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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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Evolvability.
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PubMed Journal
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3311 Kirschner, M., Gerhart, J., and Mitchison, T.
(2000).
Molecular 'vitalism'.
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PubMed Journal
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(1975).
Localization of tropomyosin in mouse embryo fibroblasts.
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PubMed
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(1975).
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(1975).
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PubMed
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(1974).
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3278 Weisenberg, R. C.
(1972).
Microtubule formation in vitro in solutions containing low calcium concentrations.
Science 177, 1104-1105.
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3279 Borisy, G. G. and Olmsted, J. B.
(1972).
Nucleated assembly of microtubules in porcine brain extracts.
Science 177, 1196-1197.
PubMed
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(1973).
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J. Cell Biol. 64, 146-158.
PubMed
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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
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3283 Carlier, M. F. and Pantaloni, D.
(1981).
Kinetic analysis of guanosine 5'-triphosphate hydrolysis associated with tubulin polymerization.
Biochemistry 20, 1918-1924.
PubMed
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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
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3286 Wegner, A.
(1976).
Head to tail polymerization of actin.
J. Mol. Biol. 108, 139-150.
PubMed
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(1997).
Microtubule treadmilling in vitro.
Science 275, 215-218.
PubMed Journal
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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
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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
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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
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3291 Rodionov, V., Nadezhdina, E., and Borisy, G.
(1999).
Centrosomal control of microtubule dynamics.
Proc. Natl. Acad. Sci. USA 96, 115-120.
PubMed Journal
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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
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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
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3294 Mitchison, T. and Kirschner, M.
(1984).
Microtubule assembly nucleated by isolated centrosomes.
Nature 312, 232-237.
PubMed
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3295 Mitchison, T. and Kirschner, M.
(1984).
Dynamic instability of microtubule growth.
Nature 312, 237-242.
PubMed
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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
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3297 Horio, T. and Hotani, H.
(1986).
Visualization of the dynamic instability of individual microtubules by dark-field microscopy.
Nature 321, 605-607.
PubMed
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3298 Schulze, E. and Kirschner, M.
(1986).
Microtubule dynamics in interphase cells.
J. Cell Biol. 102, 1020-1031.
PubMed
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3299 Schulze, E. and Kirschner, M.
(1988).
New features of microtubule behaviour observed in vitro.
Nature 334, 356-359.
PubMed Journal
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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
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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
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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
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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
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3304 Drechsel, D. N. and Kirschner, M. W.
(1994).
The minimum GTP cap required to stabilize microtubules.
Curr. Biol. 4, 1053-1061.
PubMed Journal
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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
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3306 Muller-Reichert, T., Chretien, D., Severin,
F., and Hyman, A. A. (1998). Structural changes at microtubule
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hydrolyzable analogue of GTP, guanylyl
(alpha,beta)methylenediphosphonate. Proc. Natl. Acad. Sci.
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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
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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
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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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©Jones and Bartlett Publishers (2007)
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