Proc. Natl. Acad. Sci.
USA
Vol. 74, No. 8, pp. 3466-3470, August 1977
Cell Biology
Addition of colchicine-tubulin complex to microtubule ends:
The mechanism of substoichiometric colchicine poisoning
(protein self-assembly/drug binding)
ROBERT L. MARGOLIS AND LESLIE WILSON
Department of Biological Sciences, University of California, Santa Barbara, Santa Barbara, California 93106
Communicated by Daniel Mazia, June 8, 1977
ABSTRACT Colchicine blocks microtubule polymerization
by an unusual substoichiometric poisoning mechanism. We
have investigated the mechanism by which this poisoning oc-
curs with several experimental approaches,-and have found that
colchicine acts by addition to microtubule ends as a colchi-
cine-tubulin complex.
Colchicine, a potent drug that interferes with microtubule as-
sembly both in vivo and in vitro, binds to the dimeric subunit
of the microtubule, tubulin, with a stoichiometry of 1 mol/
mol of dimer (1-6). In assembled microtubules most drug
binding sites are blocked (ref 5; W. Rodgers, R. Margolis, and
L. Wilson, unpublished data). However, it has been impossible
to rule out the binding of colchicine to a limited number of sites,
for instance, those uniquely exposed on either of the two mi-
crotubule ends.
The mechanism by which colchicine poisons microtubule
assembly has remained to be elucidated. Olmsted and Borisy
(4) showed that amounts of colchicine sufficient to bind to only
a small percentage of soluble dimers could effectively poison
microtubule assembly in vitro. It may also be inferred that a
similar poisoning event occurs during exposure in living cells.
Taylor has estimated mitotic blockage occurs when only 3-5%
of the tubulin is complexed with colchicine in KB cells (2).
There are two attractive mechanisms that might account for
the substoichiometric poisoning of microtubule assembly with
colchicine. Tubulin molecules on the microtubule free ends may
have a much higher affinity for the drug than does soluble tu-
bulin, thereby permitting a preferential binding of colchicine
to the microtubule ends; or colchicine may bind first to a limited
number of soluble dimers, then these colchicine dimer com-
plexes (CD complexes) add to the microtubule ends during
assembly, thus blocking further dimer addition.
We report here that the latter mechanism appears correct.
Colchicine poisons microtubule assembly by first binding to the
soluble 6S dimer, which then adds to the growing microtubule
as a colchicine-dimer camplex during the normal process of
assembly. Once added, the CD complex effectively "caps" the
microtubule and aborts further polymerization. Of the free
dimers in solution, only a small percentage need be complexed
with colchicine for assembly to be blocked.
MATERIALS AND METHODS
Tubulin Preparation. Beef brain tubulin was prepared by
three cycles of polymerization-depolymerization using a
modification (C. F. Asnes and L. Wilson, to be published) of the
Borisy et al. procedure for porcine brain (7), in an assembly
The costs of publication of this article were defrayed in part by the
payment of page charges from funds made available to support the
research which is the subject of the article. This article must therefore
be hereby marked "advertisement" in accordance with 18 U. S. C.
§1734 solely to indicate this fact.
3466
buffer composed of 20 mM sodium phosphate, 100 mM sodium
glutamate, 2.5 mM GTP, 1 mM MgCl2, and 0.5 mM [eth-
ylenebis(oxyethylenenitrilo)]tetraacetate (EGTA) at pH 6.75.
All experiments with beef brain tubulin were carried out in this
buffer unless indicated otherwise. The beef brain microtubule
protein preparation contained approximately 75% tubulin, and
25% high-molecular-weight microtubule-associated proteins.
Chick brain tubulin was extracted at 00 from 13- to 16-day
embryos by Dounce homogenization in 1 ml per brain of buffer
composed of 100 mM piperazine-N,N'-bis(2-ethanesulfonic
acid) (Pipes) (Calbiochem.), 1.0 mM MgCl2, and 10 mM EGTA,
pH 6.9. After centrifugation at 30,000 X g for 10 min at 20,
GTP (final concentration 2.5 mM) was added to the superna-
tant, which was centrifuged at 2° at either 30,000 X g for 20
min, or at 200,000 X g for 60 min. Resulting supernatants were
used for the electron microscopy (EM) experiments. All ex-
periments with chick brain microtubule protein were carried
out with the above buffer.
Total protein was determined by the method of Lowry et al.
(8), and tubulin concentration was determined by measurement
of areas under curves from 640-nm scans of fast-green-stained
sodium dodecyl sulfate/polyacrylamide gels (9).
Electron Microscope Assay of Assembly Blockage. Chick
embryo brain tubulin contained in a 30,000 X g supernatant
(total protein 10 mg/ml) was polymerized for 4 min at 300 to
obtain microtubule fragments and then placed on a 0.5%
Formvar/carbon-coated EM grid. Those fragments that ad-
hered to the grid in 20 sec were used as seeds for further as-
sembly. Grids were rinsed with chick brain buffer and dipped
for 10 sec in buffer solution containing DEAE-dextran at 0.7
mg/ml (Sigma) (10) then rinsed with buffer at 220. Grids were
then everted onto a 200,000 X g supernatant containing 2.5 mM
GTP (total protein 6 mg/ml) and incubated an additional 4 min
at 220. Individual colchicine incubations were performed as
described in Results, at a final concentration of 100,uM. Grids
were then negatively stained with 1% uranyl acetate according
the procedure of Olmsted et al. (10), and observed with a
Philips 300 electron microscope.
Light Scattering Assay of Assembly Blockage. Preparation
of CD Complex. CD complex was formed by incubation of
three-times-recycled beef brain microtubule protein (total
protein 1 mg/ml, in 20 mM sodium phosphate/100 mM sodium
glutamate, pH 6.75) with 100 ,gM colchicine for 30 min at 300.
The solution was chilled to 00 and CD complex and unbound
tubulin were separated from free colchicine by gel filtration
on a 1 X 18-cm column of Bio-Gel P-10 (Bio-Rad Laboratories).
The quantity of CD complex used in these experiments was
ascertained by inclusion of [3H]colchicine (New England Nu-
Abbreviations: CD complex, tubulin dimer containing bound colchi-
cine; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetate; EM, elec-
tron microscopy.
 Cell Biology: Matgolis and Wilson Proc. Natl. Acad. Sci. USA 74 (1977) 3467

A 3

r.
;'1.:

-Jr-

.
he ;.

*;
_.

an
An..
<_dS +
FIG. 1. Chick embryo brain 30,000 X g supernatant was incubated for 4 min at 300 to form microtubules. These short microtubules were affixed
to EM grids, exposed to colchicine as described in the text, decorated with DEAE-dextran, and tested for colchicine blockage by exposure to
200,000 x g superhatants containing 6S tubulin under assembly conditions. Chick embryo brain microtubules do not depolymerize upon drug
exposure in vitro (ref. 12; K. Anderson and L. Wilson, unpublished data). Similarly, the grid-bound microtubule fragments that we observed
appeared, by EM, to be stable to all our experimental manipulations. (A) Two blocked fragments with double wall decorations, after exposure
to colchicine in the presence of tubulin, are shown. Bar = 0.2 ,m. (B) Higher magnification of a fragment blocked by CD complex. Decorations,
with this technique, were uniform and highly visible. Bar = 0.1 jsm. (C) A control microtubule with a long undecorated tail (single wall) emerging
from an end decorated region (arrow). Bar = 0.2 Mm.

clear) (6). Undt-r the incubation conditions used, approximately
50% of the tubulin isolated from the column was complexed
with colchicine. The half-time for dissociation of the complex
was no less thall 22 hr under the conditions employed. Thus,
no more than 1.5% of the complex could have dissociated during
the course of these experiments.
Quantitation of the Effect of CD Complex on Microtubule
Polymerization. Microtubule assembly at 300 was followed
with use of light scattering at 350 nm or by viscometry (4, 11).
For light scattering measurements a Gilford recording spec-
trophotometer (model 2400) with a constant-temperature cu-
vette chamber was used. Whenever colchicine was present
during incubation in high concentration, measurements were
made by viscometry, because the drug absorbs strongly at 350
nm. Total protein concentrations in these experiments were 1
mg/ml (2 mg/ml for viscometry).
RESULTS
Either colchicine binds directly to the microtubule end, or the
drug binds first to a dimer, which then adds to the microtubule
end and "caps" it. The following experiments were designed
to distinguish between these two possibilities.
Electroh Microscopy Visualization of Poisoning by CD
Complex. Tubulin contained in a chick embryo brain 30,000
X g supernatant was polymerized to form short microtubule
fragments, which were then placed on an EM grid. The ad-
hering fragments were coated by a 10-sec exposure to DEAE-
dextran. Further incubation with tubulin created a "decoration"
(10) which distinctly marked that portion of the microtubule
that had been exposed to DEAE-dextran. Thus, the decoration
served as a marker for the microtubule's competency to as-
semble before and after DEAE-dextran exposure. The grid was
then exiosed to tubulin in a 200,000 X g supernatant, which
polymerized only onto preformed microtubule fragments (10).
The typical result for such a control was a population of mi-
crotubules, most of which had short (0.25 i 0.02 Am) decorated
regions and long (1.72 ± 0.28 ,um) undecorated "tails" (Fig. 1C).
Decorated regions formed by this grid incubation technique
were uniformly distinct and uninterrupted.
To determine if colchicine adds to microtubule fragments
and prevents their further polymerization, we exposed mi-
crotubule fragments on grids, rinsed free of dimers, to 100 AM
colchicine (sufficient to totally block assembly), decorated them,
then challenged their polymerization-seeding capacity by in-
cubation for 4 min at 220 with 200,000 X g supernatant protein.
Alternatively, to determine if free dimers are a necessary in-
termediate in colchicine addition to microtubules, short mi-
crotubule segments were made, as above, and colchicine (100
AM) was added to the dimer microtubule mixture for an ad-
ditional 2-min (30°) incubation. Microtubule fragments that
resulted Were then allowed to adhere to grids and assayed as
above for additions to decorated regions.
The results were quantified by scanning grids and scoring
every decorated region seen in a random scan for presence or
3468 Cell Biology: Margolis and Wilson
.0.10 -
0 'I
0.05 / .,
0-
8 16 24 32 40
Min
FIG. 2. The effect of added CD complex on microtubule assembly
was assayed by turbidity measurements of recycled beef brain tubulin
polymerization at 300 (see Materials and Methods). Relative to
control assembly ( ), the assembly rate of tubulin containing 2.6%
CD complex (---) was inhibited 41%. When CD complex (2.6% of the
zero-time free tubulin concentration) was added at 16 min (- -),
immediate blockage occurred. The control contained total protein
at 1 mg/ml, tubulin at 0.75 mg/ml. CD complex was added in 100 JI,
containing protein at 0.5 mg/ml, to a total final volume of 0.85 ml.
Therefore, dilution of the total incubation volume was only 12% and
dilution of total protein concentration was 6%.
absence of an undecorated tail at least the length of the deco-
rated region. We found that 19.7% of microtubules on control
grids did not elongate. If microtubules were exposed to col-
chicine in the absence of dimers, 15.4% did not elongate.
Therefore, the presence of colchicine, by itself, was not suffi-
cient to block assembly. If microtubules were exposed to the
drug in the presence of dimers, 65.2% of the microtubules were
blocked from further assembly. Fig. 1 compares decorated
fragments blocked by CD complex (Fig. 1 A and B) with a
typical augmented microtubule (no colchicine, Fig. IC).
Because microtubules or microtubule-dimer mixtures were
exposed to colchicine for only 2 min in the EM experiment, we
tested the binding efficiency of 100 ,gM colchicine to tubulin
in this time period, using a Bio-Gel P-10 column to separate
[3H]colchicine-CD complex from free [3H]colchicine. Nine
percent of the dimers in solution had bound the drug in this
time.
CD Complex Poisoning of Assembly. If dimers mediate
colchicine action in poisoning microtubule assembly, CD
complex in the absence of free colchicine should exhibit the
capacity to poison assembly. CD complex, when isolated from
free colchicine on a Bio-Gel P-10 column, showed the expected
biochemical activity. Microtubule assembly, as assayed by light
scattering, had a lesser rate and extent of polymerization when
approximately 1 of 40 dimers in solution was complexed to
colchicine (Fig. 2). In replicate experiments, a CD complex to
free tubulin ratio of 0.026 (10.001) yielded 0.41 (+0.02) of the
control polymerization rate. The poisoning effect was more
pronounced if CD complex was added later during polymer-
ization (Fig. 2), possibly because the ratio of CD complex to free
tubulin was much higher in late stages of polymerization.
Addition of CD Complex to Preformed Microtubules.
When CD complex was added to a solution of beef brain tubulin
after polymerization had reached a plateau, there was no
measurable depolymerization. In fact, all that was observed was
a limited and transient dilutiqn effect upon addition of CD
complex (Fig. 2). Viscometry measurements of the effect of free
colchicine addition to tubulin after polymerization was com-
plete showed a similar resistance to disassembly (Fig. 3). A
concentration of colchicinre' sufficient to totally block assembly
(100 ,uM) produced only limited depolymerization of mi-
Proc. Natl. Acad. Sci. USA 74 (1977)
0.80
0 ,,
0.60 a 0
, , 0
0.40
0.20 00
10 20 30 40 50
Min
FIG. 3. Specific viscosity (%p) measurements were made of the
beef brain microtubule response to colchicine in vitro. When colchi-
cine (100 ,sM) was added at the start of 300 incubation (@-@), no
assembly was evident. Two other samples, assembled to plateau before
colchicine (100 AM) was added at 18 min (---A), or at 42 min
(0- - -0), showed limited disassembly. In these experiments, 20
,l of colchicine solution was added to a sample volume of 2.0 ml.
crotubules once formed. EM samples of this material showed
intact microtubules still present after prolonged incubation with
drug. The quantity of microtubules could not be visually dis-
tinguished from controls. Recycled beef brain microtubules
were similarly stable to podophyllotoxin (50,uM), vinblastine
(100 AM), and GDP (1 mM), all at concentrations sufficient to
totally block assembly.
Potentiation of Poisoning by Preincubation. Colchicine,
at low concentrations, binds slowly to tubulin (6). Under
polymerization conditions, the drug must compete with the
assembly reaction for free dimers. Microtubules are relatively
insensitive to colchicine-induced depolymerization in vitro,
thus, assembly permanently removes dimers from access to
colchicine. Therefore, if the CD complex mediates colchicine
poisoning, one will obtain an inaccurately low inhibition con-
stant, and preincubation of tubulin with a low concentration
of colchicine, before assembly is allowed to occur, should po-
tentiate the effect of the drug. Beef brain microtubule protein
(protein at 1 mg/ml, 6.8 MM tubulin) was polymerized in the
presence of colchicine and the assembly was followed with light
scattering. Results (Fig. 4A) where there was no preincubation
A 0.06 B
0.15
No Pre-incubation Pre-incubation
0.04
51 0.10
0
005 ~~~~~~~~0.02
0 0 -
--
0 4 8 12 16 20 0 4 8 12 16 20
Min
FIG. 4. The substoichiometric poisoning effect of colchicine on
recycled beef brain tubulin assembly was assayed by turbidity mea-
surements at 300. (A) The protein was not preincubated with col-
chicine before the assembly reaction. A limited substoichiometric
poisoning effect of colchicine on assembly was seen. (B) If the tubulin
was preincubated for 30 min at 300 with colchicine before assembly
was initiated by the addition of GTP, the poisoning effect was sub-
stantially potentiated. Total protein concentration was 1 mg/ml.
Control; ---, 1 gM colchicine; - - -, 0.2 sM colchicine.
 Cell Biology: Margolis and Wilson
showed an 8% inhibition at 0.20AM and 23% inhibition at 1.0
MM colchicine. Preincubation with drug for 30 mim fat 3086,
before GTP was added and polymerization proceeded, yielded
(Fig. 4B) 60% inhibition at 0.20AM and 100% inhibition at 1.0
MM colchicine (measured relative to a preincubated control).
The molar ratio (0.034) of colchicine to tubulin [CD complex
to tubulin ratio of 0.023 as determined by dextran-charcoal
colchicine binding assay (6)] that yielded 60% inhibition of
assembly following preincubation was in close agreement with
the molar ratio of CD complex to tubulin (0.026) that yielded
60% inhibition in the CD complex poisoning experiment (Fig.
2). In that experiment, we determined the half time of disso-
ciation of cokhicine from tubulin to be 1400 min at 00, and thus
estimate no more than 1.5% of the CD complex reverted to free
dimer and drug in the 30 min it took to isolate CD complex and
begin kinetic measurements. We therefore believe the CD
complex to tubulin ratios derived from the two different ex-
perimental approaches are reasonably accurate, and that the
close agreement between them is real.
DISCUSSION
Three independent experimental results each indicate that
colchicine inhibits microtubule assembly by means of a CD
complex. Colchicine first binds to free dimers, and the infre-
quent dimers that bind the drug add to the microtubule ends
during the course of assembly. These CD complexes then cap
the microtubules, making further assembly impossible. The
substoichiometric blockage of microtubule assembly (4) by
colchicine is explained by the mechanism of poisoning revealed
in these studies.
Other drugs that interfere with microtubule assembly, vin-
blastine and podophyllotoxin, also poison polymerization sub-
stoichiometrically (12). The possibility exists that these drugs
poison microtubule assembly through a similar mechanism.
It is reported that microtubules are in a steady-state condition
during assembly in vitro, depolymerization occurring simul-
taneously with polymerization (13). If such a steady-state
condition exists, microtubule poisoning by an infrequent CD
addition necessitates that disassembly not occur at the site of
CD complex addition. If 1 of every 40 dimers contained col-
chicine, each CD complex released from a microtubule would
allow an average of 39 dimers to add before blockage again
occurred. The net effect, a poisoning of assembly, would be
nullified.
One or more of three possibilities can explain the success of
the in vitro poisoning phenomenon: (i) the CD complex may
have an increased affinity for the microtubule end as compared
with an uncomplexed dimer, making dissociation of the capping
complex unusually slow or perhaps nonexistent; (ii) depoly-
merization may not normally occur at the site of CD complex
addition; or (iii) a steady-state may not exist under our assembly
conditions in vitro.
A conformational shift that might block depolymerization
seems plausible. The existence of a true "cap" that freezes the
microtubule end is an attractive concept, although it is seem-
ingly contradicted by evidence that colchicine causes some
depolymerization in vitro until a new "steady state" is attained
(4) (see Fig. 3).
One alternate possibility is that disassembly is not occurring
at the site of assembly. Microtubule assembly occurs as a biased
polar phenomenon (10, 14). This means that one end of the
microtubule (designated the X end) augments to a much greater
extent per unit time than does the other (Y end). Suppose that
the end opposite the primary addition site is the primary de-
polymerization site (Y). Then a colchicine poisoned population
Proc. Natl. Acad. Sci. USA 74 (1977) 3469
of- microtuibules will reach a new steady state determined by
euIfibrum conditions at the primary site of depolymerization
(Y). If the CD complex is sterically unable to add to the mi-
crotubule Y end, and the X end is CD capped, a new equilib-
rium will be determined by concentrations of free tubulin and
Y ends, independent of the CD complex population. The Y end
is capable of net assembly (10, 14) and conceivably could reach
a new steady state in the presence of CD complex, yielding a
new microtubule population plateau.
The third possibility for the mechanism of substoichiometric
CD complex poisoning of assembly, that a true equilibrium is
not present in our in vitro microtubule system, also remains
viable.
Perhaps, the response of the microtubule population in vitro
to drug reflects a combination of preferential Y end depoly-
merization and regional blockage to depolymerization along
the microtubule length. It has been reported that microtu-
bule-associated proteins interfere with drug-induced depoly-
merization in vitro (15). Drug-poisoned microtubules possibly,
therefore, depolymerize until a region of high concentration
of microtubule-associated protein is reached.
The situation within the cell is evidently different. Cyto-
plasmic and mitotic apparatus microtubules are quite labile to
colchicine and other drugs. It is reasonable to assume that col-
chicine and other drugs act in the cell as they do in vitro, by
poisoning assembly substoichiometrically. Podophyllotoxin, a
drug that resembles colchicine and shares a binding site on
tubulin with colchicine, poisons microtubule assembly in vivo.
Knowing the concentration of free tubulin in a sea urchin em-
bryo (25 ,M) (16), and the binding constant for podophyllotoxin
at 130 (0.76 MAM), one can estimate the ratio of podophyllo-
toxin-dimer complex to free dimer present when mitosis is
inhibited by 50% (at a podophyllotoxin concentration of 0.096
uM). Less than 1 of 250 dimers need be drug bound for mitotic
blockage to occur (C. Rauch and L. Wilson, unpublished ob-
servations). This low ratio is similar to that required to block in
vitro assembly of brain microtubules (12).
Microtubules extensively depolymerize in cells when poi-
soned substoichiometrically with drug. This phenomenon
presents most clearly the paradox outlined above, that disas-
sembly cannot occur if it first releases the drug-dimer complex,
because this releases the microtubule for further assembly.
Poisoning can only occur if the drug caps the microtubule end
and alters its ability to disassemble, or if disassembly prefer-
entially occurs at the end opposite the assembling end under
physiological conditions.
These results strongly suggest that microtubule disassembly
following exposure of cells to drug occurs only when each mi-
crotubule is in active equilibrium, both assembling and disas-
sembling in the absence of drug. Thus differences in microtu-
bule sensitivity to mitostatic poisons (17) may reflect in part
different intrinsic equilibria in different microtubule popula-
tions.
We thank W. Rodgers for excellent technical assistance. This work
was supported by American Cancer Society Grant CH4B, U.S. Public
Health Service Grant NS13560, and Anna Fuller Fund Postdoctoral
Fellowship 443 to R.L.M. A preliminary report on this work was pre-
sented at the 61st Annual Meeting of the Federation of American So-
cieties for Experimental Biology (18).
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