Brief Report
Structural Transitions at Microtubule Ends Correlate with Their Dynamic
Properties in Xenopus Egg Extracts
Isabelle Arnal,*‡ Eric Karsenti,* and Anthony A. Hyman‡
*Cell Biology Program, E uropean Laboratory of Molecular Biology, 69117 H eidelberg, G ermany; and ‡Max Planck Institut for
Molecular Cell Biology and G enetics, D resden D -01307, G ermany
the number of frayed ends. Interestingly, we also find
that more dynamic populations contain more blunt
ends, suggesting that these are a metastable intermediate between shrinking and growing microtubules. O ur
results demonstrate for the first time that microtubule
assembly in physiological conditions is a two-dimensional process, and they suggest that the two-dimensional sheets stabilize microtubules against catastrophes. We present a model in which the frequency of
catastrophes is directly correlated with the structural
state of microtubule ends.
Key words: microtubules • dynamic instability •
X enopus egg extracts • electron cryomicroscopy •
protofilament sheets
Introduction
Microtubules are dynamic polymers that switch stochastically and infrequently between growing and shrinking
states (Walker et al., 1988). This unusual behavior, called
dynamic instability (Mitchison and Kirschner, 1984; H orio
and H otani, 1986), allows rapid spatial changes of the microtubule cytoskeleton during the cell cycle. A particularly
striking example of such a rearrangement is the dramatic
reorganization of microtubules during the interphase–
mitosis transition (H yman and Karsenti, 1996).
Many studies have been performed with pure tubulin to
investigate the basic mechanism underlying dynamic instability. Microtubules elongate by the addition of tubulin
dimers, which rapidly hydrolyze one of their two bound
G TP molecules (Carlier, 1989). The energy coming from
tubulin-G TP hydrolysis is essential to destabilize the miA ddress correspondence to A nthony A . H yman, E MBL, Meyerhofstrasse
1, 69117 H eidelberg, G ermany. Tel.: 49-6221-387-337. Fax: 49-6221-387512. E -mail: hyman@embl-heidelberg.de
crotubule lattice and allow its fast depolymerization (H yman et al., 1992). For many years, the most popular model
proposed that growing microtubules are stabilized by a
terminal cap of unhydrolyzed G TP subunits (for review
see E rickson and O ’Brien, 1992), the loss of which would
result in a sudden change between growing and shrinking
states (termed a catastrophe). H owever, no G TP-tubulin
has been detected at the present in the lattice of dynamic
microtubules, and the G TP cap model remains controversial.
More recently, structural approaches using E M analysis
of pure tubulin polymerization have shown that the regulation of both microtubule assembly and dynamics involves changes in their end structure. Two-dimensional
sheets of tubulin are observed at the end of growing microtubules, whereas shrinking microtubules display curved
protofilaments peeling out from their ends (E rickson,
1974; Kirschner et al., 1974, 1975; Simon and Salmon,
1990; Mandelkow et al., 1991; Chrétien et al., 1995; Tran et
The Rockefeller University Press, 0021-9525/2000/05/767/8 $5.00
The Journal of Cell Biology, Volume 149, Number 4, May 15, 2000 767–774
http://www.jcb.org
767
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Abstract. Microtubules are dynamically unstable polymers that interconvert stochastically between growing
and shrinking states by the addition and loss of subunits
from their ends. H owever, there is little experimental
data on the relationship between microtubule end
structure and the regulation of dynamic instability. To
investigate this relationship, we have modulated dynamic instability in X enopus egg extracts by adding a
catastrophe-promoting factor, O p18/stathmin. U sing
electron cryomicroscopy, we find that microtubules in
cytoplasmic extracts grow by the extension of a twodimensional sheet of protofilaments, which later closes
into a tube. Increasing the catastrophe frequency by the
addition of O p18/stathmin decreases both the length
and frequency of the occurrence of sheets and increases
Materials and Methods
Purification of Recombinant Op18/Stathmin
R ecombinant O p18/stathmin with a 6-histidine tag was purified from
E scherichia coli as follows. 5 h after induction by 0.2 mM isopropylb-D -thiogalactopyranoside at 378C, the cells were pelleted by centrifugation at 48C and resuspended in buffer A (20 mM Tris and 100 mM NaCl,
pH 6.8) supplemented with PMSF (1 mM) and protease inhibitor (leupeptin, pepstatin, and aprotinin, 100 mg/ml). The cells were lysed using the
French Press, the extract was clarified at 17,000 rpm for 30 min at 48C, and
the supernatant was incubated with a metal affinity resin (Talon™ IMA C
R esin; CLO NTE CH ) for 1 h at 48C. A fter washing the resin with buffer A
and buffer A with 10 mM imidazole, the bound protein was eluted with
100 mM imidazole and dialyzed against BR B80 buffer (80 mM potassiumPipes, 1 mM MgCl2, and 1 mM E G TA , pH 6.8). Protein concentration was
measured using a Bradford assay with BSA as a reference (calculated molecular mass of recombinant O p18/stathmin, 18,359 kD ).
addition of 0.4 mM calcium and 200 mg/ml cycloheximide and incubation
for 40 min at room temperature.
Video Microscopy and Data Analysis
Microtubule dynamics were measured in 10,000 g frog egg extracts supplemented with human purified centrosomes (2 3 108 centrosomes/ml; Bornens et al., 1987), rhodamine tubulin (H yman et al., 1991), saturated hemoglobin, and anti-Fade solution as described before (Tournebize et al.,
1997). D ynamics were recorded using a Z eiss A xioskop, a 1003 A pochromate lens (NA 1.4), and a rhodamine filter cube. Images were recorded
every 4 s on an 8-bit black and white camera (Sony SSC-M370CE ) with an
image processor (model A rgus 10; H amamatsu) and stored on a Macintosh using NIH -Image software. D ata analysis was done with a Microsoft
E xcel Macro to determine the growth rate, the shrinkage rate, and the catastrophe frequency of microtubules (Tournebize et al., 1997). For the experiments with O p18/stathmin, extracts supplemented with 3, 6, or 7.5 mM
of the recombinant protein were similarly analyzed. Four experiments
(0 mM O p18/stathmin) and two experiments (3, 6, and 7.5 mM O p18/stathmin), which were made in different extracts, were averaged. D ifferences
between experiments with and without O p18/stathmin were evaluated using a t test with a confidence level of 95% . The percentage of time spent in
the growing and shrinking states was determined by dividing the time recorded in growing (or shrinking) state by the total time recorded.
Specimen Preparation and Electron Cryomicroscopy
Vitreous ice–embedded samples were prepared as reported previously
(D ubochet et al., 1985). 15 ml of interphasic extract were mixed on ice
with 5 ml of purified human centrosomes (2 3 108 centrosomes/ml). 2-ml
samples were pipetted onto a holey carbon grid and incubated for 20 s in a
humid atmosphere at 25–268C (Chrétien et al., 1992). The droplet was
blotted and plunged quickly into liquid ethane. For the experiments with
O p18/stathmin, we followed the same procedure using interphasic extracts
mixed on ice with centrosomes and different amounts of recombinant
O p18/stathmin. The assembly time on the holey carbon grid was the same
(20 s) except with 7.5 mM O p18/stathmin, for which the sample was incubated for 1 min (because of the shorter microtubules obtained in this case;
see R esults). Specimens were stored in liquid nitrogen and observed in a
Philips CM 20 operating at 160 kV. Images were recorded under low dose
conditions at a magnification of 27,500 and z2.5 mm underfocused.
Image Analysis
Micrographs were printed and microtubule end structures were analyzed
directly on these prints. Three types of extremities were observed: (1)
blunt ends (straight protofilaments or very short protofilament protrusions
,30 nm); (2) extensions between 30 nm and 1.5 mm; and (3) spread-out
ends (separated protofilaments). The percentage of each type of microtubule ends were calculated with respect to the total number of extremities
that we could identify (235 with 0 mM O p18/stathmin, 84 with 3 mM O p18/
stathmin, 63 with 6 mM O p18/stathmin, and 90 with 7.5 mM O p18/stathmin). A lot of extremities could not be classified because of the high background of granules and vesicles, which often hide tips of microtubules. E xtension lengths were measured on the prints. We used the contrast present
on microtubule images to localize the beginning of the extension, i.e., the
point where the constant contrast and width of the complete cylinder is
interrupted because of the apparition of one-sided curved extensions
(Chrétien et al., 1995). The contrast of complete microtubules observed by
electron cryomicroscopy was also used to determine the protofilament
number (Chrétien and Wade, 1991).
Results
CSF X enopus extracts (arrested in metaphase II of meiosis) were prepared as described before (Murray, 1991) and cycled into interphase by
Two mechanisms have been proposed for the elongation
of microtubules. O ne involves the helical addition of subunits to the tip of a growing microtubule (Chen and H ill,
1985; Bayley et al., 1990; A mos, 1995), whereas the other
proposes the formation of two-dimensional protofilament
sheets, which subsequently close into a tube (E rickson,
1974; Kirschner et al., 1975; Simon and Salmon, 1990;
Chrétien et al., 1995). To determine the structural nature
of microtubule ends elongating under physiological condi-
The Journal of Cell Biology, Volume 149, 2000
768
Preparation of Extracts
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al., 1997a; Müller-R eichert et al., 1998). Therefore, the conversion between growing and shrinking events involves a
large structural change at the microtubule ends. O ne recent model to explain microtubule dynamics is based on
the elastic properties of the polymer (Chrétien et al., 1995;
Jánosi et al., 1998): a two-dimensional tubulin sheet at the
end of the microtubule would act as a structural cap to stabilize it in a growing state. The complete closure of this
sheet into a tube would induce shrinking events by promoting the release of intrinsically curved protofilaments
(Kirschner et al., 1974; H oward and Timasheff, 1986; Melki
et al., 1989; Mandelkow et al., 1991; H yman et al., 1995;
Tran et al., 1997a; Müller-R eichert et al., 1998). H ow the
biochemical properties of tubulin contribute to this mechanism is still a matter of debate.
To understand the relationship between end structure
and dynamics, it is important to look at a population of microtubules undergoing dynamic instability. In a population
of microtubules growing in vitro, there are very few catastrophes, making it difficult to correlate growing and
shrinking microtubules with their end structure (Chrétien
et al., 1995). In vivo, microtubules are much more dynamic
(Sammak and Borisy, 1988; Belmont et al., 1990; Simon et
al., 1992), but to date no studies of microtubule end structure have been performed under physiological conditions.
To investigate the structural basis of dynamic instability
under physiological conditions, we analyzed microtubule
end structure and dynamics in X enopus egg extracts. We
find that physiological microtubule assembly occurs by the
growth of two-dimensional sheets of tubulin, which later
close into a tube. To correlate potential changes in end
structures with dynamics, we increased the catastrophe
frequency by adding the destabilizing factor O p18/stathmin (Belmont and Mitchison, 1996) to extracts. The results show that the increase in the catastrophe frequency
induced by O p18/stathmin is accompanied by a decrease
in both the length and proportion of the sheets and a concomitant increase in blunt and frayed ends. These results
allow us to propose a structural model to explain dynamic
instability and its possible relationship with G TP hydrolysis.
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Figure 1. (a) Vitreous ice–embedded microtubules observed in interphasic extracts supplemented with isolated centrosomes and incubated for 20 s at 258C. E , extensions observed at microtubule ends; and C, centriole. (b and c) Vitreous ice–embedded microtubules observed in interphasic extracts in the presence of 7.5 mM recombinant O p18/stathmin. A ddition of O p18/stathmin in this condition induces the disappearance of sheets at microtubule ends (black arrows). F, frayed end; and B, blunt end. Bar, 200 nm.
tions, we examined microtubules nucleated off centrosomes in interphasic X enopus egg extracts. E xtracts supplemented with purified human centrosomes were frozen
in liquid ethane after 20 s at room temperature, and the
frozen specimens were observed by electron cryomicroscopy. A s shown in Fig. 1 a, the microtubules of the centrosomal aster are visible despite the high background
from vesicles and granules present in these dense cytoplas-
Arnal et al. Microtubule End Structure and Dynamic Instability
769
mic extracts. A ll microtubules observed have 13 protofilaments. Some of the microtubules show long extensions at
their plus ends, which are easily recognized because they
are curved and only one edge is visible, unlike the two parallel dark edges of the complete tube. These end structures
are similar to those previously observed at the ends of microtubules assembled off centrosomes using pure tubulin
and represent two-dimensional sheets of tubulin protofilaments (Chrétien et al., 1995).
Fig. 2 shows more detailed views of different end struc-
tures observed in extracts: microtubule extremities vary
from blunt ends (Fig. 2 b) to extensions of variable lengths
(Fig. 2, c–e). The average length is z440 nm with a distribution similar to the one described in vitro (Chrétien et
al., 1995). Microtubules with frayed ends (split protofilaments) are also observed in interphasic extracts (Fig. 2 a).
Microtubules with such end structures are most likely in a
shrinking state (discussed further below; see Fig. 4). No
microtubule ends were observed with tubulin oligomers,
rings, or long curled protofilaments in extracts. These
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Figure 2. D etailed views of
microtubule end structures in
interphasic extracts. (a) Microtubule end with peeling
protofilaments (frayed end).
(b) Blunt end. (c–e) E xtensions with variable lengths.
Bar, 100 nm.
types of structures were mostly reported at the end of microtubules induced to shrink rapidly by a high calcium
or magnesium concentrations or by cooling the sample
(Kirschner et al., 1974; Simon and Salmon, 1990; Mandelkow et al., 1991; Tran et al., 1997a; Müller-R eichert et
al., 1998).
These results establish that under physiological conditions, microtubules grow by elongation of a two-dimensional sheet that closes into a tube as previously proposed
on the basis of experiments performed with pure tubulin
(E rickson, 1974; Kirschner et al., 1975; Simon and Salmon,
1990; Chrétien et al., 1995).
To analyze the relationship between the structure of a
microtubule end and its dynamic nature, we manipulated
microtubule dynamics and assessed the consequences on
end structure. X enopus extracts are an excellent system to
pursue such an investigation because the growing rate and
catastrophe frequency can be independently manipulated
by the exogenous addition of regulatory factors. To vary
the catastrophe frequency, we added O p18/stathmin, which
increases the catastrophe frequency of pure microtubules
(Belmont and Mitchison, 1996). The addition of O p18/
stathmin reduces the average length of microtubules nucleated off centrosomes in interphase extracts in a dosedependent manner (Fig. 3 a). Specifically, the addition of
3 mM O p18/stathmin does not change any of the dynamic
properties of microtubules (Fig. 3 b, Table I). In contrast,
Arnal et al. Microtubule End Structure and Dynamic Instability
771
Table I. Addition of Recombinant Op18 in Interphasic Extracts
Increases the Catastrophe Frequency
Growth rate
Shrinkage rate
mm/min
mm/min
Stathmin 0 mM
11 6 4 (95)
11.51 6 4.9 (29)
Stathmin 3 mM 10.87 6 3.44 (48) 13.3 6 6.4 (19)
Stathmin 6 mM
9.57 6 3 (57)
12.94 6 5.68 (38)
Stathmin 7.5 mM 7.57 6 2.7 (59)
10.0 6 5.4 (49)
Catastrophe
frequency
events/min
0.34 6 0.05 (34)
0.33 6 0.07 (20)
0.69 6 0.09 (48)
1.43 6 0.17 (66)
Values represent the mean value 6 SD of the mean (number of microtubules for the
growth and shrinkage rate, number of events for the catastrophe frequency). The rescue frequency is not reported because the number of events recorded was too low in
most of the conditions.
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Figure 3. (a) O p18/stathmin
shortens microtubules in interphasic extracts. Images were
taken from videos at the same
time point after the beginning of
the recording in the presence of
0, 3, 6, and 7.5 mM of recombinant O p18/stathmin added in interphasic extracts. (b) O p18/
stathmin increases the catastrophe frequency in interphasic extract. The growth rate, shrinkage
rate, and catastrophe frequency
were determined from the movies recorded over time in interphasic extract with a different
amount of O p18/stathmin. The
rescue frequency is not reported
because the number of events recorded was too low in most of the
conditions.
the addition of 6 mM O p18/stathmin and 7.5 mM O p18/
stathmin increases the catastrophe rate twofold and fourfold, respectively. The shrinkage rate is not significantly
different from unperturbed extracts at either 6 or 7.5 mM
O p18/stathmin, whereas the growth rate is slightly decreased for the highest concentration used. We analyzed
microtubule end structure by electron cryomicroscopy in
the presence of 0, 3, 6, and 7.5 mM O p18/stathmin (Figs. 1
b and 4 a). Increasing the O p18/stathmin concentration reduces the proportion of sheets and increases the proportion of blunt and frayed microtubule ends (Fig. 4 a). The
proportion of blunt ends increases transiently for interme-
diate concentrations of O p18/stathmin (3 and 6 mM) and
decreases as more microtubules exhibit frayed ends. The
average length of sheets decreases from 440 nm without
O p18/stathmin to 283 nm with 3 mM O p18/stathmin and
varies between 120 and 208 nm at higher concentrations
(these last values are only an estimate given the very low
numbers of sheets observed with 6 and 7.5 mM O p18/stathmin, Fig. 4 a).
Next, we correlated the changes in the microtubule end
structure with changes in the dynamic properties of microtubules after addition of O p18/stathmin (Fig. 4 b). In the
absence of added O p18/stathmin, 73% of the microtubules
are in a growing state (Fig. 4 b), but only 54% of them display sheets at their extremity (Fig. 4 a). These values suggest that not all growing microtubules have sheets at their
ends. H owever, the combined percentage of sheets and
blunt ends correlates precisely with the percentage of
growing microtubules at different O p18/stathmin concentrations (Fig. 4 b, violet curves), suggesting that blunt ends
also correspond to the growing microtubules. This suggestion is considerably strengthened by the strong correlation
between the percentage of frayed ends and that of shrinking microtubules (Fig. 4 b, green curves). These results
lead us to conclude that in X enopus egg extracts, microtubules with protofilament sheets or blunt ends are in the
growing state, whereas those with frayed ends are in the
shrinking state.
O ne interesting observation we made is that the increase in catastrophe frequency is associated with an increase in the blunt end proportion (6 mM O p18/stathmin;
Fig. 4 a) followed by a strong increase in the frayed end
proportion (7.5 mM O p18/stathmin; Fig. 4 a). This result
suggests that blunt ends are more unstable compared with
the ends with sheets at their extremities. Blunt ends could
either grow or transit to shrinkage with a certain probabil-
The Journal of Cell Biology, Volume 149, 2000
772
Figure 4. (a) Microtubule end structure in the presence of a different amount of recombinant O p18/stathmin added in interphasic extracts. Microtubule ends are represented as the percentage
of the total number of extremities. Values are the mean of four
(control) or two (3, 6, and 7.5 mM O p18/stathmin) experiments
made in different extracts. For each condition, the mean value is
represented by a closed symbol and the minimum and the maximum values by open symbols. (b) Microtubule end structure of
growing and shrinking microtubules. The percentage of time
spent in the growing state (light violet curve) as well as the combined proportion of sheets and blunt ends (dark violet curve) decrease with increased amount of recombinant O p18/stathmin
added in interphasic extracts caused by the higher level of catastrophes. The percentage of time spent in the shrinking state (dark
green curve) and the frayed end proportion (light green curve)
follow the inverse change versus the O p18/stathmin concentration.
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Figure 5. Structural model explaining dynamic instability and its
possible relationship with the G TP cap model. (a) Microtubules
with two-dimensional sheets at their end are in a stable growing
state. G TP-tubulin (in green) contained in the sheet could explain part of its stabilization capacity. When the tube closes, a
few subunits in a G TP state at the extremity would be sufficient
to maintain the microtubule in a growing state. (b) A s soon as the
G TP is hydrolyzed (c), the microtubule will explode because of
the weak lateral interaction between G D P-tubulin (d). Blunt
ends (b and c) are unstable because the loss of the G TP-tubulin
subunits triggers them in a shrinking state, whereas sheets are always stable.
ity, whereas all microtubules with visible sheets at their
ends are in a stable growing state. In apparent contradiction with this idea, at 3 mM O p18/stathmin, the proportion
of blunt ends is greater than at 0 mM, but there is no significant increase in the catastrophe frequency. O ne possibility is that the probability of blunt ends to transit to the
shrinking state is different at 3 and 6 mM O p18/stathmin
despite their similar proportion. This is reflected in the
greater number of frayed ends at 6 mM O p18/stathmin.
A nother possibility is that we may have classified as blunt
ends some very short sheets made of a few protofilaments
(as O p18/stathmin decreases the sheet length). In this
case, a structural transition would occur between 3 and 6
mM O p18/stathmin, allowing the complete closure of the
tube and increasing the probability of a shrinking event.
Discussion
Arnal et al. Microtubule End Structure and Dynamic Instability
We are grateful to R ebecca H eald (U niversity of California, Berkeley,
Berkeley, CA ) for her gift of the X enopus O p18/stathmin construct. We
would like to thank D enis Chrétien (City of R ennes, France) and A rshad
D esai (E MBL, G ermany) for helpful discussion. We also thank Vladimir
R ybin (MPI, G ermany) for his help with the statistical analysis.
This work was supported by the E uropean Molecular Biology O rganization.
Submitted: 28 February 2000
R evised: 5 A pril 2000
A ccepted: 6 A pril 2000
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(Fig. 5). Nevertheless, such a mechanism does not explain
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