distribution of microtubules maintained in the cytoplasm? How are the temporal events of microtubule assembly-disassembly regulated? How is the polarity of microtubules determined? What is the function(s) of the CMTC and how does it relate to the organization of microfilaments and other cytoskeletal components? These are questions for investigators to ponder during the next decade, but already significant progress has been made. In vitro assays for the analysis of MTOCs have been developed and progress has been made in the biochemical characterization of these components. Newer techniques have enabled investigators to determine the polarity and directionality of assembly of microtubules in the spindle and CMTC. Calcium, along with the calcium binding protein calmodulin, has been implicated in the control of microtubule assembly and disassembly (Marcum et al., 1978). Recently, several laboratories have reported the cloning of tubulin genes, and the amino acid sequence of at least one a and p tubulin has been determined (Valenzuela et al., 1980). After three decades, microtubule research is still booming, but surprisingly, much remains to be learned, not the least of which is how microtubules actually function to achieve any one of their numerous roles in cells.
11. Microfilaments One of the most significant episodes in modern cell biology began with the recognition of major musclelike proteins in the cytoplasm of nonmuscle cells-
1.
THE CYTOSKELETON: A PERSPECTIVE
5
even plant cells! Clearly, this area of cytoskeletal research is still enjoying exponential growth and much progress has been due to the availability of familiar techniques derived from an earlier decade of muscle biochemistry and physiology. Initially, electron microscopists recognized microfilaments as a somewhat heterogenous class of thick (>10 nm) and thin (4-6 nm) fibrous elements displaying various levels of organization and association in the cytoplasm. Sorting out the microfibrils into functional groups became an impossibility because of an identity problem until Ishikawa et al. (1969) adapted the heavy meromyosin labeling procedure of Huxley for “decorating” actin microfibrils in glycerolextracted cells. Through this procedure actin filaments formed “arrowheads ” displaying a pointed end and a barbed end. In every case, the thin 4-6 nm filaments were decorated with the heavy meromyosin or S1. This procedure not only has aided in the identification of a specific class of microfibrils, but has provided valuable information on the polarity of F-actin. For example, actin filaments always decorate with their barbed ends facing the plasma membrane. Through the use of a combination of techniques, including biochemistry, electron microscopy, and immunocytochemistry , much progress has been made in understanding the organization and function of actin in nonmuscle cells. Muscle and nonmuscle actin are very similar, as indicated by the fact that they differ in only 6% of their amino acid residues. Moreover, antibodies raised against vertebrate skeletal muscle actin cross-react with most forms of nonmuscle actin. On closer inspection, however, the nonmuscle (p- and a-isoactins) proteins are seen to differ significantly from muscle (a-actin) proteins in their isoelectric points. Mammalian a-actin displays 25 sequence differences from Physarum actin. Actins from vertebrate brain, platelet, and Acanthamoeba and Physarum have threonine at position 129, whereas heart and skeletal muscle actins substitute the hydrophobic residue valine (Geisow, 1979). Recent studies also suggest that the eukaryotic genome may contain multiple genes for actin-a surprising finding that may also apply to the tubulins. Obviously, recombinant DNA technology will provide much-needed information about the molecular structure, genetics, and evolution of cytoskeletal proteins. Although much has been learned about the structure, biochemistry, and localization of actin filaments, little is yet known about how they are organized into functional units in the cytoplasm of nonmuscle cells. The functional analogy with the sarcomere and contraction in skeletal muscle may be an oversimplification. A survey of motility in nonmuscle cells, including amoeboid movement, chromosome movement, endocytosis, exocytosis, cleavage, and cell surface receptor mobility, suggests that a variety of functional associations have evolved. Although the characters of the play have been identified for the most part, the plot is yet to be revealed. For example, force production ranges from the explosive polymerization of G-actin in the acrosome reaction of echinoderms (Tilney ef ul., 1973) to an apparent sliding filament mechanism in microfibril bundles such
6
B . R. BRINKLEY
as that seen in microvilli, the contractile ring, and stress fibers. The latter have received much attention and through immunofluorescence studies appear to contain F-actin, myosin, a-actinin, tropomyosin, filamin, and calmodulin. Unlike the sarcomere, microfilament bundles are frequently labile and may be maintained by a steady-state polymerization of G-actin. Although ionic conditions in the cytoplasm favor the assembly of actin, much of the actin exists in nonfilamentous (G-actin) form. In nonmuscle cells, a protein called profilin binds to G-actin and prevents its nucleation into F-actin. Interesting differences also exist in the mode of calcium-activated contraction. In the sarcomere actin binds to troponin C causing a conformation change in the troponin-tropomyosin complex that enables actin to bind to myosin, thereby inducing myosin ATPase activation and contraction. In nonmuscle actin, activation of myosin ATPase apparently requires the phosphorylation of myosin light chain by a calmodulin-activated light chain kinase. Thus in muscle, calcium binding releases an inhibition; in nonmuscle cells calcium binding to calmodulin activates myosin light chain kinase that activates myosin. Through studies of actin gels in vitro, progress has been made in identifying a variety of proteins that bind to actin and probably regulate the various structural and functional levels of organization of microfibrils Seen in the cytoskeleton (Bryan, in Volume 25). Such in vivo models are invaluable in determining the function of actin microfibrils in vitro. As yet, surprisingly little is known about the interaction of actin microfibrils with other cytoskeletal components and with various cell components such as membrane proteins. The viable proposal that actin may bind to or interact with transmembrane proteins and control their movements on the cell surface seems highly probable but is yet to be convincingly proved. The finding that both actin and tubulin co-cap with immunoglobulin in membranes of mouse B lymphocytes (Gabbiani et al., 1977) and the strong association of actin with the histocompatability H-2 antigen in spontaneously shed plasma membrane (Koch and Smith, 1978) are examples of experiments that support the notion that the cytoskeleton interacts with membrane proteins to regulate their movement and perhaps controls transmembrane regulation of events in the cytoplasm and nucleus.
111. Intermediate Filaments Intermediate filaments constitute another ubiquitous population of microfibrils in the cytoplasm. These 10-nm filaments were originally thought to be disaggregated forms of either microtubules or myosin, but more recent biochemical and immunocytochemical studies indicate that they are a distinct class of heterogenous proteins. Intermediate filaments may be classified into five major groups: ( 1 )
1.
THE CYTOSKELETON: A PERSPECTIVE
7
desmin filaments, found predominantly in skeletal, cardiac, and smooth muscle; (2) keratin filaments (tonofilaments), found largely in epithelial cells; (3) vimentin filaments, found in mesenchymal cells; (4) neurofilaments, found in neurons; and ( 5 ) glial filaments, found exclusively in glial cells. Such classification is useful in terms of identifying specific proteins associated with these filaments. It is known, however, that more than one class of intermediate filaments may be found in a single cell, and more than one protein may constitute a filament. At this juncture, less is known about the subunit structure, assembly, and distribution of intermediate filaments than about any other of the microfibrils of the cytoskeleton. They exist in both muscle and nonmuscle cells and seem to have a major function as mechanical integrators of the other cytoskeletal elements (Lazarides, 1980). Other roles are likely to be found with additional research. All intermediate filament proteins identified so far can be phosphorylated in v i m and may be regulated in vivo by this mechanism. Little is known about their assembly in vivo and as yet no specific intermediate filament initiation sites or organizing center has been found. Their involvement with other cytoskeletal proteins is not well understood. Their distribution in cells as shown by indirect immunofluorescence is greatly altered by agents that disrupt cytoplasmic microtubules. This fact, along with the finding by Goldman and co-workers (1980) that intermediate filaments associate with centrioles, suggests a very close association of intermediate filaments with microtubules and tubulin initiation sites. Perhaps one of the most important tasks in cytoskeletal research will be to determine how intermediate filaments, microtubules, actin microfibrils, myosin, and various regulatory components interact and are modulated as an integrated unit in cells. Much emphasis has been placed on the components of the cytoskeleton as individual units and little is known about how overall coordination is carried out. In conclusion, much has been learned about the filaments and tubules in the cytoskeleton. Have we now identified all of the major structural components of the cytoskeleton? Analysis of whole-mount and sectioned cells by high-voltage electron microscopy indicates a structural entity in cytoplasmic ground substance that Porter and co-workers (Wolosewick and Porter, 1979) call the microtrabecular lattice. Utilizing several fixation schedules and following vigorous protocols that minimize structural artifact, these investigators have described an ordered lattice composed of slender strands or microtrabeculae that interconnect membranes, polysomes, cytoskeletal elements, and other cell components Whether or not the microtrabecular lattice constitutes a separate set of cytoplasmic proteins and structural components, is an artifact, or merely represents extensions of the familiar cytoskeletal elements remains to be determined. At the present it is not unreasonable to assume that new, uncharted dimensions of cytoplasm exist and that even more complicated levels of molecular organization will be found in cells. The rapid development of new technology for cell research and its applica-
8
B . R . BRINKLEY
tion to studies of the cytoskeletal structure should make the next decade a decisive interval in defining the structure of cytoplasm.
REFERENCES Amos, L. A., and Klug, A. (1974). J. Cell Sci. 14,523-549. Borisy, G. G . . and Taylor, E. W. (1967a). J. Cell Biol. 34, 525-533. Borisy, G. G., and Taylor, E. W. (1967b). J. Cell Biol. 34, 535-548. Brinkley, B. R., Fuller, G. M., and Highfield, D. P. (1975). Proc. Natl. Acad. Sci. U . S . A . 72, 4981-4985. Bryan, J . , and Wilson, L. (1971). Proc. Natl. Acad. Sci. U.S.A. 68, 1762-1766. Chasey, D. (1972). Exp. Cell Res. 74, 140-146. Coons, A. H., Creech, H. J., and Jones, R. N. (1941). Proc. SOC. Exp. Biol. Med. 47, 200. Erickson, H. P. (1974). J . Cell Biol. 60, 153-167. Fawcett, D. W . , and Porter, K . R. (1954). J. Morphol 94, 221-281. Gabbiani, G. (1977). Nature (London) 269, 697. Geisow, N. (1979). Nature (London) 278, 507-508. Goldman, R. D., Hill, B. F., Steinert, P., Whitman, M. A., and Zackroff, R . B. (1980). In “Microtubules and Microtubule Inhibitors” (M. De Brabander and J. De Mey, eds.), pp. 91-102 Elsevier/North-Holland, Amsterdam. Inoue, S., and Ritter, H. (1975). In “Molecules and Cell Movements” (S. Inoue and R. Stephens, eds.), pp. 3-30. Raven, New York. Inoue, S., and Sato, H. ( 1 967). J . Gen. Physiol. 50, 259-292. Ishikawa, H., Bischoff, R., and Holtzer, H. (1969). J. Cell Biol. 43, 312-328. Koch, G. L. E., and Smith, M. J. (1978). Nature (London) 273, 274. Lazarides, E. (1980). Nature (London) 283, 249-256. Ledbetter, M. C., and Porter, K. R. (1964). Science 144, 872-874. Mandelkow, E., Thomas, E., and Bensch, K. G. (1977). Proc. Nail. Acad. Sci. U . S . A . 74, 3370-3374. Manton, I., and Clarke, R. (1952). J. Exp. Bot. 3, 265-275. Marcum, J. M., Dedman, J. R., Brinkley, B. R., and Means, A. R. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 377 1-3775. Mohri, H. (1968). Nature (London) 217, 1053-1054. Roth, L. E., and Daniels, E. W. (1962). J. Cell Biol. 12, 57-78. Sabatini, D. D., Bensch, K., and Barmett, R. J. (1963). J. Cell Biol. 17, 19-58. Shelanski, M. L., and Taylor, E. W. (1967). J. Cell Biol. 38, 304-315. Tilney, L. G., Bryan, J., Bush, D. J . , Fujiwara, K., and Mooseker, M. (1973). J. Cell Biol. 59, 109- 126. Valenzuela, P., Zaldivar, J . , Quiroga, M., Rutter, W., Cleveland, D., and Kirschner, M. (1980). Eur. J . Celt Biol. 22, 14. (Abstr.) Weisenberg. R. C. (1972). Science 177, 1104-1105. Weisenberg, R. C., Borisy, G. G., and Taylor, E. W . (1968). Biochemistry 7, 4466-4467. Wilson, L., and Friedkin, M. (1967). Biochemistry 6, 3126-3135. Wolosewick, J . J., and Porter, K. R. (1979). J. Cell Biol. 82, 114-139.
The Cytoskeleton: A Perspective B. R. BRINKLEY Department of Cell Biology Baylor College of Medicine Houston, Texas
I. Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11. Microfilaments . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ill. Intermediate Filaments . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 4 6 8
This timely two-part volume on the cytoskeleton appears at the end of a productive decade in cell biology, accentuated by enormous progress in understanding the structural order and functional complexities of cytoplasm. The old concept of cytoplasm as an amorphous colloidal mass containing a suspension of organelles and inclusions must now be abandoned in favor of a more dynamic view in which organized arrays of fibrous elements interact to form a highly integrated structural network called the cytoskeleton. The capacity of cytoplasmic proteins such as tubulin, actin, and intermediate filament proteins to form polymers, to depolymerize, to intertwine and interact, and to elongate and contract in conjunction with subtle cell movements and shape changes gives cytoplasm its unique properties of life. Although the term cytoskeleton has become widely accepted, it is not totally descriptive. The expression applies to both muscle and nonmuscle cells and encompasses all of the fibrous elements of cytoplasm, including filamentous actin, myosin, intermediate filaments, microtubules, and a myriad of other proteins that anchor, cross-link, bind, or otherwise regulate the fibrous network in the cytoplasm. As the chapters in this volume will attest, however, the cytoskeleton provides a much more extensive function in cells than merely maintaining skeletal support and cytoplasmic consistency. Indeed, it appears to be actively involved in both force production and force transduction in various forms of cell motility, including cytoplasmic 1 METHODS IN CELL BIOLOGY, VOLUME 24
Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-564124-9
2
8 . R . BRINKLEY
streaming, organelle movement, cytokinesis , phagocytosis , secretion, axonal transport, and cell surface modulation. The suggested linkage of cytoskeletal components to transmembrane protein receptors implies a role in peptide hormone action. Components of the cytoskeleton may also be involved in mitogenesis and control of cell proliferation, but here the evidence is too new and incomplete to be fully convincing.
I.
Microtubules
Like other developments in the field of cell biology, advances in knowledge of the cytoskeleton were largely made possible by a series of technical achievements dating back over two decades. Although a fibrous network was detected in silver-stained neurons by Cajal in the nineteenth century, we generally credit early electron microscopists with the discovery of microtubules and microfilaments. The vast improvements in fixation afforded by glutaraldehyde (Sabatini et al., 1963) led to the widespread recognition of microtubules in most all eukaryotic cells. Prior to the glutaraldehyde era, discrete “filaments” were recognized in the mitotic spindles of amoebas (Roth and Daniels, 1962), and even earlier, fibrous elements were faintly seen to form a 9 2 pattern in dismembered and sectioned cilia (Manton and Clark, 1952; Fawcett and Porter, 1954). A significant contribution to our knowledge of microtubule substructures came from EM studies in meristematic cells of juniper. As a result of the natural electron opacity afforded by the cell wall material of this plant, Ledbetter and Porter (1964) identified 13 globular subunits in the walls of microtubules, a finding that in recent years has been widely confirmed in many diverse species using tannic acid as a stain (Tilney et al., 1973). Much of our current understanding of how subunits are arranged in the microtubule surface lattice has come from optical diffraction studies of negatively stained flagellar microtubules using optical filtering techniques and computer methods of image analysis (Amos and Klug, 1974; Erickson, 1974; Chasey, 1972). In addition, X-ray diffraction studies of unfixed, hydrated material has largely confirmed early electron microscopic studies (Mandelkow et al., 1977). In retrospect and considering all of the elegant electron microscopic studies, it is significant to point out that evidence for microtubules in living cells came a full decade before the EM era. By observing dividing marine oocytes through a polarizing microscope equipped with rectified, strain-free optics, Inoue detected weak form birefringence in the mitotic apparatus that was later shown to be due to highly oriented microtubules. Through a series of experimental manipulations Inoue and co-workers (1967, 1975) proposed that soluble subunits were in a dynamic equilibrium with their polymeric forms contained in spindle fibers. Moreover, Inoue proposed that the assembly was entropy driven and that the
+
1.
THE CYTOSKELETON: A PERSPECTIVE
3
subunits were maintained in the polymer by weak hydrophobic bonds. This important study has now been confirmed by three decades of research, including in vitro assembly experiments using purified microtubule protein. Currently, renewed interest is being placed on studies of the cytoskeleton in living cells using microinjection techniques and videomicroscopy. Thus Inoue ’s pioneering experiments were well in advance of their time. Although it is inappropriate to dwell excessively on microtubules in an overview of the cytoskeleton, I would be remiss in ignoring the exciting era of microtubule biochemistry that followed closely, and to some extent paralleled, the morphological studies of the 1960s and 1970s and continues unabated today. I need not elaborate the details here, since much of it will be covered in the chapters that follow. However, it should be pointed out that through the use of [3H]colchicine as a probe and the specificity of binding of this drug to microtubule protein, E. W . Taylor and his students (Shelanski and Taylor, 1967; Weisenberg ef al., 1968; Borisy and Taylor, 1%7a,b) and Wilson and Friedkin (1967) were able to isolate and characterize a single protein with a sedimentation coefficient of 6 S and a molecular weight of 110,000-120,000. Later it was shown that denaturation of the protein with guanidine hydrochloride produced two similar 55,000 M , a and /3 subunits. It was concluded that in aqueous solutions the larger colchicine-binding molecules existed as a dimer that came to be known as tubulin (Mohri, 1968). It was also discovered that a- and P-tubulin subunits existed in a constant I:] molar ratio in microtubules forming an (YP heterodimer (Bryan and Wilson, 1971). The 1970s saw the rise of tubulin biochemistry as a bona fide discipline with worldwide participation. One of the more significant developments came when Weisenberg (1972) succeeded in attaining microtubule assembly in vifro. He demonstrated that microtubules would form spontaneously from supernatants of brain homogenates when the solution was warmed to 37°C in the presence of GTP and magnesium. The key to his success was the addition of the calcium chelating agent EGTA to the reassembly mixture. He concluded that free calcium concentrations as low as 6 p M could inhibit the in vifro assembly of microtubules, an observation that may have physiological relevance concerning cellular control of microtubule assembly. For assembly to occur, a “critical concentration” of tubulin (0.2 mg/ml) was essential. This concentration is the lowest level at which cooperative association of subunits occurs forming structures that nucleate the assembly of microtubules. Thus assembly appears to be a two-step condensation-polymerization process in which nuclei (seeds) form and then elongate. At steady state polymerized tubulin is in equilibrium with a critical concentration of soluble tubulin. The in vitro assembly procedure of Weisenberg as utilized and modified by many other laboratories has contributed significantly toward an understanding of the biology and biochemistry of microtubules in eukaryotic cells. It has also provided a convenient means of purifying tubulin from a variety of tissues.
4
B . R. BRINKLEY
The availability of highly purified cytoskeletal proteins has enabled numerous investigators to produce monospecific antibodies to these proteins and to use them as immunofluorescent and immunoelectron microscopic probes in cells. The procedure of indirect immunofluorescence originally introduced by Coons and co-workers (1941) has had a popular rebirth in cytoskeleton research. As will be discussed in the chapters by Lazarides and by Osborn and Weber, this method has had a major impact on our understanding of the holistic organization of cytoskeletal elements in cells. For example, through the use of tubulin antibodies and indirect immunofluorescence, two microtubule arrays have been identified in proliferating cells: the mitotic spindle and a delicate lacework of tubules in interphase cells called the cytoplasmic microtubule complex or CMTC (Brinkley et al., 1975). The microtubules of the spindle and CMTC are morphologically and immunocytochemically similar and are both organized around discrete microtubule organizing centers. The two arrays differ, however, in overall morphology, numbers and length of microtubules, response to drugs and physical agents, time of appearance in the cell cycle, and, of course, function. These facts raise a number of new questions concerning regulation. Are there multiple populations of tubulin and microtubules in cells? How is the length, number, and distribution of microtubules maintained in the cytoplasm? How are the temporal events of microtubule assembly-disassembly regulated? How is the polarity of microtubules determined? What is the function(s) of the CMTC and how does it relate to the organization of microfilaments and other cytoskeletal components? These are questions for investigators to ponder during the next decade, but already significant progress has been made. In vitro assays for the analysis of MTOCs have been developed and progress has been made in the biochemical characterization of these components. Newer techniques have enabled investigators to determine the polarity and directionality of assembly of microtubules in the spindle and CMTC. Calcium, along with the calcium binding protein calmodulin, has been implicated in the control of microtubule assembly and disassembly (Marcum et al., 1978). Recently, several laboratories have reported the cloning of tubulin genes, and the amino acid sequence of at least one a and p tubulin has been determined (Valenzuela et al., 1980). After three decades, microtubule research is still booming, but surprisingly, much remains to be learned, not the least of which is how microtubules actually function to achieve any one of their numerous roles in cells.
11. Microfilaments One of the most significant episodes in modern cell biology began with the recognition of major musclelike proteins in the cytoplasm of nonmuscle cells-
1.
THE CYTOSKELETON: A PERSPECTIVE
5
even plant cells! Clearly, this area of cytoskeletal research is still enjoying exponential growth and much progress has been due to the availability of familiar techniques derived from an earlier decade of muscle biochemistry and physiology. Initially, electron microscopists recognized microfilaments as a somewhat heterogenous class of thick (>10 nm) and thin (4-6 nm) fibrous elements displaying various levels of organization and association in the cytoplasm. Sorting out the microfibrils into functional groups became an impossibility because of an identity problem until Ishikawa et al. (1969) adapted the heavy meromyosin labeling procedure of Huxley for “decorating” actin microfibrils in glycerolextracted cells. Through this procedure actin filaments formed “arrowheads ” displaying a pointed end and a barbed end. In every case, the thin 4-6 nm filaments were decorated with the heavy meromyosin or S1. This procedure not only has aided in the identification of a specific class of microfibrils, but has provided valuable information on the polarity of F-actin. For example, actin filaments always decorate with their barbed ends facing the plasma membrane. Through the use of a combination of techniques, including biochemistry, electron microscopy, and immunocytochemistry , much progress has been made in understanding the organization and function of actin in nonmuscle cells. Muscle and nonmuscle actin are very similar, as indicated by the fact that they differ in only 6% of their amino acid residues. Moreover, antibodies raised against vertebrate skeletal muscle actin cross-react with most forms of nonmuscle actin. On closer inspection, however, the nonmuscle (p- and a-isoactins) proteins are seen to differ significantly from muscle (a-actin) proteins in their isoelectric points. Mammalian a-actin displays 25 sequence differences from Physarum actin. Actins from vertebrate brain, platelet, and Acanthamoeba and Physarum have threonine at position 129, whereas heart and skeletal muscle actins substitute the hydrophobic residue valine (Geisow, 1979). Recent studies also suggest that the eukaryotic genome may contain multiple genes for actin-a surprising finding that may also apply to the tubulins. Obviously, recombinant DNA technology will provide much-needed information about the molecular structure, genetics, and evolution of cytoskeletal proteins. Although much has been learned about the structure, biochemistry, and localization of actin filaments, little is yet known about how they are organized into functional units in the cytoplasm of nonmuscle cells. The functional analogy with the sarcomere and contraction in skeletal muscle may be an oversimplification. A survey of motility in nonmuscle cells, including amoeboid movement, chromosome movement, endocytosis, exocytosis, cleavage, and cell surface receptor mobility, suggests that a variety of functional associations have evolved. Although the characters of the play have been identified for the most part, the plot is yet to be revealed. For example, force production ranges from the explosive polymerization of G-actin in the acrosome reaction of echinoderms (Tilney ef ul., 1973) to an apparent sliding filament mechanism in microfibril bundles such
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as that seen in microvilli, the contractile ring, and stress fibers. The latter have received much attention and through immunofluorescence studies appear to contain F-actin, myosin, a-actinin, tropomyosin, filamin, and calmodulin. Unlike the sarcomere, microfilament bundles are frequently labile and may be maintained by a steady-state polymerization of G-actin. Although ionic conditions in the cytoplasm favor the assembly of actin, much of the actin exists in nonfilamentous (G-actin) form. In nonmuscle cells, a protein called profilin binds to G-actin and prevents its nucleation into F-actin. Interesting differences also exist in the mode of calcium-activated contraction. In the sarcomere actin binds to troponin C causing a conformation change in the troponin-tropomyosin complex that enables actin to bind to myosin, thereby inducing myosin ATPase activation and contraction. In nonmuscle actin, activation of myosin ATPase apparently requires the phosphorylation of myosin light chain by a calmodulin-activated light chain kinase. Thus in muscle, calcium binding releases an inhibition; in nonmuscle cells calcium binding to calmodulin activates myosin light chain kinase that activates myosin. Through studies of actin gels in vitro, progress has been made in identifying a variety of proteins that bind to actin and probably regulate the various structural and functional levels of organization of microfibrils Seen in the cytoskeleton (Bryan, in Volume 25). Such in vivo models are invaluable in determining the function of actin microfibrils in vitro. As yet, surprisingly little is known about the interaction of actin microfibrils with other cytoskeletal components and with various cell components such as membrane proteins. The viable proposal that actin may bind to or interact with transmembrane proteins and control their movements on the cell surface seems highly probable but is yet to be convincingly proved. The finding that both actin and tubulin co-cap with immunoglobulin in membranes of mouse B lymphocytes (Gabbiani et al., 1977) and the strong association of actin with the histocompatability H-2 antigen in spontaneously shed plasma membrane (Koch and Smith, 1978) are examples of experiments that support the notion that the cytoskeleton interacts with membrane proteins to regulate their movement and perhaps controls transmembrane regulation of events in the cytoplasm and nucleus.
111. Intermediate Filaments Intermediate filaments constitute another ubiquitous population of microfibrils in the cytoplasm. These 10-nm filaments were originally thought to be disaggregated forms of either microtubules or myosin, but more recent biochemical and immunocytochemical studies indicate that they are a distinct class of heterogenous proteins. Intermediate filaments may be classified into five major groups: ( 1 )
1.
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desmin filaments, found predominantly in skeletal, cardiac, and smooth muscle; (2) keratin filaments (tonofilaments), found largely in epithelial cells; (3) vimentin filaments, found in mesenchymal cells; (4) neurofilaments, found in neurons; and ( 5 ) glial filaments, found exclusively in glial cells. Such classification is useful in terms of identifying specific proteins associated with these filaments. It is known, however, that more than one class of intermediate filaments may be found in a single cell, and more than one protein may constitute a filament. At this juncture, less is known about the subunit structure, assembly, and distribution of intermediate filaments than about any other of the microfibrils of the cytoskeleton. They exist in both muscle and nonmuscle cells and seem to have a major function as mechanical integrators of the other cytoskeletal elements (Lazarides, 1980). Other roles are likely to be found with additional research. All intermediate filament proteins identified so far can be phosphorylated in v i m and may be regulated in vivo by this mechanism. Little is known about their assembly in vivo and as yet no specific intermediate filament initiation sites or organizing center has been found. Their involvement with other cytoskeletal proteins is not well understood. Their distribution in cells as shown by indirect immunofluorescence is greatly altered by agents that disrupt cytoplasmic microtubules. This fact, along with the finding by Goldman and co-workers (1980) that intermediate filaments associate with centrioles, suggests a very close association of intermediate filaments with microtubules and tubulin initiation sites. Perhaps one of the most important tasks in cytoskeletal research will be to determine how intermediate filaments, microtubules, actin microfibrils, myosin, and various regulatory components interact and are modulated as an integrated unit in cells. Much emphasis has been placed on the components of the cytoskeleton as individual units and little is known about how overall coordination is carried out. In conclusion, much has been learned about the filaments and tubules in the cytoskeleton. Have we now identified all of the major structural components of the cytoskeleton? Analysis of whole-mount and sectioned cells by high-voltage electron microscopy indicates a structural entity in cytoplasmic ground substance that Porter and co-workers (Wolosewick and Porter, 1979) call the microtrabecular lattice. Utilizing several fixation schedules and following vigorous protocols that minimize structural artifact, these investigators have described an ordered lattice composed of slender strands or microtrabeculae that interconnect membranes, polysomes, cytoskeletal elements, and other cell components Whether or not the microtrabecular lattice constitutes a separate set of cytoplasmic proteins and structural components, is an artifact, or merely represents extensions of the familiar cytoskeletal elements remains to be determined. At the present it is not unreasonable to assume that new, uncharted dimensions of cytoplasm exist and that even more complicated levels of molecular organization will be found in cells. The rapid development of new technology for cell research and its applica-
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tion to studies of the cytoskeletal structure should make the next decade a decisive interval in defining the structure of cytoplasm.
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