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  1. 1. MILESTONES M I L E S TO N E 1 To see them contract for the first time Our present understanding of the mechanism Straub joined Szent-Györgyi at about this of contraction is based on fundamental time, and it became clear that the difference discoveries, all of which arose from studies on between myosin A and myosin B was due to striated muscle as the early biochemists and the presence of another protein in the physiologists became (naturally) interested in myosin B preparations, which they called STOCKDISC the basic question of how muscles generate actin. Straub purified actin, and showed that movement. Much of the important work in it increased the viscosity of myosin A and this area took advantage of the beautiful, made it contractile. Straub also discovered regular organization of muscle, as well as the that actin existed in two forms: in the …many of the early abundance of material available for absence of salt the actin was globular milestones for the experimentation. The modern era began with (G-actin), whereas in physiological salt cytoskeleton field the demonstration that contraction is the concentrations the actin polymerized to are the same as one result of the interaction of ATP with two form filaments (F-actin). Magnesium (Mg) proteins, actin and myosin. activated the steady-state ATPase activity of would list if making During the Second World War, and in myosin B (renamed actomyosin), but not that a historical overview complete scientific isolation in Szeged, of myosin alone. In 1950, Straub and Feuer of the muscle field. Hungary, Albert Szent-Györgyi and found that ATP was a functional group of Margaret Titus colleagues established that the myosin G-actin, and that actin hydrolysed bound originally described by Wilhelm Kühne in ATP when it polymerized. Subsequently, 1864 consisted of two proteins. The sole it was shown that actin polymerizes by a scientific instruments available to them nucleation and elongation mechanism, and ORIGINAL RESEARCH PAPERS Banga, I. & Szent-Györgyi, A. in Studies from the Institute of Medical Chemistry University were a simple Ostwald viscometer and that non-muscle cells also contain actin. Szeged Vol. 1 (ed. Szent-Györgyi, A.) 5–15 (S. Karger AG, polarizing filters to detect double refraction However, it was the behaviour of the Basel, 1941–1942) | Szent-Györgyi, A. (ed.) in Studies from the of flow. In 1942, Banga and Szent-Györgyi glycerol-extracted psoas muscle preparation Institute of Medical Chemistry Univ. Szeged Vol. 1 17–26 (S. Karger AG, Basel, 1941–1942) | Needham, J. et al. Is muscle reported that exposure of ground muscle to described by Szent-Györgyi that provided contraction essentially an enzyme–substrate combination? a high salt concentration for 20 min led to conclusive evidence that the interaction of Nature 150, 46–49 (1942) | Straub, F. B. in Studies from the Institute of Medical Chemistry University Szeged Vol. 2 (ed. the extraction of a protein of low viscosity, ATP with actomyosin was the basic Szent-Györgyi, A.) 3–15 (S. Karger AG, Basel, 1942) | Straub, myosin A, whereas the protein extracted contractile event. Upon addition of Mg-ATP, F. B. in Studies from the Institute of Medical Chemistry overnight, myosin B, had a high viscosity. the preparation develops a tension that is University Szeged Vol. 3 (ed. Szent-Györgyi, A.) 23–37 (S. Karger AG, Basel, 1943) | Szent-Györgyi, A. Studies on Addition of ATP reduced the viscosity of comparable to that in living muscle. muscle. Acta Physiol. Scand. 9 (Suppl. 25), 1–115 (1945) | myosin B, whereas the viscosity of myosin Moreover, the preparation behaves like Szent-Györgyi, A. Free energy relations and contraction of A remained essentially unaffected. In 1942, actomyosin to some extent. The actomyosin. Biol. Bull. 96, 140–161 (1949) | Straub, F. B. & Feuer, G. ATP, the functional group of actin. Biochim. Biophys. the effect of ATP on K hne’s myosin was demonstration that contraction can be Acta 4, 455–470 (1950) independently discovered by Needham et al. reproduced in vitro by two proteins, actin FURTHER READING Kühne, W. Untersuchungen uber das Protoplasma und die Contractilitat (W. Engelmann, Leipzig, Szent-Györgyi found that the threads and myosin, opened up the modern phase of 1864) | Kasai, M., Askura, S. & Oosawa, F. The cooperative prepared from myosin B in physiological salt muscle biochemistry. nature of G–F transformation of actin. Biochim. Biophys. Acta solutions shortened on addition of boiled Soon afterwards, myosin was also isolated 57, 22–30 (1960) | Hatano, S. & Oosawa, F. Isolation and characterization of plasmodium actin. Biochim. Biophys. muscle juice, whereas fibres of myosin A from non-muscle cells, followed by pioneering Acta 127, 488–498 (1966) | Ishikawa, H., Bischoff, R. & remained unchanged. The shortening was work on muscle contraction (see Milestone 3 Holtzer, H. Formation of arrowhead complexes with heavy apparently due to the exclusion of water and Milestone 9). meromyosin in a variety of cell types. J. Cell Biol. 43, 312–328 (1969) | Wegner, A. Head to tail polymerization of actin. from the threads, and the active material in Ekat Kritikou, Senior Editor, J. Mol. Biol. 108, 139–150 (1976) the boiled extract was identified as ATP. Nature Reviews Molecular Cell Biology NATURE MILESTONES | CYTOSKELETON DECEMBER 2008 | S5
  2. 2. DIGITAL VISION sion and withdrawal took place at the leading edge of the advancing cell. These motile sheet-like projections Although this were defined as lamellipodia. Further paper was analyses revealed that the rates of protrusion and withdrawal were published more similar, and that forward movement than half a century resulted from the greater proportion ago, it discusses M I L E S TO N E 2 of time that a cell spent protruding. contact inhibition, Membrane ruffles, which were visual- which is very ized by phase-contrast microscopy as On the move dark waves arising at the leading edge of the cells, were found to form mainly important in the field of not only during the switch from protrusion to cell biology, but withdrawal, and to move centripetally also pathology. Michael Abercrombie was a pioneer cells to restrain each other’s movement towards the cell body. In addition, the Yoshimi Takai in the study of cell behaviour. By was defined as ‘contact inhibition’, and retrograde movement of particles on setting up some of the first time-lapse its implications for processes such the dorsal surface of the lamellipodia experiments with chicken fibroblasts as the organization of tissues during was noted. Together, these findings and a phase-contrast microscope, development, wound healing and the led to the idea that cell movement he described the cell-motility cycle, formation of metastasis were outlined. requires the rapid insertion of new which is the basis of our current In the 1970s, Abercrombie, material at the leading edge, which understanding of how cells migrate. Heaysman and Pegrum published causes the excess surface to move In 1953, Abercrombie and five seminal papers that led to several backwards steadily. Heaysman first described how contact hypotheses on the mechanisms of cell Detailed examination of lamellipo- with neighbouring cells negatively migration. The authors described how dia by electron microscopy revealed regulates cell progression. The ability of repeated cycles of membrane protru- the presence of discrete accumulations M I L E S TO N E 3 Relaxed Z disc A band Muscle sliding filaments Thick myosin filament Thin actin filament Two ground-breaking papers, published In interference microscopy, the 1 band H zone back-to-back in Nature 54 years ago, reference beam does not cross through Contracted A band constant independently showed that muscle the specimen, allowing the striations shortens as a result of the sliding within it (A and I bands) to be identified between the thick and thin filaments of unambiguously and their lengths to be the fibres (muscle cells, each of which measured when the fibres are stretched, consists of many parallel myofibrils). stimulated or otherwise manipulated; 1 band shortens H zone shortens Although these papers reported work the resultant changes in the width of the that was done independently, both sets striations can be measured. In muscle of authors discussed their results before cells, the A and I bands are organized into In their accompanying paper, Hugh The width of ‘A bands’ in submitting their manuscripts, and basic structural units, the sarcomeres, muscle fibres remains Huxley and Jean Hanson, who had constant during contraction referred to each other’s studies in the which repeat along the length of the previously shown that myosin is located suggesting a ‘sliding filament’ Nature papers. myofibril, so measurements of a single in the thick filaments of the A band and model in which myosin filaments run the length of the In the experiments reported in the sarcomere can be extrapolated to a that actin is located in the thin filaments A band and actin filaments first of these two papers, Andrew whole muscle’s action. of the I band, reported their studies of slide into the A band. 2004 Huxley and Rolf Niedergerke used Huxley and Niedergerke found that Nature Publishing Group. myofibrils using light microscopy. In their interference microscopy to show that the ratio of widths of the A and I band experiments, they mounted a suspension the width of ‘A bands’ (thick filaments depend simply on the length of the of myofibrils on a microscope slide under consisting of the protein myosin) in fibre, and are unaffected by tension a coverslip. When they found a fibril with muscle fibres remains constant during development. The length of the A band one end embedded in a fibre fragment contraction, implying that during muscle is constant. The natural conclusion from adhering to the coverslip and the other contraction, the actin-containing thin these results is that the myosin in the end in a fragment attached to the slide, filaments of the ‘I band’ are drawn into A bands is in the form of submicroscopic they moved the coverslip slightly to the A band. In order to perform their (in 1950s terms) rods of definite length. make the fibril stretch, observing the desired experiments, the authors had During contraction, the actin filaments behaviour of the A and I bands during to design an interference microscope are drawn into the A bands, between the the process. During this stretching of the and have it built to their specifications. rodlets of myosin. single myofibrils, the A band remained S6 | DECEMBER 2008
  3. 3. MILESTONES of dense material, which constitute cell motility are still subject of intense M I L E S TO N E 4 sites of attachment to the substratum, investigation. and longitudinal filaments (see Milestone 12). This first glimpse into Monica Hoyos-Flight, Associate Editor, Nature Reviews Neuroscience and Nature Beating a path to Reviews Drug Discovery the cytoarchitecture of the leading edge raised the idea that adhesion to ORIGINAL RESEARCH PAPERS Abercrombie, M. success the substratum provided a means of & Heaysman, J. E. Observations on the social Stoc k behaviour of cells in tissue culture. I. Speed of Com traction, which together with contrac- movement of chick heart fibroblasts in relation to From spermatozoa motility to the passage of tile fibrils allowed a cell to pull itself their mutual contacts. Exp. Cell. Res. 5, 111–131 mucus through the airways, the movement of forward. Indeed, subsequent studies (1953) | Abercrombie, M., Heaysman, J. E. & Pegrum, cilia and flagella is vital for numerous physiological functions S. M. The locomotion of fibroblasts in culture. I. have shown that both the continuous Movements of the leading edge. Exp. Cell Res. 59, and has long fascinated biologists (see Milestone 22). Efforts addition of membrane at the leading 393–398 (1970) | Abercrombie, M., Heaysman, J. E. to understand this motility led to some of the fundamental edge and the generation of tractional & Pegrum, S. M. The locomotion of fibroblasts in discoveries in cell biology in the twentieth century. culture. II. “Ruffling”. Exp. Cell Res. 60, 437–444 force at sites of adhesion enable cell (1970) | Abercrombie, M., Heaysman, J. E. & Pegrum, In the 1950s, the blossoming field of electron microscopy had movement. Furthermore, a key role S. M. The locomotion of fibroblasts in culture. III. allowed the structure of the axoneme — the structural core of for microtubules in the stabilization Movements of particles on the dorsal surface of cilia and flagella — to be visualized, revealing the now familiar the leading lamella. Exp. Cell Res. 62, 389–398 of the leading edge and the generation arrangement of nine microtubule doublets, linked by protein ‘arms’, (1970) | Abercrombie, M., Heaysman, J. E. & Pegrum, of directed motility has also emerged S. M. The locomotion of fibroblasts in culture. IV. surrounding a central microtubule pair. However, little was known Electron microscopy of the leading lamella. Exp. about the protein components of the axoneme. (see Further reading). Cell Res. 67, 359–367 (1971) | Abercrombie, M., Axoneme-isolation studies had demonstrated that axoneme motility Despite powerful imaging, genetic Heaysman, J. E. & Pegrum, S. M. Locomotion of required an ATPase. In 1965, Gibbons and Rowe identified an ATPase and computational methods, many fibroblasts in culture. V. Surface marking with concanavalin A. Exp. Cell Res. 73, 536–539 (1972) with enzymatic and structural properties matching those of the questions regarding the molecular FURTHER READING Vasiliev, J. M. et al. Effect of axoneme arms within the ciliated protozoon Tetrahymena pyriformis. mechanisms that underlie cell migra- colcemid on the locomotory behaviour of They named the protein dynein (from the Greek dyne, meaning fibroblasts. J. Embryol. Exp. Morphol. 24, 625–640 tion remain unanswered. In particular, (1970) | Izzard, C. S. & Lochner, L. R. Formation of force). This marked the discovery of the first microtubule motor the mechanisms of adhesion assembly cell-to-substrate contacts during fibroblast protein and 20 years would pass before another — kinesin — would and disassembly and the precise motility: an interference-reflexion study. J. Cell be identified (see Milestone 15). Three years after the identification of Sci. 42, 81–116 (1980) regulation of the cytoskeleton during dynein, another component of the axoneme was identified by Mohri, who isolated and characterized the protein subunit of sea urchin spermatozoa microtubules and named it tubulin. at constant length and the actin in these two 1954 papers: the force In subsequent years, attention turned to the mechanism of filaments were pulled or ‘folded-up’ between actin and myosin is generated cilia bending. The ‘sliding filament model’, which proposed that into the A band. They photographed in the region of overlap between the microtubules actively slide along each other rather than individually myofibrils contracting either freely or thick and thin filaments, sliding the contracting, was gaining support, largely due to the demonstration while held at both ends, without or with filaments together and shortening the by Satir that microtubule length remains constant during the addition of various concentrations muscle fibre. bending. In 1971, Summers and Gibbons provided a notable visual of ATP. They found that the I bands Maxine Clarke, Publishing Executive demonstration of the model in action. shortened from ~0.8 μm at resting Editor, Nature Summers and Gibbons were studying the sea urchin spermatozoa length to zero during contraction, axoneme. Trypsinization sensitized the isolated axoneme so that whereas the A bands remained at a ORIGINAL RESEARCH PAPERS Huxley, A. F. & the addition of ATP caused it to disintegrate rapidly. Using dark-field constant length of ~1.5 m. Niedergerke, R. Structural changes in muscle microscopy, Summers and Gibbons recorded the disintegration, The authors suggested, on the basis during contraction: interference microscopy of living muscle fibres. Nature 173, 971–973 revealing sliding movements between microtubule doublets, and the of these and other results described in (1954) | Huxley, H. E. & Hanson, J. Changes in protrusion and expulsion of individual doublets from the axoneme. the paper, that the driving force for the the cross-striations of muscle during These experiments helped to ensure the firm acceptance of the process of contraction is the formation contraction and stretch and their structural interpretation. Nature 173, 973–976 (1954) | sliding filament model. of actin–myosin linkages when ATP Huxley, A. F. Muscle structure and theories of Today, it is known that microtubule-mediated transport is crucial is split by the myosin enzyme. The contraction. Prog. Biophys. Biophys. Chem. 7, for many aspects of cellular function. In addition to the large family enzymatically generated movement of 255–318 (1957) | Huxley, H. E. The double array of dyneins that drive axonemal motility, cytoplasmic dynein, which of filaments in cross-striated muscle. J. Biophys. the linkages or crossbridges represents Biochem. Cytol. 3, 631–648 (1957) was discovered in 1987, has multiple roles throughout the cell, from the molecular ‘working stroke’ that FURTHER INFORMATION Huxley, H. E. the control of mitosis to the transport of cargo. drives muscle contraction. Electron microscope studies on the structure Katherine Whalley, Senior Editor, Nature Reviews Neuroscience of natural and synthetic protein filaments In his retrospective essay “A from striated muscle. J. Mol. Biol. 7, 281–308 personal view of muscle and motility (1963) | Huxley, H. E. Structural difference mechanisms”, Hugh Huxley recalls how between resting and rigor muscle; evidence ORIGINAL RESEARCH PAPERS Gibbons, I. R. & Rowe, A. J. Dynein: a protein with from intensity changes in the low-angle adenosine triphosphatase activity from cilia. Science 149, 424–426 (1965) | Mohri, H. he and Hanson estimated at the time equatorial X-ray diagram. J. Mol. Biol. 37, Amino-acid composition of ‘tubulin’ constituting microtubules of sperm flagella. that under maximum load, the actin 507–520 (1968) | Huxley, A.F. and Simmons, Nature 217, 1053–1054 (1968) | Summers, K. E. & Gibbons, I. R. Adenosine filaments needed to be pulled along R.M. Proposed mechanism of force generation triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin by ~100 Å (0.01 μm) each time about in striated muscle. Nature 233, 533–538 (1971) | sperm. Proc. Natl Acad. Sci. USA 68, 3092–3096 (1971) Huxley, A. F. Reflections on Muscle. The FURTHER READING Machin, K. E. Wave propagation along flagella. J. Exp. Biol. 35, one-third of the myosin molecules split Sherrington Lectures XIV (Liverpool Univ. Press, 796–806 (1958) | Satir, P. Studies on cilia. 3. Further studies on the cilium tip and a ATP, to create the sliding force needed 1981) | Huxley, H. E. A personal view of muscle “sliding filament” model of ciliary motility. J. Cell Biol. 39, 77–94 (1968) | Goodenough, to explain muscle contraction. and motility mechanisms. Ann. Rev. Physiol. 58, U. W. & Heuser, J. E. Substructure of the outer dynein arm. J. Cell Biol. 95, 798–815 1–19 (1996) (1982) | Paschal, B. M., Shpetner, H. S. & Vallee, R. B. MAP 1C is a microtubule-activated Hence, the ‘sliding filament’ proposal ATPase which translocates microtubules in vitro and has dynein-like properties. emerged from the work described J. Cell Biol. 105, 1273–1282 (1987) NATURE MILESTONES | CYTOSKELETON DECEMBER 2008 | S7
  4. 4. MILESTONES M I L E S TO N E 5 Mitosis: a dynamic view Chromosomes (white) segregated by microtubules (stained with anti-tubulin, (red)), illustrating the dynamics of mitosis The fundamental question of how the mitotic spindle forms that Inoue deduced from the birefringent and functions to capture, align and segregate chromosomes observation of spindle fibres. Courtesy of Z. Yang and C. L. Rieder, Wadsworth Center, into two daughter cells dates back to 1882 and the Albany, NY, USA. microscopic observations that were made by Walther Flemming of the changes in spindle morphology seen at cornerstone by presenting a model of mitotic spindle different stages of mitosis. Despite their fundamental dynamics and their role in chromosome movements. They importance, these findings were limited by the use of fixed proposed that the birefringent fibres could reversibly …protein cell preparations, which would not allow detailed analysis of polymerize and depolymerize during normal mitosis. In polymerization the mechanisms that are involved in spindle assembly or its their ‘dynamic equilibrium model’, the spindle fibres were dynamics drive ability to control chromosome behaviour. described as orientated polymers in equilibrium with a pool morphogenesis A decisive step towards the solution to this problem was of 22S particles that were found around that time, by realized in the 1950s, when polarized light microscopy and Robert Kane, to be the major proteins extractable from an of, and force live-cell imaging allowed the limitations of fixed samples to isolated spindle. Tubulin was then identified as the protein production be overcome, and opened the way for a dynamic view of that comprises the spindle fibres or microtubules (see by, the mitotic biological processes. During the following two decades, Milestone 6). Inoue showed that the equilibrium could be spindle… Shinya Inoue and his co-workers pioneered live-cell shifted towards depolymerization by low temperatures and Tim Mitchison imaging by developing microscopes that allowed them to colchicine, or towards polymerization by treatment with visualize parallel spindle fibres that polarized the light heavy water, and that fibre reassembly from the soluble thanks to their birefringent properties. Crucially, pool occurred in the absence of de novo protein synthesis. birefringence could be measured and correlated with Inoue proposed that, throughout mitosis, the fibre structural alterations occurring in the fibres during mitosis dynamics were controlled by the activity of ‘orientating or in response to given experimental conditions. centres’ (centrioles, kinetochores and the cell plate) and by In 1967, in a seminal paper based on the discussion of the concentration of the free subunits. their own observations as well as those of several other A second fundamental observation made by Inoue and investigators, Inoue and Hidemi Sato laid a fundamental co-workers was that the chromosome movements during the kinetics of colchicine binding to M I L E S TO N E 6 cells could be modelled by a single class of binding sites, indicating that Building blocks a unique target might exist. Gary Borisy embarked on the project to identify it. By adding radiolabelled colchicine to a range of extracts from cells and tissues, he found a single In the early 1960s, microtubules confusing body of literature described 6S component co-purifying with were known to be constituents other cellular and physiological effects. colchicine. Importantly, this binding of the mitotic spindle fibres (see In 1967, Edward Taylor reported that activity was high in tissue-culture Milestone 5) and the 9+2 array of cells, sea urchin eggs, isolated mitotic filaments that are observed in cilia and spindles and brain tissue — all of which spermatozoa tails (see Milestones 4). The are rich in microtubules. Borisy and identification of tubulin as the basic Taylor therefore proposed that the 6S subunit of microtubules opened up protein was the microtubule subunit, these structures to molecular analysis although the name tubulin was coined and demonstrated that microtubules only in a later report by Hideo Morhi from different sources had the same on the biochemical composition of composition. The drug colchicine spermatozoa flagella. In addition to the played a key role in this discovery. discovery of tubulin, the work by Today, colchicine, together with Borisy and Taylor established the colcemid and nocodazole, is commonly powerful approach of using specific used in the laboratory to block drugs to probe the function of the microtubule polymerization; these cytoskeleton. drugs bind to tubulin and prevent its Efforts to isolate tubulin and to study addition to growing microtubule ends. its assembly properties ensued. In In the 1960s, although colchinine was 1972, Richard Weisenberg and Borisy known to destroy the mitotic spindle, a re-assembled microtubules from tubulin S8 | DECEMBER 2008
  5. 5. BANANASTOCK mitosis were controlled by the shortening and lengthening of the fibres. Experiments involving fibre depolymerization by treatment with colchicine or gradual cooling led them to the groundbreaking hypothesis that spindle dynamics can generate forces capable of pushing and pulling chromosomes. It took another 20 years, and the development of biochemical assays and labelling techniques in the 1980s, to start to uncover the mechanistic details of how the spindle assembles and M I L E S TO N E 7 controls chromosome movements (see Milestone 14). Nevertheless, the work of Inoue was an early description of a new form of biological motility driven by the assembly and disassembly of a biological polymer. Inoue and Sato The in-between further speculated that such motility could be used to move organelles other than chromosomes or to deform By the late 1960s, many investigators had observed unknown filaments in developing the cell surface, paving the way for the modern view of muscle cells that did not appear to be either actin or myosin filaments — the two the crucial role of cytoskeleton polymerization dynamics major cytoskeletal elements in muscle. In 1968, on the basis of electron-microscopy in cellular morphogenesis and force generation. studies of cultured differentiating muscle and fibroblast cells, Holtzer and colleagues Silvia Grisendi, Associate Editor, reported a third type of filament. The key experiment was to treat dividing cells, Nature Cell Biology which have few identifiable actin microfilaments, with mitotic inhibitors to break down spindle microtubules. The remaining predominant filament type was novel ORIGINAL RESEARCH PAPER Inoue, S. & Sato, H. Cell motility by labile with a diameter (10 nm) in between that of actin (~6 nm) and myosin (~15 nm) — association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J. Gen. Physiol. 50, 259–292 (1967) hence, the name intermediate filaments (IFs). FURTHER READING Mazia, D., Mitchison, J. M., Medina, H. & Harris, P. The direct The Holtzer team recognized that the 10-nm filaments might, in fact, represent a isolation of the mitotic apparatus. J. Biophys. Biochem. Cytol. 10, 467–474 (1961) | heterogeneous class, which turned out to be correct (see Further reading). Yet, how Kane, R. E. The mitotic apparatus. Identification of the major soluble component of the glycol-isolated mitotic apparatus. J. Cell Biol. 32, 243–253 (1967) | Gorbsky, were these histologically diverse IFs unified at the molecular level? In the early 1980s, G. J., Sammak, P. J. & Borisy, G. G. Microtubule dynamics and chromosome motion Weber and Geisler sequenced several IFs and discovered visualized in living anaphase cells. J. Cell Biol. 106, 1185–1192 (1988) | that they share a conserved structural domain that forms Mitchison, T. J. Polewards microtubule flux in the mitotic spindle: evidence from a double-stranded coiled coil of -helices. The variety in Interestingly, the photoactivation of fluorescence. J. Cell Biol. 109, 637–652 (1989) size was shown to arise from the N-terminal and C-terminal name “intermediate extensions flanking the coiled-coil structure. filaments” was given Insight into the possible roles of IFs came from several to these filaments by studies, which showed that IFs are also present in the Holtzer because they purified from rat brain homogenates, nucleus. Initially, Gerace, Blum and Blobel showed that the were intermediate showing that the process required three predominant polypeptides present in a fraction from in diameter between The identification a calcium chelator. This key study actin filaments and rat liver nuclei localize exclusively at the nuclear periphery demonstrated that the capacity for myosin filaments, not of tubulin as the and coincide with the nuclear lamina — a protein meshwork microtubules, as it assembly resided in the tubulin subunit that underlies the inner nuclear membrane and is associated basic subunit has come to be itself and did not require a separate with nuclear pore complexes. They also noticed that, of microtubules known. polymerase. Work by Weisenberg also concomitant with the disassembly of the nuclear envelope in Gregg Gundersen opened up provided a protocol for microtubule prophase, the major lamina polypeptides (or lamins) become these structures self-assembly, opening the door to dispersed, until telophase, when the nuclear envelope research into its mechanisms (see reassembles. In a follow-up study, Gerace and Blobel showed to molecular Milestone 14). that the disassembly of the nuclear lamina results from the reversible depolymerization analysis… Christina Karlsson Rosenthal, Locum of the lamins, which is correlated with their reversible phosphorylation. In 1986, Associate Editor, Nature Cell Biology Aebi and colleagues reported the structural and assembly properties of lamins, and confirmed by sequence analysis that they are in fact a type of IF. Together, these discoveries marked the identification of the third cytoskeletal ORIGINAL RESEARCH PAPERS Borisy, G. G. & Taylor, E. W. The mechanism of action of filamentous system. colchicine. Binding of colchicine-3H to cellular Arianne Heinrichs, Chief Editor, protein. J. Cell Biol. 34, 525–534 (1967) | Borisy, Nature Reviews Molecular Cell Biology G. G. & Taylor, E. W. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J. Cell Biol. 34, ORIGINAL RESEARCH PAPERS 535–548 (1967) | Weisenberg, R. C., Borisy, G. G. Ishikawa, H., Bischoff, R. & Holtzer, H. Mitosis and intermediate filament-sized filaments in developing skeletal & Taylor, E. W. The colchicine-binding protein of muscle. J. Cell Biol. 38, 538–555 (1968) | Gerace, L., Blum, A. & Blobel, G. Immunocytochemical localization of the mammalian brain and its relation to major polypeptides of the nuclear pore complex-lamina fraction. J. Cell Biol. 79, 546–566 (1978) | Gerace, L. & microtubules. Biochemistry 7, 4466–4479 (1968) Blobel, G. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell 19, 277–287 (1980) | FURTHER READING Weisenberg, R. C. Aebi, U. et al. The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323,560–564 (1986) Microtubule formation in vitro in solutions FURTHER READING Small, J. V. & Sobieszek, A. Studies on the function and composition of the 10-nm containing low calcium concentrations. Science (100-Å) filaments of vertebrate smooth muscle. J. Cell Sci. 23, 243–268 (1977) | Franke, W. W. et al. Different 177, 1104–1105 (1972) | Borisy, G. G. & intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc. Natl Acad. Sci. USA 75, Olmsted, J. B. Nucleated assembly of 5034–5038 (1978) | Steinert, P. M. et al. Ten-nanometer filaments of hamster BHK-21 cells and epidermal microtubules in porcine brain extracts. Science keratin filaments have similar structures. Proc. Natl Acad. Sci. USA 75, 6098–6101 (1978) | Aebi, U. et al. The 177, 1196–1197 (1972) fibrillar substructure of keratin filaments unraveled. J. Cell Biol. 97, 1131–1143 (1983) | Weber, K. & Geisler, N. Intermediate filaments: structural conservation and divergence. Ann. NY Acad. Sci. 455, 126–143 (1985) NATURE MILESTONES | CYTOSKELETON DECEMBER 2008 | S9
  6. 6. MILESTONES in sea urchin eggs. He observed that fragment of muscle myosin that M I L E S TO N E 8 the contractile ring was composed of interacts with actin filaments. He microfilaments that were similar to also observed that the contractile Belting up muscle actin filaments. Formation of the contractile ring coincided with the onset of cytokinesis, its spatial location ring contained another component, besides the actin microfilament, which was not marked by HMM, coincided with that of the cleavage and proposed that it could be an furrow and the filaments disappeared oligomeric form of myosin. Indeed, when cytokinesis was over, which in 1976, using myosin-specific In a landmark study published in 1972, indicated that the contractile ring might antibodies that were coupled with Thomas Schroeder provided the first be responsible for cytokinesis. fluorescent dyes, Fujiwara and Pollard ultrastructural description of the To test this hypothesis, Schroeder demonstrated that myosin-II was contractile ring during cell cleavage treated cells with the drug cytochalasin abundant in the contractile ring. B, which was later found to inhibit actin It was therefore reasonable to polymerization. The microfilaments postulate that actin and myosin-II rapidly depolymerized, the contractile interact to produce the force of ring disassembled and the cleavage ring constriction, and the first furrow regressed. By contrast, demonstration of this came in 1977, microtubules of the mitotic apparatus with the work of Mabuchi and Okuno. were unaffected. So, the contractile They microinjected antibodies against ring drives cytokinesis independently of starfish egg myosin-II into eggs, and mitosis. Schroeder also showed that the found that this blocked cytokinesis volume of the contractile ring declined but did not interfere with the function as the furrow constricted, and proposed of the mitotic spindle. So, during that the filaments disassemble as the cytokinesis, myosin-II powers the ring contracts. ‘belting up’ of the contractile ring A year later, Schroeder confirmed composed of actin filaments. the presence of actin in the contractile Francesca Cesari, Associate Editor, ring by treating cells with heavy Nature Reviews Felinda | meromyosin (HMM), which is a tryptic Molecular Cell Biology M I L E S TO N E 9 (PAK) kinase. This new ATPase, named myosin-I, could bind to and The unconventional ones bundle filamentous actin. The novel myosin was small com- pared with the conventional muscle myosin; in addition, whereas muscle Hundred years after the discovery Pollard and Korn were convinced myosin was a dimer that contained of muscle myosin (see Milestone 1), that amoeboid movement was two ATPase domains, myosin-I was Pollard and Korn uncovered a second driven by a motility system based This was the active as a monomer with a single type of myosin, which exhibits nota- on actin filaments associated with ATPase region. These features gener- first hint that ble differences from the classic form the plasma membrane. Using the ated much scepticism within the in its molecular organization and its knowledge of the enzymatic proper- there may be community: was myosin-I simply effects on actin filaments. ties of the muscle myosin, they set more myosins a catalytic degradation product of out to isolate an actin-dependent than those a traditional myosin more like the motor from Acanthamoeba castel- muscle form? Thirteen years later, lanii biochemically. They purified an found in muscle. Hammer and colleagues cloned the ATPase with properties that had pre- Kathleen Trybus gene that encodes myosin-I, provid- viously been associated with muscle ing definitive evidence for the exist- myosin: its activity in non-physio- ence of this unusual myosin. logical high-salt concentrations was Following the bloom of sequenc- increased by potassium and inhib- ing analysis, research in the field ited by magnesium (Mg), whereas flourished and many unconventional the physiological Mg-ATPase was myosins were identified, each with activated by actin. They purified distinctive molecular properties. two light chains associated with the Dimeric myosins do not necessarily novel enzyme, as well as the first form filaments as muscle myosin does, cofactor that enhances the ATPase and some others, like myosin-VI, activity of a myosin, which was move in the opposite direction along later identified as the p21-activated the actin filaments. Unconventional S10 | DECEMBER 2008
  7. 7. MILESTONES M I L E S TO N E 1 0 NEIL SMITH …showed that cytokinesis Sharing the depends on a myosin-powered actin’ limelight contraction of a ring of actin We take for granted our ability to see various filaments. components of the cytoskeletal network; however, back in 1969, the presence of actin Tom Pollard in cells other than muscle cells came as a surprise. The ability of heavy meromyosin (HMM) to ORIGINAL RESEARCH PAPERS Schroeder, T. E. bind to isolated actin filaments and form The contractile ring. II. Determining its brief arrowhead complexes was shown by Huxley in existence, volumetric changes, and vital role in cleaving Arbacia eggs. J. Cell. Biol. 53, 419–434 1963. Ishikawa, Bishoff and Holtzer used this (1972) Schroeder, T. E. Actin in dividing cells. information to investigate whether intermediate Contractile ring filaments bind heavy filaments were related to actin filaments in meromyosin. Proc. Natl Acad. Sci. USA 70, 1688–1692 (1973) Fujiwara, K. & Pollard, T. D. sections of myotubes. Their electron micrographs Fluorescent antibody localization of myosin in showed that these filaments did not bind HMM, the cytoplasm, cleavage furrow and mitotic and so were unlikely to contain actin. However, spindle of human cells. J. Cell. Biol. 71, 848–875 (1976) Mabuchi, I. & Okuno, M. The effect of they noticed that fibroblasts, which were also myosin antibody on the division of starfish present, seemed to have a filamentous network in blastomeres. J. Cell. Biol. 74, 251–263 (1977) FURTHER READING Rappaport, R. which arrowhead-complex formation was evident. Experiments concerning the cleavage They verified this finding in chondrocytic cells and stimulus in sand dollar eggs. J. Exp. Zool. 148, reasoned that these ‘mesenchymal-like cells’ 81–89 (1961) might require such filaments for amoeboid movement. However, the addition of HMM to epithelial cell preparations confirmed a similar network. Therefore, actin was not restricted to myosins turned out to be involved in contractile or motile cells. cytoskeleton that could be effectively viewed a wide range of functions that extend Visualization of the spatial arrangement of the using scanning or high-voltage electron well beyond those envisaged when actin network was achieved in 1974, thanks to microscopy. This made possible the detailed Pollard and Korn embarked on their Lazarides and Weber. They purified actin from analysis of the cytoskeleton in specific areas of search for the motor responsible for mouse fibroblasts and used it to raise an antibody. the cell, and furthered our understanding cell motility. Indirect immunofluorescence revealed the now of its importance in all aspects of cellular Nathalie Le Bot, Associate Editor, famous actin stress-fibre network that is common function. Nature Cell Biology to cells in tissue culture, as well as several of what Nicola McCarthy, Chief Editor, ORIGINAL RESEARCH PAPERS Pollard, T. D. & the authors termed ‘focal points’ where actin fibres Nature Reviews Cancer Korn, E. D. Acanthamoeba myosin. I. Isolation from converge. Although indirect immunofluorescence Acanthamoeba castellanii of an enzyme similar to muscle myosin. J. Biol. Chem. 248, 4682–4690 (1973) had already been used to show the localization of ORIGINAL RESEARCH PAPERS Ishikawa, H., Bischoff, R. & | Pollard, T. D. & Korn, E. D. Acanthamoeba myosin. II. myosin and troponin, this paper demonstrated Holtzer, H. Formation of arrowhead complexes with heavy mero- Interaction with actin and with a new cofactor the ease with which it is possible to visualize the myosin in a variety of cell types. J. Cell Biol. 43, 312–328 (1969) | protein required for actin activation of Mg2+ Lazarides, E. & Weber, K. Actin antibody: the specific visualization adenosine triphosphatase activity. J. Biol. Chem. actin network. of actin filaments in non-muscle cells. Proc. Natl Acad. Sci. USA 248, 4691–4697 (1973) Examination of the intact cytoskeleton at the 71, 2268–2272 (1974) | Heuser, J. E. & Kirschner, M. W. Filament FURTHER READING Hammer, J. A., Jung, G. & electron microscopic level remained difficult organization revealed in platinum replicas of freeze-dried Korn, E. D. Genetic evidence that Acanthamoeba cytoskeletons. J. Cell Biol. 86, 212–234 (1980) myosin I is a true myosin. Proc. Natl Acad. Sci. USA owing to disruption of the network by chemical FURTHER READING Fuller, G. M., Brinkley, B. R. & Boughter, J. M. 83, 4655–4659 (1986) | Fukui, Y., Lynch, T. J., fixation; however, in 1980, Heuser and Kirschner Immunofluorescence of mitotic spindles by using monospecific Brzeska, H. & Korn, E. D. Myosin I is located at the leading edges of locomoting Dictyostelium — through the use of rapid freezing in liquid antibody against bovine brain tubulin. Science 187, 948–950 (1975) | Weber, K., Bibring, T. & Osborn, M. Specific visualization of amoebae. Nature 341, 328–331 (1989) | Johnston, helium, freeze drying and rotary platinum–carbon tubulin-containing structures in tissue culture cells by G. C., Prendergast, J. A. & Singer, R. A. The coating — produced an exact replica of the immunofluorescence. Cytoplasmic microtubules, vinblastine- Saccharomyces cerevisiae MYO2 gene encodes induced paracrystals, and mitotic figures. Exp. Cell Res. 95, an essential myosin for vectorial transport of 111–120 (1975) | Euteneuer, U. & McIntosh, J. R. Structural polarity vesicles. J. Cell Biol. 113, 539–551 (1991) | of kinetochore microtubules in PtK1 cells. J. Cell Biol. 89, 338–345 Espindola, F. S. et al. Biochemical and (1981) | Small, J. V. Organization of actin in the leading edge of immunological characterization of p190– calmodulin complex from vertebrate brain: a These papers gave the textbook cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks. J. Cell Biol. 91, novel calmodulin-binding myosin. J. Cell Biol. 118, view of the cell cytoskeleton. 695–705 (1981) | Svitkina, T. M., Verkhovsky, A. B. & Borisy, G. G. 359–368 (1992) | Wells, A. L. et al. Myosin VI is an Plectin sidearms mediate interaction of intermediate filaments actin-based motor that moves backwards. Nature Jonathon Howard with microtubules and other components of the cytoskeleton. 401, 505–508 (1999) J. Cell Biol. 135, 991–1007 (1996) NATURE MILESTONES | CYTOSKELETON DECEMBER 2008 | S11
  8. 8. MILESTONES behave similarly to unlabelled actin M I L E S TO N E 1 1 both in vitro and in vivo — it polym- Live actin brightens up erized normally, activated myosin ATPase and was incorporated into contracted pellets in motile cell extracts to the same extent as endog- enous actin. Furthermore, the ability of labelled actin to form filamentous cells has become routine. However, bundles when microinjected into the until the late 1970s, experimental slime mold Physarum polycephalum designs relied mostly on static tools demonstrated that, besides retaining such as electron microscopy and its biological activity, the modi- immunofluorescence techniques, fied actin could be incorporated Live fish fibroblast that expresses cyan which were inappropriate to analyse into normal structures. Another fluorescent protein dynamic processes. important contribution of the work (CFP)-fascin (blue), yellow fluorescent Cell biology drastically changed by Taylor and Wang consisted protein (YFP)-actin with the development of ‘molecular of defining the controls that are (green) and cytochemistry’, whereby purified necessary for the use of fluorescent mCherry-paxillin (red). Courtesy of M. cellular components are covalently analogues; these include comparing Nemethova and V. labelled with fluorescent probes and, the biochemical activity, subcellular Small, Austrian Academy of Sciences after being tested for their function localization and in vivo stability of (IMBA) Austria. in vitro, are reintroduced into living the analogue with the properties of cells. This experimental approach native molecule, all of which were Are you not eager to see how your was first introduced by Taylor considered when developing mod- favourite molecule behaves in a and Wang, who used a reactive ern probes such as GFP. cell? With the current microscopy fluorescent dye — 5-iodoacetamido- These experiments opened up technologies and the availability of fluorescein (IAF) — to label purified the study of cytoskeleton dynamics, fluorescent probes such as green actin, and then directly microin- leading to exciting findings, such as fluorescent protein (GFP), the jected the actin derivative into cells. actin treadmilling — the continuous visualization of molecules in living IAF-labelled actin was shown to removal of actin monomers from M I L E S TO N E 1 2 First (focal) contact The attachment of cells to the protein -actinin localized at actin extracellular matrix (ECM) underpins filament termini. A few years later, activities from embryogenesis to Geiger et al. found that the cytoskeletal tumorigenesis. In the late 1970s and protein vinculin co-localized with early 1980s, advances in microscopy -actinin at focal adhesions, indicating allowed researchers to visualize the the importance of these two proteins focal adhesions that link cellular in attaching actin filaments to the ECM. actin microfilaments to the ECM (see A link between intercellular proteins Milestone 2). The initial identification and the ECM was identified that of focal contact proteins, and early same year, when Hynes and Destree insights into cell–substratum detected the ECM protein fibronectin attachment, laid the groundwork for at actin microfilament termini. This was continuing studies into cell adhesion later confirmed by electron microscopy and migration. (see Further reading). However, it In 1978, Heath and Dunn provided the was the work of Horwitz et al. in 1986 first evidence that actin microfilament that elucidated how cytoplasmic bundles terminated at a focal contact actin fibres made contact with Paxillin (green), a marker of adhesions, and the with the ECM. The identification of extracellular fibronectin. They found myosin regulatory light chain (red) in a chinese focal adhesion proteins proceeded that cell-surface receptors known as hamster ovary cell; note the actomyosin concurrently, as Lazarides and Burridge filaments that link adhesions. Image courtesy of integrins, which had previously been M. Vicente-Manzanares and A. F. Horwitz, reported in 1975 that the cytoskeletal shown to bind to fibronectin, also University of Virginia, USA. S12 | DECEMBER 2008
  9. 9. MILESTONES the pointed ends of filaments and M I L E S TO N E 1 3 their reincorporation at barbed ends — at the leading edge of cells and These papers microtubules exhibiting dynamic instability in vivo. However, the opened up the study of the I can see clearly now use of different fluorescent probes dynamics of combined with later advances in microscopy proved molecular cyto- cytoskeletal chemistry to be a tool that is more proteins in living generally applicable to the analysis cells. of different structures and processes Gregg Gundersen in living cells. Importantly, this also led to the development of additional techniques to measure protein dynamics. Kim Baumann, Online Editor, Cell Migration Gateway ORIGINAL RESEARCH PAPERS Taylor, D. L. & An absolutely critical technological Wang, Y.-L. Molecular cytochemistry: advance for the field. This technology incorporation of fluorescently labeled actin into allowed investigators to clearly resolve living cells. Proc. Natl Acad. Sci. USA 75, 857–861 (1978) | Taylor, D. L. & Wang, Y.-L. Fluorescently and record movements of organelles and labelled molecules as probes of the structure and visualize microtubules. Margaret Titus function of living cells. Nature 284, 405–410 (1980) FURTHER READING Wang, Y.-L. Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling. J. Cell Biol. 101, 597–602 (1985) | Sammak, P. J. & Borisy, G. G. In the early 1980s, the decreasing cost of video cameras, tape recorders and Direct observation of microtubule dynamics in other ‘consumer electronics’ meant that it was possible for cell biologists to living cells. Nature 332, 724–726 (1988) | Axelrod, D. & Omann, G. M. Combinatorial microscopy. incorporate electronic equipment into their daily experiments. Nature Rev. Mol. Cell Biol. 7, 944–952 (2006) In 1981, Inoué and Allen et al. separately reported the successful pairing of a video camera with a microscope. These two papers revolutionized the field of cell biology, because they made it possible to ‘watch’ microscopic cellular events for extended periods of time. In addition, it was possible to ‘freeze’ a frame of the movie, giving the biologist a ‘snapshot’ of a cellular event, and to enhance the analogue video signal electronically, yielding an image that effectively had a interacted with the cytoskeletal protein talin, which in turn bound higher contrast than images obtained through the use of a camera. vinculin. These observations Benny Geiger and The Inoué paper included images of a broad range of biological events, and provided the first model of the Keith Burridge he showed that it was possible to use differential interference contrast (DIC) focal adhesion as a multiprotein microscopy to film a sea cucumber spermatozoan extending its acrosomal launched the complex in which integrins link process. Inoué probed the kinetics of this biological event, during which the molecular era of acrosomal process can become up to 90 m long in <10 seconds. Allen et al. actin-associated cytoplasmic proteins to the ECM. focal adhesion described a new method — called Allen video-enhanced contrast (AVEC)–DIC Emily J. Chenette, Associate Editor, research. — that they used to examine transport along microtubules in a foraminifer; UCSD–Nature Signaling Gateway Rick Horwitz their images showed that cytoplasmic organelles were able to move along the microtubules in either direction and that they stopped moving if they ‘fell off’ the microtubule. These two papers paved the way for further studies that used AVEC–DIC ORIGINAL RESEARCH PAPERS Lazarides, E. & Burridge, K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell microscopy, for example, in applications ranging from observing fast axonal 6, 289–298 (1975) | Heath, J. P. & Dunn, G. A. Cell to substratum contacts of chick transport in the giant axon of a squid to monitoring microtubule dynamics in fibroblasts and their relation to the microfilament system. A correlated interference- the newt lung epithelium, which conclusively showed that dynamic instability reflexion and high-voltage electron-microscope study. J. Cell Sci. 29, 197–212 (1978) | Hynes, R. O. & Destree, A. T. Relationships between fibronectin (LETS protein) and occurred in living cells (see Further reading). In addition, DIC microscopy was actin. Cell 15, 875–886 (1978) | Geiger, B. et al. Vinculin, an intracellular protein instrumental to the discovery of kinesin (see Milestone 15). localized at specialized sites where microfilament bundles terminate at cell membranes. Proc. Natl Acad. Sci. USA 77, 4127–4131 (1980) | Horwitz, A. et al. Joshua M. Finkelstein, Senior Editor, Nature Interaction of plasma membrane fibronectin receptor with talin—a transmembrane linkage. Nature 320, 531–533 (1986) ORIGINAL RESEARCH PAPERS Allen, R. D., Allen, N. S. & Travis, J. L. Video-enhanced contrast, differential FURTHER READING Geiger, B. A 130K protein from chicken gizzard: its localization at interference contrast (AVEC–DIC) microscopy: a new method capable of analyzing microtubule-related the termini of microfilament bundles in cultured chicken cells. Cell 18, 193–205 (1979) | motility in the reticulopodial network of Allogromia laticollaris. Cell Motil. 1, 291–302 (1981) | Inoué, S. Video Singer, I. I. The fibronexus: a transmembrane association of fibronectin-containing image processing greatly enhances contrast, quality, and speed in polarization-based microscopy. J. Cell Biol. fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell 16, 89, 346–356 (1981) 675–685 (1979) | Burridge, K. & Connell, L. Talin: a cytoskeletal component FURTHER READING Allen, R. D., Metuzals, J., Tasaki, I., Brady, S. T. & Gilbert, S. P. Fast axonal transport in concentrated in adhesion plaques and other sites of actin-membrane interaction. squid giant axon. Science 218, 1127–1129 (1982) | Brady, S. T., Lasek, R. J. & Allen, R. D. Fast axonal transport in Cell Motil. 3, 405–417 (1983) extruded axoplasm from squid giant axon. Science 218, 1129–1131 (1982) | Cassimeris, L., Pryer, N. K. & Salmon, E. D. Real-time observations of microtubule dynamic instability in living cells. J. Cell Biol. 107, 2223–2231 (1988) NATURE MILESTONES | CYTOSKELETON DECEMBER 2008 | S13
  10. 10. MILESTONES M I L E S TO N E 1 4 Key instability Following the description by Inoue came when Mitchison and Kirschner with the latter depolymerizing rapidly Fluorescently-labelled of the dynamic nature of spindle combined biochemical assays with to provide new subunits for growth. microtubules growing and shrinking in Xenopus laevis egg microtubules (see Milestone 5), microscopy to uncover the coexistence Given that long microtubules eventually extracts. Image courtesy of researchers in the field actively of growing and shrinking microtubules disappeared, they concluded that N. Le Bot. sought to understand how tubulin in vitro, in a state of ‘dynamic instability’. transitions between polymerization and polymerization could produce this Previous studies had inferred the depolymerization were probably rare. behaviour. It was initially thought behaviour of individual microtubules The authors coined the term ‘dynamic that microtubule polymerization by from the biochemical properties instability’ to describe these properties GTP-linked tubulin subunits followed a of the bulk polymer. Mitchison and of microtubule polymerization. treadmilling model, according to which Kirschner used microtubule seeds The main differences between the microtubule length would result from incubated in solutions of various dynamic instability and treadmilling GTP-tubulin being added to one end tubulin concentrations and, in addition models are the transitions from and GDP-tubulin dissociating from the to assessing these properties, they growth to shrinkage (catastrophe) or other. However, it was possible to test visualized the microtubules at fixed from shrinkage to growth (rescue) at this model only after Weisenberg and time points using microscopy. They the same end of the microtubule. To This work was Borisy achieved efficient microtubule observed that although the total explain these transitions, Mitchison and polymerization in vitro in 1972 (see polymer mass reached a plateau and Kirschner postulated that GTP-bound an instant Milestone 5). This assay provided a remained constant, the microtubule tubulin subunits were added during classic, defining starting point for the purification of population did not consist of a fixed polymerization and that GTP then the dynamic microtubule-associated proteins, which number of microtubules of the hydrolysed to GDP, resulting in the properties of were later shown to influence polymer same length. Instead, the number of presence of a GTP-tubulin cap at the dynamics and organization, and for the microtubules decreased with time end of a growing microtubule. They microtubules. isolation of molecular motors (see also and their mean length increased. This surmised that this cap was more stable Erika Holzbaur Milestone 15). But, the key insight into demonstrated the coexistence of than the GDP-tubulin lattice; therefore, microtubule polymerization properties growing and shrinking microtubules, at low concentrations of GTP-tubulin M I L E S TO N E 1 5 Brand new motor In the early 1980s, the molecular video and electron microscopy, they mechanisms for directed transport of succeeded in identifying these fila- organelles remained elusive. Of par- ments as single microtubules. Vale ticular interest was the process of fast and colleagues also observed that, axonal transport, in which organelles like organelles, small plastic beads STOCKBYTE are actively and rapidly transported coated with axonal cytosol could in both anterograde and retrograde move along microtubules and that Using squid axons, Vale and col- directions in neuronal axons. In 1985, glass coverslips coated with the leagues isolated the motor protein an amazing series of articles culmi- same cytosol supported microtubule … laid the by using AMP-PNP to stabilize the nated in the identification of kinesin gliding across the surface — both of ground work for motor–microtubule interaction as as the molecular motor responsible for which are assays for motor activity a first-affinity purification step, fol- the fast axonal transport of organelles that remain in common use today. people to start lowed by column chromatography. along microtubules. Raymond Lasek and Brady then dem- thinking in terms During the purification, they assayed Ron Vale and colleagues adopted onstrated that the non-hydrolysable of ‘many motors’. the fractions for motor activity using an assay for the real-time observa- ATP analogue - -imidoadenosine William Bement microscopy-based in vitro assays. The tion of fast axonal transport in vitro 5 -triphosphate (AMP-PNP) led to purified protein that powered motility that was developed by Robert Allen the stabilization of microtubule– in vitro contained polypeptides of and Scott Brady (see Milestone 13), organelle complexes, indicating 110–120 kDa and 60–70 kDa, which and showed that organelle transport that the ATPase responsible for fast were much smaller than the main occurred in an ATP-dependent axonal transport could be locked onto polypeptide of dynein — the only manner and at a uniform rate along microtubules, thereby providing a other microtubule-dependent motor the isolated filaments. By combining useful tool for purification. known at the time. Hence, a new S14 | DECEMBER 2008