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. 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 www.nature.com/milestones/cytoskeleton

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. 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 www.nature.com/milestones/cytoskeleton

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)
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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 | Dreamstime.com
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
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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)
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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.
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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)
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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 www.nature.com/milestones/cytoskeleton