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GREAT EXPERIMENTS
Dynamic instability of microtubules

Marc Kirschner and Tim Mitchison

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Background

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The cytoskeleton as a concept was in a sense created by the development of antibodies to tropomyosin (3274), tubulin (3275; 3276), and actin (3277) in the early 1970s. Immunofluorescence staining using these antibodies revealed a striking pattern of filaments that spanned long distances within the cell and were organized into networks that extended throughout the cytoplasm. The organization of filaments was highly variable from cell to cell, but was closely associated with the individual cell morphologies. These properties made the filaments good candidates for organizing cell shape and behavior. Before that time, the possibility of global organizing structures common to all types of cells had not been generally appreciated. Cell structure had been in the hands of cytologists, who had had no way of visualizing individual types of filaments, and electron microscopists, who had been unable to appreciate the global organization of cellular anatomy through reconstruction of thin sections.

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With the discovery that simple polymers of actin and tubulin underlay cell morphology, biochemical approaches to the problem became tractable. With them, several profound questions needed to be addressed. What were the organizing principles that determined the global placement of cytoskeletal polymers in the cell? How could the same simple polymers be employed to form structures as different as the axon and the mitotic spindle? How could the dynamic structure of a dividing cell or a moving cell be explained? How could cells of very different sizes and shapes reliably carry out tasks like mitosis or cell movement? It was a fair bet even then that the answers to these questions would involve extremely complex networks of interactions among many different types of proteins, each capable of acting in multiple ways. Although this was a safe conclusion it was self defeating to focus initially on the complexity of the interactions that must be involved. A more optimistic approach was to try to reconstruct revealing properties of the system from its central components, the molecules which form the polymers.

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Marc Kirschner had started his career studying the structure and dynamics of allosteric enzymes; when he finished his degree in 1971, he sought a more challenging problem. Although the concept of the cytoskeleton was still unappreciated at the time, the importance of some of its components, such as microtubules, was coming into focus. Microtubules had long been known as components of the mitotic spindle, and electron microscopy had demonstrated their presence in nonmitotic cells as well. As the nature of the cytoskeleton became clearer over the next decade, the questions that would emerge about tubulin and other cytoskeletal polymers were how they assembled and how they related to cell organization. In John Gerhart's lab at Berkeley, Kirschner started with the question of how microbutules polymerize, with the assumption that the larger questions were indeed very difficult, perhaps beyond biochemical analysis. At that time tubulin was known as a protein that bound the antimitotic drug colchicine, and was thus considered the best candidate for the basic subunit of microtubules. However, no one had been able to assemble it into microtubules and there was even some doubt developing that it played that role. Kirschner's experimental approach was to try to reconstruct the assembly of tubulin by microinjection of radioactively labeled tubulin into frog eggs, monitoring its incorporation into the mitotic spindle. On arriving at Princeton a year later in his first independent position, he was confronted with Dick Weisenberg's reconstitution of microtubule assembly from crude brain extracts (3278) and, in quick order, Gary Borisy's elegant (but ultimately completely erroneous) pathway of assembly (3279), Mike Shelanski's purification of active tubulin (3280), and Weisenberg's demonstration of nucleated assembly in clam extracts (3281). To a young assistant professor, it seemed that the leaders in the field had made major advances on the problem using straightforward biochemical approaches. Although frog eggs later would loom very large in the Kirschner lab for understanding both microtubule assembly and control of the cell cycle, the most immediate approach seemed to be to explore biochemical reconstitution — if there was anything left to do.

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One peculiarity of both tubulin and actin was that both seemed to require energy merely to assemble the polymer. Most protein complexes, even large structures like viruses and ribosomes, assemble without hydrolyzing GTP or ATP. A possible role of energy in the assembly of microtubules always intrigued Kirschner. Tubulin has two sites for GTP binding and initially there were several baroque pathways suggested involving enzymology at both sites; however, experiments by Bruce Spiegelman in Kirschner's lab conclusively proved that one of the GTPs was metabolically inert (3282). The other was hydrolyzed as a result of assembly, with exactly 1 mole of GTP being hydrolyzed to GDP for each mole of tubulin incorporated into microtubules (3283). Despite earlier reports that energy was required for assembly, Penningroth and Kirschner were able to demonstrate conclusively that nonhydrolyzable GTP analogs could promote assembly (3284); we now know that hydrolysis of GTP follows assembly and that the energy is later used to disassemble the polymer.

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Figure 1  
A treadmilling polymer constantly gains subunits at one end and loses them at the other. As a result the subunits move through the filament.

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The role of nucleotide hydrolysis in polymer formation was also receiving scrutiny in the actin field, where ATP hydrolysis accompanies actin assembly in a remarkable case of convergent evolution (3285). Albrecht Wegner had analyzed the role of hydrolysis in thermodynamic terms and demonstrated indirectly that ATP hydrolysis drove rapid exchange of subunits in and out of the polymer, which he interpreted as "treadmilling" (3286). As illustrated in Figure 1, treadmilling is a process where at steady state subunits would add to one end of an actin filament and come off the other end, the polymer remaining the same length. Later, the term treadmilling was used in a broader sense, to describe any situation where one end of a cytoskeleton polymer or network disassembles and the other assembles, independent of mechanism (3287). Here we use the term only in its original definition, where ATP or GTP hydrolysis within the polymer drives the process. Working with microtubules, Leslie Wilson had devised GTP exchange experiments that he also interpreted as indicating treadmilling (3288). This interpretation ultimately turned out to be incorrect, the results later clearly being demonstrated to be due to dynamic instability (3289). However, at the time, these experiments and the idea of treadmilling, though a flawed explanation, nevertheless served to help focus our attention on the dynamic properties of microtubules. But what was the purpose of these dynamics? What purpose would it serve to have subunits go on one end and come off the other end, with no change in polymer length?

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Two more features have to be considered before we confront the real role of energy in the morphogenesis of the cytoskeleton. Kirschner was asked to teach some lectures to a biophysics class at Berkeley in 1979 and chose the Wegner and Wilson papers. On thinking further about them, he realized that in a closed system like the cell treadmilling could ensure that only microtubules attached to the centrosome would persist, with any formed randomly in the cytoplasm being unstable and rapidly disappearing (3290), a prediction recently demonstrated experimentally in cells (3291). Kirschner also interested Terrill Hill in further studies of the theory of ATP and GTP hydrolysis and polymer assembly, which resulted in a paper relating polymer stability, kinetics, and the capacity of polymerizing and depolymerizing systems to do work (3292), now a revitalized subject as well (3293). Discussion and publications with Hill continued through the period of the discovery of dynamic instability. Thus, a more and more theoretically rigorous picture of the thermodynamics of assembly accompanied the novel experiments that were about to unfold. This environment of rigorous theory in the Kirschner lab was an essential ingredient in the discovery of dynamic instability. When Mitchison first observed microtubule behavior that appeared inconsistent with treadmilling theory, he realized not only its importance but also its potential to upset a large apple cart. Finally, in Kirschner's mind it seemed time to look at microtubule assembly in a more natural context, nucleation of microtubules from centrosomes. It had been known since the days of late nineteenth century histologists that mitotic spindle fibers emanated from centrosomes, and most nonmitotic cells also had microtubule arrays organized by centrosomes. It seemed clear that it would be important to determine what effect they had on microtubule dynamics. Work in the Kirschner lab had already shown that in vitro preparations of active centrioles could be produced, setting the stage for what was initially intended to be a biochemical study of the role of centrosomes in microtubule assembly.

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The experiment

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Tim Mitchison joined the Kirschner lab as a graduate student in 1982. He focused on the biochemistry of nucleation of microtubules and the purification of active centrosomes from mammalian cells. This was a difficult task for a young graduate student. The centrosome is embedded deep in vesicular and cytoskeletal elements, and there is no obvious criterion of purification. In addition, the assays are difficult and nonquantitative. However, after about 2 years, Mitchison had developed a good purification for crude centrosomes that was to be the basis for the discovery of dynamic instability. Given that his partially purified centrosome preparation was achieved just before his qualifying examination, the examining committee asked Mitchison how he planned to follow up this encouraging work. Experiments could have proceeded in many directions. One examiner stressed that the centrosome with its pericentriolar material was a structural enigma, and the purified material could be used as an object of state-of-the-art image reconstruction. Another pointed out that functional assays in vitro were hard to relate to in vivo function, so the work might be better pursued in a genetically tractable system. Another argued that the most obvious direction at the time was to attempt a biochemical description of the centrosome. However, preliminary biochemical investigation of centrosomal proteins and RNA revealed how difficult these directions would be. Indeed, they are only now gearing up seriously. Instead, Mitchison argued for in vitro experiments to ask how centrosomes affected the polymerization of tubulin. Although his committee passed him, every member but one came to Kirschner afterward to protest Mitchison's answer to that important question about future directions and to say that it brought them close to failing him.

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Figure 2  
Purified centrosomes were capable of nucleating large numbers of microtubules. The resulting arrays (often called "asters") were collected by sedimentation and viewed either by immunofluorescence with an antibody against tubulin (left) or by electron microscopy (right). In both cases individual microtubules could be distinguished. Reprinted from (3294) by permission from Nature (http://www.nature.com/nature).

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The first type of experiments (3294) is shown in Figure 2. They involved incubating partially purified centrosomes with pure tubulin and GTP and measuring the number and length of microtubules nucleated from each centrosome by electron microscopy or immunofluorescence. Significantly, the assays we used allowed us to look at individual microtubules rather than at a bulk measure of polymerization (such as the turbidity of the solution). The quantitative results were at first glance unremarkable. Both the number of microtubules nucleated per centrosome and their growth rate increased as we increased the tubulin concentration. This monotonic relationship might have gone without further examination, but three aspects bothered us. First, why did the number of microtubules increase over a very broad concentration range? Did this imply that the nucleation sites within the centrosomes were heterogeneous and, if so, why? Second, at low tubulin concentrations the lengths of the nucleated microtubules were heterogeneous, as if nucleation were a slow and stochastic process. We expected the opposite, that the centrosome, as a nucleation site, would facilitate microtubule growth so that nucleation would be extremely rapid compared to the rate of assembly. Hence all microtubules should have been the same length. (There would have been some statistical variation, but for microtubules 10 microns long the standard deviation should have been less than one percent.) However, most peculiar was that some microtubules were assembled even below the minimum tubulin concentration that treadmilling predicted would be required for nucleated microtubules to grow. When we looked closely, these "unremarkable" findings challenged every idea we had about how polymers grow.

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Figure 3  
After microtubules were grown off centrosomes at high tubulin concentration, the tubulin concentration was lowered and the number and length of the remaining microtubules was determined. Reprinted from (3294) by permission from Nature (http://www.nature.com/nature).

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A simple test of the idea that heterogeneous growth at low concentrations was due to slow and stochastic nucleation would be to grow microtubules at high tubulin concentration, where they all grew uniformly from the centrosome, and then reduce the tubulin concentration by diluting them. The expectation would be that, since they had already been nucleated, the further growth of each microtubule would be the same, governed by the concentration of tubulin. Figure 3 shows that, instead, dilution reproduced the kind of length heterogeneity seen in the initial growth experiments at low tubulin concentrations. Apparently, on dilution some microtubules continued to grow, while others shrank quickly and disappeared. We realized that this result was impossible on the basis of all outstanding ideas of polymer growth. Simple polymers, uncomplicated by nucleotide hydrolysis, must all grow or shrink at the same time since their ends must be in equilibrium with free subunits. Even were tubulin to treadmill, the free ends of all nucleated microtubules would have to behave equivalently. Though it was possible that the centrosome could nucleate more than one type of microtubule, this seemed very improbable. More likely was the possibility that microtubules could exist in two different and slowly interconvertible states with different properties, allowing one to grow and the other to shrink under the same conditions. The proportion in each state would depend on the concentration of tubulin, as would the frequency of transitions between growth and shrinking.

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Figure 4  
Pure tubulin was polymerized to the point at which the total amount within microtubules did not change with time. From that point on, the number of microtubules per volume and their mean length were determined by sedimenting them onto coverslips and visualizing them by immunofluorescence. Fields from samples taken at the times indicated are shown at the bottom. In order to facilitate the analysis, the microtubules were sheared at the point indicated by an open triangle in the uppermost graph. Reprinted from (3295) by permission from Nature (http://www.nature.com/nature).

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A more rigorous test of whether we were observing an inherent property of microtubules or a property conferred on them by centrosomes came when we examined the properties of pure tubulin. Figure 4 illustrates the experiment. We polymerized pure tubulin in the absence of centrosomes and again followed the reaction by using immunofluoresence to visualize the individual microtubules after we had sedimented them onto cover slips. We still found that microtubules existed in two slowly interconverting populations, one that grew slowly and persistently and the other that shrank rapidly (3295). This had been overlooked up to that time because no one in the microtubule field had previously looked carefully at the behavior of the individual microtubules within a population. Instead, they had monitored microtubule polymerization by methods, such as light scattering, sedimentation, or viscometry, which measure properties of the population as a whole and are much less sensitive to changes in the lengths of the microtubules. Using these bulk techniques, they found that polymerization invariably started with a lag phase, went through an exponential phase, and finished with a plateau, where polymer was thought to be at equilibrium. But when we looked at the individual microtubules it was clear that the "equilibrium" or plateau phase was not even a steady state. In this pseudo-plateau, where the total amount of polymer was constant, most of the polymers grew slowly while a minority depolymerized rapidly, generating subunits that added on to the growing ones.

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Figure 5  
Microtubules grow or shrink depending on the condition of their ends. Conversion between the two states is infrequent enough so that a microtubule can grow or shrink across large distances within a cell.

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We also addressed the potential mechanism of these novel polymerization kinetics (3295). Marie-France Carlier and Dominique Pantaloni, along with Terrell Hill, were already looking at the effect of the heterogeneity of subunits that occurs in an actin polymer when actin is polymerized rapidly (3296). This heterogeneity is the result of actin's ATPase activity and its stimulation as the result of the assembly of actin; when hydrolysis lags behind assembly, there should be a length of ATP actin subunits capping the end of the polymer, while the interior should contain ADP actin. This produced a situation in which the reactions which assembled and disassembled an actin filament were not the reverse of one another. The polymerization phase should reflect ATP actin binding to the ATP actin end, while in the shrinking phase the ADP actin end would be exposed and ADP actin subunits would be dissociating. Because of the energy released by the ATP hydrolysis, the rates of these two reactions would not have to be related as would be the association and dissociation reactions of a simple equilibrium binding reaction. Among other possibilities was that the dissociation reaction could be much faster than it would otherwise be. Indeed, Carlier, Pantoloni, and Hill observed for actin, as we did for tubulin, a huge discontinuity between the expected depolymerization rate (based upon extrapolation of the rates of polymer growth) and the actual depolymerization rate from the diphosphate ends. This led us to postulate that the unusual kinetics of microtubules were due to two populations of polymers, some that were growing with a cap of GTP-containing tubulin and others that had lost the cap and were simply depolymerizing with a GDP end exposed. Interconversion, which was later termed "catastrophe" for the loss of the cap and "rescue" for regaining of the cap, would depend on several conditions including the concentration of tubulin. We called this process of using GTP energy to create two slowly interconverting phases of growth and shrinkage at steady state "dynamic instability." The critical properties that create dynamic instability are shown in Figure 5.

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Figure 6  
Growing and shrinking microtubules explore in all directions within a cell. Microtubules which happen to grow in a particular direction encounter a specialized structure and are selectively stabilized, polarizing the cell's microtubule array. The stabilized microtubules can be used to localize activities, such as the delivery of materials required to expand the cell.

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These experiments suggested a purpose for dynamic instability that transcended the mere assembly of the polymer. What this behavior made possible is shown in Figure 6. In the cell, the nucleating center would be the source of microtubules that would grow very rapidly in random directions, many reaching all the way to the periphery of the cell before randomly transiting to the depolymerizing phase and shrinking all the way back to the centrosome, to be quickly replaced by other microtubules polymerized in unrelated directions. GTP hydrolysis was required for this process and kept the individual microtubules very dynamic, while maintaining a high steady state level of polymer. As a result, microtublules would constantly explore the interior of the cell and could be stabilized by interactions at their ends. Hence, morphogenesis could occur by distal interactions that would selectively stabilize a few microtubules from a large variable population. Selective stabilization could eventually transform the randomly polymerized array into a new polarized configuration. Hence, energy was required for the formation of an exploratory array of microtubules, easily adaptable to new conditions and modifiable for new tasks.

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Subsequent work

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Support and elaboration of the basic idea of dynamic instability came quickly in some areas, slowly in others; many elements of the process are still not understood. Within little over a year after we put the idea forward, Hotani demonstrated by direct observation using darkfield microscopy that microtubules in vitro undergo excursions of growth, shrinkage, catastrophe, and rescue as predicted by the model (3297). Eric Schulze in the Kirschner lab showed that biotin-labeled tubulin injected into fibroblasts rapidly assembled onto the ends of a subset of microtubules, and that microtubules attached to the centrosome were replaced one at a time, as expected for a process of growth, intermittent catastrophic disassembly back to the centrosome and repolymerization (3298). Soon real-time observations of fluorescently labeled microtubules in cells demonstrated dynamic instability directly (3299; 3300; 3301). Dynamic instability became the obvious model to account for rapid microtubule turnover in the mitotic spindle observed by McIntosh and Salmon (3302). Although McIntosh first wondered if the original results were a photobleaching artifact, that was not the case. We were able to infer that the fast turnover of spindle microtubules was due to dynamic instability by pulse labeling cells with biotin-labeled tubulin and observing the growth of microtubules by electron microscopy (3303). Due to the high density of microtubules in the structure, dynamic instability has still not been observed directly in the mitotic spindle, but individual microtubules in mitotic extracts and cells show the requisite fast turnover rate.

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While the phenomenon of dynamic instability was relatively quickly verified, the underlying molecular changes in tubulin that allow it, and the other components that supported its biological role in morphogenesis, took much longer. David Drechsel in the Kirschner lab showed that the microtubules could be stabilized by a very small cap of GTP subunits (3304). The true size of the GTP cap has yet to be measured, but many now suspect that hydrolysis occurs shortly after the exchangeable GTP binding site is buried in the lattice. Salmon's group tested the prediction that exposing GDP subunits should cause immediate rapid depolymerization by severing microtubules with a laser beam, exposing the internal subunits; both new ends quickly depolymerized, as predicted (3305). Most recently, Tony Hyman found a structural difference between the GTP and GDP microtubule lattices, which might explain the rapid shrinking of microtubules with exposed GDP ends (3306). Yet, though a structure of tubulin exists at the atomic level, there is still no clear picture as to how GTP hydrolysis destabilizes the polymer.

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What has been lacking for a long time is proof of components that stabilize the polymer from the free end, as required for a model of cell morphogenesis by selective stabilization of microtubules. In vivo growth experiments with tagged tubulin by Schulze, Evans, and Mitchison in the Kirschner laboratory confirmed that microtubules emerge from the centrosome in mitosis with no preferred orientation (3303). Yet ultimately the majority of microtubules focus on the central spindle, suggesting that the morphogenesis of the spindle is determined not by preferred direction of nucleation but by later stabilization. Consistent with this, we showed that isolated metaphase chromosomes stabilize the ends of microtubules (3307). Despite this demonstration of the validity of the model, the chemistry of stabilization remained primitive until recent experiments in budding yeast demonstrated that specific proteins docked near the bud neck stabilize microtubules and allow the mitotic spindle to be positioned correctly before division (3308).

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Many components that interact with microtubules have been identified since our 1984 papers on dynamic instability. There are fascinating proteins that bundle, sever, stabilize, and destabilize microtubules. A vast array of motor proteins play roles in sliding materials on microtubules and sliding microtubules against each other. One might have said that the study of microtubule dynamics in 1984 was premature because the entire cast of characters was unknown. Yet the core properties of tubulin itself form the basis of much of microtubule function. The dynamics of pure tubulin are very similar to the dynamics observed in vivo. Addition of only two other proteins in vitro restores quantitatively the in vivo dynamics (3309). Many of the other proteins that interact with microtubules perturb the basic assembly properties but do not radically change them.

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Dynamic instability has potential evolutionary implications. Like tubulin itself, this fundamental microtubule behavior has proven to be highly conserved. Nature has presumably conserved the core process of dynamic instability because it is able to support many diverse morphologies and physiologies. Such robust processes are very adaptable in evolution (3310; 3311).

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The authors

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Marc Kirschner (on the left) is the chairman of the Department of Cell Biology at the Harvard Medical School, where he is the Carl W. Walter Professor of Cell Biology. A native of Chicago and a graduate of Northwestern University, he received his Ph.D. from the University of California, Berkeley. Prior to moving to Harvard he was a professor at the University of California, San Francisco. Along with John Gerhart, he is the author of Cells, Embryos, and Evolution (Blackwell, 1997).

Dr. Kirschner is a Foreign Member of both the Royal Society of London and the Academia Europaea. He was the 2001 recipient of the William C. Rose Award, presented by the American Society for Biochemistry and Molecular Biology. He also received the 2001 International Award by the Gairdner Foundation of Toronto. He is a member of the National Academy of Sciences and the American Academy of Arts and Sciences. He has served on the Advisory Committee to the Director of the National Institutes of Health and is a Past-President of the American Society of Cell Biology.

The Kirschner laboratory investigates three diverse areas: regulation of the cell cycle, the role of the cytoskeleton in cell morphogenesis, and mechanisms of establishing the basic vertebrate body plan.


Marc W. Kirschner
Department of Cell Biology
Harvard Medical School
240 Longwood Avenue
Boston, MA 02115
E-mail: marc@hms.harvard.edu

Timothy J. Mitchison (Ph.D., F.R.S.) (on the right) is Professor of Cell Biology at the Harvard Medical School. He obtained his Ph.D. in 1984 from UCSF, where he worked with Marc Kirschner on the mechanism of microtubule polymerization dynamics. That work included the discovery of dynamic instability, and led to a lasting interest in cytoskeletal dynamics and the spatial organization of the cytoplasm. In 1987 he joined the faculty at UCSF as an assistant professor of pharmacology. His group started work on the dynamics of the cytoskeleton using a combination of biochemistry and imaging, with the long-term goal of understanding how protein polymerization dynamics are used as an organizing and force-producing principle by cells. His group has worked on these problems since, focusing on the mechanism of mitosis and later the mechanism by which an intracellular pathogen, Listeria, moves through the cytoplasm. Notable progress by his group includes the establishment of cell-free systems for mitosis (1991) and Listeria motility (1994), and discovery of a number of important mechanisms and protein factors involved in those processes. These include microtubule dynamics at kinetochores, microtubule depolymerizing proteins, the chromosome assembly factor condensin, and the arp2/3 complex as the factor that nucleates a Listeria actin tail. In 1997 he moved to the Harvard Medical School to establish and codirect, with Stuart Schreiber, the Institute of Chemistry and Cell Biology. That laboratory is dedicated to developing synthetic organic chemistry as a research approach in cell biology, with one goal being discovery of useful small molecule tools for cell biology research. Recent progress in the small molecule area includes discovery of useful inhibitors of motor proteins from both the kinesin and myosin families. He also maintains a summer research lab at Marine Biological Laboratory, Woods Hole, where he is pursuing the mechanochemistry of mitosis in collaboration with Ted Salmon.


Timothy J. Mitchison
Harvard Medical School
Institute of Chemistry and Cell Biology (ICCB)
SGM 604
250 Longwood Avenue
Boston, MA 02115
E-mail: timothy_mitchison@hms.havard.edu

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Last Revised on September 10, 2004

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reviews
  • 3285 Mitchison, T. J. (1995).  Evolution of a dynamic cytoskeleton.  Philos. Trans. R. Soc. Lond. B Biol. Sci. 349, 299-304.  PubMed  
  • 3310 Kirschner, M. and Gerhart, J. (1998).  Evolvability.  Proc. Natl. Acad. Sci. USA 95, 8420-8427.  PubMed   Journal
  • 3311 Kirschner, M., Gerhart, J., and Mitchison, T. (2000).  Molecular 'vitalism'.  Cell 100, 79-88.  PubMed   Journal
reviews
  • 3274 Jorgensen, A. O., Subrahmanyan, L., and Kalnins, V. I. (1975).  Localization of tropomyosin in mouse embryo fibroblasts.  Am. J. Anat. 142, 519-525.  PubMed  
  • 3275 Weber, K., Pollack, R., and Bibring, T. (1975).  Antibody against tuberlin: the specific visualization of cytoplasmic microtubules in tissue culture cells.  Proc. Natl. Acad. Sci. USA 72, 459-463.  PubMed  
  • 3276 Fuller, G. M., Brinkley, B. R., and Boughter, J. M. (1975).  Immunofluorescence of mitotic spindles by using monospecific antibody against bovine brain tubulin.  Science 187, 948-950.  PubMed  
  • 3277 Lazarides, E. and Weber, K. (1974).  Actin antibody: the specific visualization of actin filaments in non-muscle cells.  Proc. Natl. Acad. Sci. USA 71, 2268-2272.  PubMed  
  • 3278 Weisenberg, R. C. (1972).  Microtubule formation in vitro in solutions containing low calcium concentrations.  Science 177, 1104-1105.  PubMed  
  • 3279 Borisy, G. G. and Olmsted, J. B. (1972).  Nucleated assembly of microtubules in porcine brain extracts.  Science 177, 1196-1197.  PubMed  
  • 3280 Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973).  Microtubule assembly in the absence of added nucleotides.  Proc. Natl. Acad. Sci. USA 70, 765-768.  PubMed  
  • 3281 Weisenberg, R. C. and Rosenfeld, A. C. (1975).  in vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs.  J. Cell Biol. 64, 146-158.  PubMed  
  • 3282 Spiegelman, B. M., Penningroth, S. M., and Kirschner, M. W. (1977).  Turnover of tubulin and the N site GTP in Chinese hamster ovary cells.  Cell 12, 587-600.  PubMed   Journal
  • 3283 Carlier, M. F. and Pantaloni, D. (1981).  Kinetic analysis of guanosine 5'-triphosphate hydrolysis associated with tubulin polymerization.  Biochemistry 20, 1918-1924.  PubMed  
  • 3284 Penningroth, S. M. and Kirschner, M. W. (1977).  Nucleotide binding and phosphorylation in microtubule assembly in vitro.  J. Mol. Biol. 115, 643-673.  PubMed  
  • 3286 Wegner, A. (1976).  Head to tail polymerization of actin.  J. Mol. Biol. 108, 139-150.  PubMed  
  • 3287 Rodionov, V. I. and Borisy, G. G. (1997).  Microtubule treadmilling in vitro.  Science 275, 215-218.  PubMed   Journal
  • 3288 Margolis, R. L. and Wilson, L. (1978).  Opposite end assembly and disassembly of microtubules at steady state in vitro.  Cell 13, 1-8.  PubMed   Journal
  • 3289 Grego, S., Cantillana, V., and Salmon, E. D. (2001).  Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.  Biophys. J. 81, 66-78.  PubMed   Journal
  • 3290 Kirschner, M. W. (1980).  Implications of treadmilling for the stability and polarity of actin and tubulin polymers in vitro.  J. Cell Biol. 86, 330-334.  PubMed  
  • 3291 Rodionov, V., Nadezhdina, E., and Borisy, G. (1999).  Centrosomal control of microtubule dynamics.  Proc. Natl. Acad. Sci. USA 96, 115-120.  PubMed   Journal
  • 3292 Hill, T. L. and Kirschner, M. W. (1982).  Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work.  Proc. Natl. Acad. Sci. USA 79, 490-494.  PubMed  
  • 3293 van Oudenaarden, A. and Theriot, J. A. (1999).  Cooperative symmetry-breaking by actin polymerization in a model for cell motility.  Nat. Cell Biol. 1, 493-499.  PubMed   Journal
  • 3294 Mitchison, T. and Kirschner, M. (1984).  Microtubule assembly nucleated by isolated centrosomes.  Nature 312, 232-237.  PubMed  
  • 3295 Mitchison, T. and Kirschner, M. (1984).  Dynamic instability of microtubule growth.  Nature 312, 237-242.  PubMed  
  • 3296 Pantaloni, D., Hill, T. L., Carlier, M. F., and Korn, E. D. (1985).  A model for actin polymerization and the kinetic effects of ATP hydrolysis.  Proc. Natl. Acad. Sci. USA 82, 7207-7211.  PubMed  
  • 3297 Horio, T. and Hotani, H. (1986).  Visualization of the dynamic instability of individual microtubules by dark-field microscopy.  Nature 321, 605-607.  PubMed  
  • 3298 Schulze, E. and Kirschner, M. (1986).  Microtubule dynamics in interphase cells.  J. Cell Biol. 102, 1020-1031.  PubMed  
  • 3299 Schulze, E. and Kirschner, M. (1988).  New features of microtubule behaviour observed in vitro.  Nature 334, 356-359.  PubMed   Journal
  • 3300 Sammak, P. J., Gorbsky, G. J., and Borisy, G. G. (1987).  Microtubule dynamics in vitro: a test of mechanisms of turnover.  J. Cell Biol. 104, 395-405.  PubMed  
  • 3301 Cassimeris, L., Pryer, N. K., and Salmon, E. D. (1988).  Real-time observations of microtubule dynamic instability in living cells.  J. Cell Biol. 107, 2223-2231.  PubMed  
  • 3302 Salmon, E. D., Leslie, R. J., Saxton, W. M., Karow, M. L., and McIntosh, J. R. (1984).  Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.  J. Cell Biol. 99, 2165-2174.  PubMed  
  • 3303 Mitchison, T., Evans, L., Schulze, E., and Kirschner, M. (1986).  Sites of microtubule assembly and disassembly in the mitotic spindle.  Cell 45, 515-527.  PubMed   Journal
  • 3304 Drechsel, D. N. and Kirschner, M. W. (1994).  The minimum GTP cap required to stabilize microtubules.  Curr. Biol. 4, 1053-1061.  PubMed   Journal
  • 3305 Walker, R. A., Inoue, S., and Salmon, E. D. (1989).  Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.  J. Cell Biol. 108, 931-937.  PubMed  
  • 3306 Muller-Reichert, T., Chretien, D., Severin, F., and Hyman, A. A. (1998).  Structural changes at microtubule ends accompanying GTP hydrolysis: information from a slowly hydrolyzable analogue of GTP, guanylyl (alpha,beta)methylenediphosphonate.  Proc. Natl. Acad. Sci. USA 95, 3661-3666.  PubMed   Journal
  • 3307 Mitchison, T. J. and Kirschner, M. W. (1985).  Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation.  J. Cell Biol. 101, 766-777.  PubMed  
  • 3308 Lee, L., Tirnauer, J. S., Li, J., Schuyler, S. C., Liu, J. Y., and Pellman, D. (2000).  Positioning of the mitotic spindle by a cortical-microtubule capture mechanism.  Science 287, 2260-2262.  PubMed   Journal
  • 3309 Kinoshita, K., Arnal, I., Desai, A., Drechsel, D. N., and Hyman, A. A. (2001).  Reconstitution of physiological microtubule dynamics using purified components.  Science 294, 1340-1343.  PubMed   Journal

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