(CANCER RESEARCH 58, 1177-1184, March 15. 1998]
Low Potency of Taxol at Microtubule Minus Ends: Implications for its Antimitotic
and Therapeutic Mechanism1
W. Brent Derry, Leslie Wilson, and Mary Ann Jordan2
Department of Molecular, Cellular, anil Developmental
Biolog\, Universit\ of California Sania Barbara, Stinta Barbara, California, 93106
ABSTRACT
subunits are exposed at one end of a microtubule
In many cells, low concentrations of Taxol potently block mitosis at the
transition from metaphase to anaphase, with no change in microtubule
polymer mass and no microtubule bundling. Mitotic block ultimately
results in apoptotic cell death and appears to be the most potent antitumor
mechanism of Taxol (M. A. Jordan et al, Cancer Res. 56: 816-825,1996).
subunits are exposed at the opposite end (18).
During microtubule assembly, GTP is hydrolyzed to GDP, and the
microtubule core becomes enriched with tubulin liganded to GDP,
whereas the microtubule ends are believed to be capped by tubulin
liganded to GTP or GDP-phosphate, which stabilizes them against
Mitotic inhibition results, at least in part, from stabilization of growing
and shortening dynamics, specifically at the plus ends of microtubules, by
the binding of very few Taxol molecules to the microtubule surface (M. A.
Jordan et al, Proc. Nati. Acad. Sci. USA, 90: 9552-9556, 1993; W. B.
Derry et al, Biochemistry, 34: 2203-2211, 1995). A number of actions of
Taxol on mitotic spindle function may be due to its effects on microtubule
dynamics at the minus ends of microtubules, effects that previously have
not been described. Here, we determined the effects of Taxol on minus
ends of purified microtubules at steady state. In contrast to the strong
stabilizing effects on plus ends, substoichiometric ratios of Taxol bound to
tubulin in microtubules did not affect growing, shortening, or dynamicity
at minus ends. Thus, in blocked mitotic cells, Taxol can potently suppress
dynamics at plus ends of spindle microtubules, whereas its impotence at
minus ends permits continued microtubule depolymerization at the spin
dle poles. Differential effects of Taxol at opposite microtubule ends may
explain Taxol's actions on spindle structure and function and its unique
potent antitumor action.
INTRODUCTION
Taxol®is an important new cancer chemotherapeutic agent that is
used in the treatment of refractory ovarian cancer and shows prom
ising activity against several other carcinomas (1-5). In addition, it
has been used extensively as a tool to examine the functions of
microtubules in mitosis, secretion, signaling, migration, and other
cellular processes (6-11). Taxol is a potent inhibitor of eukaryotic cell
proliferation, blocking cell cycle progression at mitosis through its
stabilizing actions on spindle microtubules (Refs. 12-14; for brief
reviews of drug effects on microtubule dynamics, on mitosis, and in
cancer chemotherapy, see Refs. 15 and 16).
Microtubules are dynamic cytoskeletal components that function in
the development and maintenance of cell shape and polarity, in
mitosis, and in cellular movement. They are long, cylindrical poly
mers of dimeric aß-tubulin subunits arranged in parallel protofilaments that interact through lateral contacts to form the microtubule
lattice. Microtubule assembly is initiated at a critical subunit concen
tration, and elongation proceeds by the reversible addition of tubulin
dimers to the microtubule ends (17). The structural asymmetry of the
tubulin aß-heterodimer is reflected in the polar ordering of subunits in
the microtubule protofilaments. Tubulin dimers are arranged such that
the a-subunit of one dimer contacts the ß-subunitof the adjacent
dimer, giving rise to an inherent structural polarity in which a-tubulin
Received 9/26/97; accepted 1/16/98.
The costs of publication of this article were defrayed in pan by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by United States Public Health Service Grant CA57291
from the National Cancer Institute (to M. A. J. and L. W.I and the Materials Research
Laboratory National Science Foundation Grant DMR-9123048 (to W. B. D. and L. W.).
2 To whom requests for reprints should be addressed. Phone: (805)893-5317; Fax:
(805)893-4724; E-mail: [email protected].
and ß-tubulin
disassembly. The energy liberated from the hydrolysis of GTP to GDP
at microtubule ends gives rise to dynamic instability, a nonequilibrium
behavior in which the stochastic interconversion of microtubule ends
between phases of relatively slow growth and rapid shortening occurs,
presumably as a result of the gain and loss of the stabilizing cap (19,
20). The two ends of a microtubule are kinetically distinct, with one
end, the plus end, being more dynamic than the opposite, minus end
(21, 22). The inequality between kinetic parameters at opposite mi
crotubule ends at steady state results in net tubulin addition at plus
ends balanced by net loss at the minus ends, a behavior termed
treadmilling or flux (23).
Taxol binds reversibly along the surfaces of microtubules, with a
maximum stoichiometry of 1 mol of Taxol per 1 mol of tubulin in
microtubules (24, 25) and an apparent dissociation constant in the
10-nM range (26). At high concentrations. Taxol enhances microtu
bule polymerization, increasing the mass of microtubules both in cells
and in vitro. High concentrations of Taxol also induce the formation
of extensive bundles of microtubules in cells. We have recently found
that, in vitro, at low Taxol concentrations, binding of only a few
molecules of Taxol to tubulin in microtubules potently suppresses
microtubule dynamic instability at microtubule plus ends and that the
suppression is accompanied by only a marginal increase in microtu
bule polymer mass (14, 27).
In human cancer cells, we found that low concentrations of Taxol
block or slow mitosis at the transition from metaphase to anaphase
(14).3 Mitotic block is accompanied by suppression of dynamic in
stability at plus ends of the microtubules. with little or no increase in
microtubule polymer mass or microtubule bundling. Rieder et al. (6)
found that low concentrations of Taxol inhibit the metaphase oscilla
tions of chromosomes associated with the plus ends of microtubules in
spindle kinetochore fibers. These data in cells and in vitro have
indicated that the potent antimitotic and antiproliferative activity of
Taxol involves suppression of dynamics at plus ends of mitotic
spindle microtubules.
The effects of Taxol on dynamics at microtubule minus ends had
not been previously described thoroughly. In vitro, the conditions
ordinarily used to assemble microtubules result in the formation of
very few minus ends, and the dynamics of the minus ends that form
are minimal, so that it is difficult to measure any changes induced by
Taxol. In cells, minus ends are generally buried in regions of the cell
that are inaccessible to video microscopy. However, studies indicating
that Taxol suppresses dynamics at microtubule plus ends only par
tially explain the observed actions of Taxol in cells. It has long been
recognized that centrosoma! components that are normally located at
the minus ends of microtubules in the mitotic spindle often become
disorganized in the presence of Taxol (28, 29). In addition, using fixed
cells, we and others found that the lengths of microtubules in mitotic
1A-M. Yvon, P. Wadsworth. and M. A. Jordan, unpublished results.
1177
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LOW POTENCY
OF TAXOL AT MICRUTl'Hlï.l-:
spindles following Taxol incubation decreased significantly rather
than lengthening, as might be predicted from the ability of Taxol to
enhance microtubule polymerization (11. 14, 30). In living mitotic
cells. Waters et al. (7) found that minus-end microtubule disassembly
in spindles continued at control rates in the presence of Taxol at
concentrations that blocked plus end-associated chromosome oscilla
tion (6). They suggested that the minus-end disassembly was likely to
be mediated by a microtubule-severing protein like katanin (31) or a
microtubule motor like Kar3. which has been shown to induce the
disassembly of Taxol-stabili/ed microtubule minus ends in vitro (32).
Thus, it is important to examine the effects of Taxol on minus ends
of microtubules to determine whether the effects of Taxol by itself on
the minus ends might account for these cellular observations or
whether other factors, such as microtubule-associated proteins, might
be required. Here, we adopted conditions that allowed simultaneous
measurement of the effects of Taxol, both on plus and minus ends of
microtubules in vitro. We found that substoichiometric ratios of Taxol
bound to tubulin in microtubules did not suppress and. possibly, even
enhanced dynamics at microtuhule minus ends, whereas the same
ratios of bound Taxol strongly suppressed dynamics at microtubule
plus ends. Thus, low concentrations of Taxol differently effect dy
namics at opposite microtubule ends. The differential effects of Taxol
on opposite microtubule ends may play an important role in the
chemotherapeutic blocking of mitosis and cell proliferation and in the
ensuing cell death (33).
MATERIALS AND METHODS
Purification
of Tubulin and Flagellar Axonemal "Seeds." Microlubule
protein was purified from bovine hrain (34). and tubulin was purified from the
microtubule protein by phosphocellulose chromatography (20). Tubulin was
concentrated to 3 mg/ml in PEMJ al 4°C.drop-frozen in liquid nitrogen, and
stored at —7()°C.
Tubulin was thawed on the day of an experiment and
centrifuged at 48,000 x g for 15 min (4°C,Sorvall RC 5B. SS-34 rotor) to
remove any aggregated and/or denatured tubulin. Axoneme seeds were pre
pared from sea urchin sperm (SlrongyUicenlrotus purpúralas; Ref. 35). All
protein concentrations were determined using BSA as the standard (36).
Determination of Microtubule Dynamics by Video Microscopy. Grow
ing and shortening dynamics of individual microtubules were visualized at
30°Cby video-enhanced D1C microscopy. Tubulin ( 17 f¿M
in PEM plus 1 mM
GTP) was assembled using axoneme fragments as seeds. At polymer mass
steady-state (approximately 30 min alter initiation of polymerization, deter
mined by turbidimetry at 350 nm), Taxol (NSC125973: a gift from the
National Cancer Institute) in mcthunol was added to the microtubule suspen
sion (final concentration of methanol £1% (v/v)j. and incubation was contin
ued for an additional 15 min. Samples (2-fil volumes) were prepared for video
microscopy and analyzed as described previously (37). A microtubule was
considered to be in a phase of growth ¡Iits length increased by >0.2 ¡anat a
rate of >0.15 firn/min and in a phase of shortening if its length decreased by
>0.2 /im at a rate of >0.3 fim/min. Length changes of SO.2 firn over the
duration of at least six time points were considered as attenuation (pause)
phases. Between 8 and 24 microtubules from at least three separate experi
ments were measured tor each condition.
Classification of Microtubule Plus and Minus Ends. Microlubules as
sembled both at the plus and minus ends of axonemal seeds under the solution
conditions used in this study. Microlubule polarity was assigned based on the
rates of microtubule growth, the number of microtubules per axoneme end. and
microtubule length (38). Minus-end microlubules grew at slower rates and for
shorter lengths per growing event than did plus-end microtubules. Minus-end
microtubules were, on average, shorter and fewer in number than were plusend microtubules (Figs. 1 and 2).
Transition Frequencies. The transition of a microtubule end from a state
of growth (or attenuation) to a shortening phase is referred to as a "catastro4 The abbreviations used are: PEM. I(K) nisi PIPES. I mM EGTA. and
(pH 6.9); D1C. differential interference contrast.
MINUS ENDS
10
E
3
s
o>
C
0
3
.£>
2
O
4
(B
9
20
40
60
80
100
[Taxol] (nM)
Fig. I. Effects of Taxol on mean lengths at plus and minus ends of microtubules.
Purified tubulin was polymeri/ed at the ends of axoneme seeds to steady state in PEM
with I mM GTP at 30 C for 25 min. and then Taxol was added and the microtubules were
incubated for an additional 15 min. Microtubule lengths at plus (•)and minus ends (•)
were measured by D1C microscopy ("Materials and Methods"). Between 8 and 24
micrutubules from at least three separate experiments were measured for each condition.
Bars. SE.
phe." whereas the switching of a microtubule end from a shortening phase to
a phase of growth or attenuation is referred to as a "rescue" (22). The
catastrophe frequency was calculated as the total number of shortening events
divided by the total time spent growing plus the total time spent in the
attenuated stale. The rescue frequency was calculated as the total number of
transitions from shortening to either phases of growth or attenuation divided by
the total time spent shortening. The number of rescues per firn was determined
by dividing the total number of rescue events by the total length of microtubule
shortened (39). Dynamicity was calculated as the total tubulin subunit ex
change at a microtubule end during all detectable growing and shortening
phases divided by the total time of observation. Suppressivity was determined
as the absolute value of the slope correlating the magnitude of a dynamic
parameter (i.e.. shortening rate, length shortened per shortening event, or
rescue frequency) with the stoichiometry of Taxol bound to tubulin in micro
tubules (27).
Microtubule Polymer Mass and Stoichiometry of Taxol Binding to
Tubulin in Microtubules. Microtubules (17 U.Mtotal tubulin) were assem
bled to steady state as described above. Taxol was added, and incubation was
continued for 15 min. Aliquots of the suspension (100 fil) were pipetted into
Beckman microfuge tubes (5 X 20 mm) and centrifuged ( 160.(XX) X i;. 15 min
at 30°C:Beckman LS-50. SW 50.1 rotor). Supematants were aspirated, and the
microtubule pellets were solubilized in 50 fil of 0.2 M NaOH (for a2 h at
25°C).Protein concentrations were determined in both supernatant and pellet
fractions. The stoichiometries of bound Taxol used in this analysis were those
previously determined by sedimentation assay using |'H]Taxol (27). The
stoichiomelries were 1.5 ±0.2, 3.7 ±0.4. 6.6 ±0.7, and 14 ±3 molecules
per I(XX)dimers of tubulin in microtubules at added Taxol concentrations of
10, 25. 50. and 100 nM. respectively.
RESULTS
Effects of Taxol on Microtubule Polymer Mass and on the
Lengths of Minus- and Plus-End Microtubules. Polymer mass
levels were determined 25 min after addition of Taxol to steady-state
microtubules that had been preassembled using axoneme seeds (see
"Materials and Methods"). Taxol ( 10-100 nM)modestly increased the
polymer mass. For example, the microtubule polymer mass increased
from 6.6 /MMin the absence of Taxol to 8.6 /UMin the presence of 10
nMTaxol (a 1.3-fold increase) and to 9.2 /UMin the presence of 100 nM
Taxol (a 1.4-fold increase over controls; data not shown). The lengths
I mM MgSO4
of individual microtubules at plus and minus ends of axonemes were
1178
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LOW POTENCY OF TAXOL AT MICROTUBULE MINUS ENDS
16
Fig. 2. Life history traces of steady-state microtubules at opposite
ends of axoneme seeds in the absence or presence of Taxol. Shown are
typical examples of length changes of individual microtubules with
time. A, plus ends in the absence of Taxol. B. minus ends in the
absence of Taxol. C. plus ends in the presence of 50 n.vt Taxol. /),
minus ends in the presence of 50 JIM Taxol. Lengths of individual
microtubules were measured by video DIC microscopy, as described
in "Materials and Methods."
8
10 0
Time
measured using DIC microscopy. Under the conditions of these ex
periments, in controls, microtubules at the plus ends of axonemes
were slightly longer than microtubules at the minus ends (4.0 ±0.4
and 3.3 ±0.4 yum. respectively; Fig. 1). Taxol (10-KX) IIM)induced
2
468
( min )
shortening events were detectable). The kinetic parameters of micro
tubule dynamics at both plus and minus ends are summarized in Table
1. As expected, minus ends of control microtubules grew more slowly
and underwent shorter length excursions than did plus ends (Table 1).
Growing and shortening length excursions at minus ends were only
one-half and two-thirds as long, respectively, as excursions at plus
an increase in microtubule length at the plus ends. However, the
lengths of microtubules at the minus ends remained unchanged (Fig.
1). For example, at 50 nM Taxol, plus ends doubled in length from
4.0 ±0.4 firn in controls to 7.8 ±0.8 /¿m,whereas minus-end
microtubules remained constant in length at 3.2 ±0.3 /urn (Fig. 1).
The microtubule number concentration remained unchanged (data not
shown). Therefore, the modest increase in polymer mass at low Taxol
concentrations resulted from a selective increase in microtubule
length at plus ends.
Intrinsic Differences between the Dynamics of Control Micro
tubules at Plus and Minus Ends. "Life history" traces of typical
ends. The shortening rates at the two ends were not significantly
different. Dynamicity. u measure of total detectable tubulin subunit
exchange, was only half as great at minus ends as it was at plus ends.
Minus ends underwent rescue twice as often as plus ends (Table 2),
whereas they underwent catastrophe less frequently than did plus ends
(21.22.38-41).
Low Concentrations of Taxol Had No Significant Effect on
Rates or Lengths of Shortening or Growing at Microtubule Minus
Ends. Visual inspection of microtubule lite-history traces shown in
control plus- and minus-end microtubules (Fig. 2. A and B. respec
tively) show that both microtubule ends displayed characteristic dy
namic instability behavior consisting of episodes of slow growth,
rapid shortening, and attenuation or pause (when neither growing nor
Fig. 2 indicated, surprisingly, that 50 nM Taxol had little effect on
dynamics at microtubule minus ends (compare Fig. 2. B and D),
whereas it clearly suppressed dynamics at plus ends (compare Fig. 2,
A and C). As summarized in Table 1. Taxol (10-100 nM) had little
Table I Effects oj Ta\ul un kinetic parameters
tif minus- unti plus-end microtubule dynamics assembled front purified bovine brain tubulin
(nM)ParameterShortening
Taxol concentration
rale (jim/min)
±8"
±5*
±9
±9
±7
Minus ends
4''1.7
28 ±
±4*1.9±
27
±2*1.5
15
±42.0
35
endsMean
Plus
71.8
44±
length shortened (uni)
0.2
±0.3
±0.2
±0.3
±0.3
Minus ends
1.5±0.2/71.2
±0.3*0.89
1.2
0.30.78
2.8 ±
0.30.91
2.6 ±
0.40.80
2.4 ±
endsGrowing
Plus
rate (/¿m/min)
±0.1*
±0.13
0.071.4
±
±0.11
±0.09
Minus ends
0.94
O.OS''1.4
±
±0.10.97
1.3
±0.1I.I
±0.11.2
1.3
±0.11.0
1.4
endsMean
Plus
length grown (firn)
±0.2
±0.13
±0.1
±0.1
±0.1
Minus ends
1.2
±0.1*2.82.11411too39 I.I±0.1*1.91.2814
2.1
±0.21.63.219241045
1.6
±0.21.62.914192552
1.7
±0.21.73.115145033
endsDynamicity
Plus
(¿im/mint
Minus
endsPlus
endsTotal
microtubules analyzed
Minus
endsPlus
ends052
" Errors are SEs.
* Rates and lengths lhat were significantly different from controls by two-tailed t lest (95<7rconfidence level).
1179
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LOW POTENCY OF TAXOL AT MICROTUBULE MINUS ENDS
Table 2 Effects of Taxol on ¡hetransition frequencies
(nM)Transition
of minus- and plus-end microtubules
Taxol concentration
eventCatastrophe
(min"1)Minus
±0.39
±19.7
endsPlus
endsRescue
)Minus(min" '
2.2±
3.0±
2.6±
endsPlus
±0.240.107.02.0"
1.40.380.5815.36.225±0.10±0.15±4.0±
1.70.720.418.36.650±0±0100.19.12±
2.00.67
ends0.390.4711.35.30±0.09"±0.10± 1.10.360.4511.16.310±0.09±0.10±
Errors are SEs.
effect on shortening rates or on length excursions during growing and
shortening events at minus ends, but it significantly inhibited short
ening rates and excursion lengths at plus ends. For example, 25 nM
Taxol reduced the mean plus end shortening rate by 36%, whereas the
minus end shortening rate was not affected (t test, 95% confidence
level). At 100 nM Taxol, the mean plus end shortening rate was
reduced 66%, whereas the mean minus end shortening rate was
reduced only 25%. The lengths grown or shortened at minus ends
70
60
50
40
n
CC
30
c
0)
20
O
£
10
a»
3.2
ÃŽ2.8
§ >
2.4
W
2-°
C
fil
go
i-«
Q> J=-
_l fi
i-
1.2
0.8
were unaffected by 100 nM Taxol, but they were reduced 48 and 57%,
respectively, at plus ends.
Suppressivity of Taxol for Shortening Is Significantly Reduced
at Minus Ends. As shown in Fig. 3A, shortening rates decreased
linearly with increasing stoichiometry of Taxol bound to tubulin in
microtubules both at the minus ends (closed circles) and plus ends
(closed squares). However, equivalent suppression of shortening at
minus ends required a higher density of bound Taxol molecules than
was required at plus ends. Binding of only 12 Taxol molecules for
every 1000 dimers of tubulin in microtubules suppressed the mean
shortening rate at plus ends by 50% (Ref. 18; Table 1 and Fig. 3/4). In
contrast, at the same Taxol binding stoichiometry, there was only 11%
suppression of shortening rates at minus ends. The absolute value of
the slopes of the lines in Fig. 3/4 is termed the suppressivity (37),
which reflects the efficacy of bound Taxol in altering a specific
dynamics parameter, in this case, the shortening rate. The suppressiv
ity values for shortening rates at minus and plus ends were 1.78 X IO6
and 3.19 X IO6, respectively (Table 3). Thus, substoichiometric Taxol
bound to tubulin along the length of the microtubule was only 55% as
effective at suppressing the mean shortening rate at minus ends, as
compared with its efficacy at plus ends. The suppressivity for the
length shortened per shortening event at each microtubule end is
shown in Fig. 3ßand indicates that the length shortened per shorten
ing event at minus ends was 5-fold less sensitive to substoichiometri-
0
n a>
±7.6
0
2
4
6
8
101214
Bound Taxol (moles per 1000 moles tubulin in microtubules)
Fig. 3. Microtubule shortening rates and lengths shortened per shortening event as a
function of bound Taxol. A. mean shortening rates of microtubules with increasing
stoichiometries of Taxol bound to tubulin in microtubules. B. microtubule lengths short
ened per shortening event.
and —-, least squares linear regression lines for plus (•)
and minus (•)ends, respectively. Bars. SE.
endsParameterShortening
cally bound Taxol than it was at plus ends (Table 3).
Taxol Preferentially Promotes Catastrophe at Minus Ends
while Preferentially Inducing Rescue at Plus Ends. Taxol nearly
doubled the catastrophe frequency at minus ends, from 0.38 ±0.09
catastrophes/min in controls to 0.72 ±0.19 catastrophes/min at 50 nM
Taxol (Table 2). In contrast, at plus ends, the catastrophe frequency
was not altered significantly at these Taxol concentrations. Taxol
increased the rescue frequency (rescues/min) at both the plus and
minus ends (Table 2). However, from the suppressivities, at minus
ends, bound Taxol was only 42% as effective at increasing the
frequency of rescue per /n.mas it was at plus ends (Fig. 4). Thus, Taxol
differentially modulates kinetic parameters at opposite microtubule
ends.
Taxol Did Not Affect the Dynamicity or the Percentage of Time
Microtubules Remained in the Attenuated State at Minus Ends.
The dynamicity at microtubule plus ends was suppressed in a Taxol
concentration-dependent manner, whereas at minus ends, the dynamicity
Table 3 Taxol suppressivity and S! values for microtubules al plus and minus
endsSuppress!
endsr»0.95
vity°3.13
X 10~6
X 10~6
rate
Length shortened
117
0.95
26
0.78
0.22
Rescue eventsPlus
43Minus
0.97Suppressivity"1.78
18r*0.66
0.79Sr*0.57
0.42
" Suppressivity is the absolute value of the slope of the linear regression line correlating a dynamic parameter with the stoichiometry of Taxol bound to tubulin in the microtubules,
assuming a random distribution of Taxol (37). The units of suppressivity have been omitted for simplicity,
r = correlation coefficient of linear regression lines for suppressivity determinations.
S! —ratio of suppressivity values at minus and plus ends.
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LOW POTENCY OF TAXOL AT MICROTl Bll I: MINUS ENDS
1.0
E
0.8
3.
9
5.
0.6
I
in
0
a
u
«
at
E
0.4
0.2
O
Bound
2
4
6
8
101214
Taxol (moles per 1000 moles
tubulin
in microtubules)
of
Fig. 4. Effects of Taxol on rescue frequency per ^im of microlunule length shortened.
Rescue frequencies were calculated by dividing the total number of rescue events by the
total length shortened per shortening event.
and —. least squares linear regression
lines for plus (•)and minus (•)ends, respectively. Burs, SE.
was unaffected by Taxol (Table I ). Taxol also selectively increased the
percentage of time plus-end microtubules remained in the attenuated state
while having no significant effect at minus ends (Fig. 5).
DISCUSSION
Substoichiometric Ratios of Taxol Bound to Tubulin in Micro
tubules Had Little Effect on Growing, Shortening, or Dynamicity
at Minus Ends. Our goal in this work was to determine to what
extent low concentrations of Taxol, which result in the binding of
Substoichiometric ratios of Taxol to tubulin in microtubules, affect
growing and shortening dynamics at opposite microtubule ends at
steady state in vitro. Such low Taxol concentrations in HeLa cells and
other tumor cells appear to block mitosis selectively at the metaphaseanaphase transition by stabilizing spindle microtubule dynamics (6, 7,
14).3 In agreement with previous findings, at microtubule plus ends,
has been estimated to be approximately 10 nM (26). The dramatic
increase in affinity of Taxol for tubulin during polymerization indi
cates that a «informational change occurs in tubulin during polymer
ization that creates the high-affinity binding site (42). An alternative
possibility, that the high affinity Taxol binding site is formed at the
interface between adjacent dimers. cannot be formally eliminated.
High stoichiometries of Taxol bound per molecule of tubulin in a
microtubule greatly increase the polymer mass and reduce the soluble
tubulin concentration to zero or near zero, primarily by reducing the
dissociation rate constants at both microtubule ends (43. 44).
In previous work using low Taxol concentrations, we found that
very low ratios of bound Taxol (between 1:1(X)()and 1: KM)molecules
of Taxol bound per molecule of tubulin in a microtubule) strongly and
selectively suppressed the rate and extent of microtubule shortening at
steady state at plus ends (27). The lowering of the linkage tree energy
associated with the binding of Taxol to tubulin in microtubules is most
likely responsible for the stabilization. A reasonable model is that
Taxol binds with high affinity to a tubulin molecule that is not too
distant from the microtubule plus end. During a shortening excursion,
the microtubule shortens in normal fashion until the region in the
microtubule containing the rare Taxol-tubulin complex is reached.
The rate of shortening is then reduced due to (he decreased free energy
of the association of the Taxol-bound molecule of tubulin with adja
cent tubulin molecules at the plus end of the microtubule lattice.
These results demonstrate that, under conditions in which plus ends
are strongly stabilized and increase in length, the minus ends are not
kinetically stabilized and do not increase in length. Thus, the plus and
minus ends respond differently when an end shortens and reaches a
Taxol-tubulin complex. How might this be explained'.' The head-totail ordering of ur/3-tubulin dimers within a microtubule prototilamcnt
gives rise to structural polarity, where the u-tubulin subunit of each
dimer is facing one microtubule end, and the ß-Uibulinsubunit of each
dimer is facing the opposite end ( 18). The structural asymmetry built
into the microtubule must be responsible for differences in the stabil
ity of the stabilizing GTP or GDP-phosphate cap at opposite micro
tubule ends, which gives rise to the different growing and shortening
dynamics at opposite microtubule ends. One reasonable mechanism
that could account for the inability of Taxol to affect dynamics at
minus ends would be that a structural difference exists in tubulin when
it is situated at the plus or minus end that creates a difference in the
affinity for Taxol. A tubulin dimer containing bound Taxol exposed at
the minus end, with its a chain exposed, would not be in the same
structural environment as a Taxol-containing dimer exposed to sol-
low concentrations of Taxol enhanced growth, strongly suppressed the
rate and extent of shortening and the overall dynamicity, and in
creased the frequency of rescue (27). However, at minus ends, low
concentrations of Taxol exerted little or no effect, other than approx
imately doubling the catastrophe frequency at the concentrations
examined. In contrast to its ability to double the mean microtubule
70
length at plus ends. Taxol at low concentrations did not affect the
mean length of minus-end microtubules (Fig. I).
60
The single measurable effect of Taxol on minus ends was an
apparent doubling of the time-based catastrophe frequency (Table 2).
50
The catastrophe frequency is inversely proportional to the soluble
tubulin concentration (22). Because these Taxol concentrations
40
slightly but measurably increased the polymer mass at plus ends, the
increase in the catastrophe frequency at minus ends most likely results
30
from the reduced soluble tubulin concentration coupled with the lack
of stabilization of growing and shortening dynamics at minus ends
(Fig. 1). In marked contrast to the direct effect of Taxol on plus ends,
O
resulting in increased stabilization and lengths, the results indicate that
IL 1 O
low concentrations of Taxol have no direct effect at minus ends.
Taxol Mechanism. Taxol binds extremely poorly to soluble tubu
O
2
4
6
8
101214
lin, but it binds with very high affinity to polymerized tubulin, and at
Bound Taxol (moles per 1000 moles tubulin In microtubules)
high Taxol concentrations, one molecule of Taxol per molecule of
Fig. 5. Effects of bound Taxol on percentage of time in the attenuated state at opposite
tubulin can bind in microtubules (24, 42). The affinity of Taxol for
microtubule ends. The percentage of the total observation time thai microtuhulcs did not
detectably grow or shorten at plus • and minus • ends. Hurs. SI:.
tubulin in microtubules has been difficult to determine accurately but
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LOW POTENCY OF TAXOL AT MICROTUBULE MINUS ENDS
vent at the plus end, with its ß-chainexposed. The idea is that the
affinity tbrTaxol, which is created by incorporation of tubulin into the
microtubule. is considerably higher when a Taxol-containing tubulin
dimer is exposed at the plus end than at the minus end. Thus,
shortening of the microtubule at the minus end would expose a
Tuxol-tubulin dimer. but the Taxol would rapidly dissociate and be
essentially ineffective. One would not expect a difference in the
affinity for Taxol of a tubulin dimer buried in the lattice because the
a subunit of each buried dimer would be in normal contact with a
ß-chainof the adjacent dimer in each protofilament, and the ß-chain
of the dimer would be in normal contact with an «-chain in each
protofilament.
A second reasonable mechanism involves the differences in free
energy input necessary to stabilize a plus or a minus end. Following
GTP hydrolysis, tubulin subunits are thought to exist in a strained
conformation within the microtubule lattice. As microtubules shorten,
the tubulin subunits peel away from the lattice and form small curved
segments of protofilaments. They appear to be free to undergo a
conformational change as they dissociate, indicating a high change in
free energy (45-47). Growing and shortening dynamics at plus ends
are inherently more robust than those at the minus ends (21, 22. 38.
39). The free energy changes associated with microtubule shortening
may be higher at minus ends as compared with plus ends. A single
Taxol molecule binding at the minus end may be unable to counter
balance the high free energy changes associated with tubulin disso
ciation from minus ends, whereas a single Taxol molecule binding at
the plus end may transfer enough free energy of stabilization to the
lattice to stabilize the plus end.
High concentrations of Taxol appear to significantly stabilize both
microtubule ends. For example, dilution-induced disassembly of double-radiolabelcd microtubules that had been incubated with stoichiometric concentrations of Taxol indicated that the dissociation rate
constants were indistinguishable at the opposite microtubule ends
ends (44). However, it is likely that, under the conditions used. Taxol
was binding stoichiometrically to tubulin in microtubules. and the
thermodynamic disadvantage of a rare Taxol molecule at the minus
end would be nullified by the concerted activity of multiple Tuxol
molecules.
A third possible mechanism is that conformational changes in
tubulin are propagated either unidirectionally or bidirectionally
through the microtubule lattice following the binding of Taxol. Taxol
binding to microtubules induces conformational changes within the
microtubule lattice (48-50). In addition, tubulin assembled in the
presence of Taxol, both in vitro and in cells (49, 51). preferentially
forms microtubules composed of 12 rather than 13 protofilaments.
Changes in tubulin conformation or protofilament number may dif
ferentially modulate dynamic parameters at opposite microtubule ends
when low ratios of Taxol are bound. Thus, it is conceivable that Taxol
may stabilize plus ends by effecting conformational changes in tubu
lin some distance from the site of Taxol binding. A propagated
conformational change in the aß-tubulinsubunits of the polymer or in
the microtubule protofilament number may lead to unequal changes in
attractive forces at opposite ends, i.e., the attractive force at the plus
end might be enhanced by Taxol. whereas the attractive forces at the
minus end are changed very little or are even diminished.
We found that the biased suppression of dynamics at microtubule
plus ends by low ratios of Taxol binding to tubulin in microtubules
reversed the normal microtubule polarity in favor of more rapid net
tubulin exchange at minus ends than plus ends. The plus ends of
control microtubules had 2-fold higher dynamicity. they lost 1.6 times
more length per shortening event, and they underwent catastrophe 1.2
times more often than minus ends. However, at 100 nM Taxol. the
minus ends had 1.6-fold higher dynamicity, lost 1.2 times more length
per shortening event, and underwent catastrophe 1.7 times more often
than did plus ends (Tables 1 and 2 and Fig. 4). By exploiting the
structural and kinetic differences at the opposite microtubule ends, it
is clear that laterally interacting ligands such as Taxol (and perhaps
microtubule-associated proteins) can differentially modulate dynam
ics at opposite ends. Interestingly, we found that vinblastine, a mol
ecule that binds preferentially to microtubule ends rather than along
their surfaces, also reversed the kinetic polarity of the microtubule at
steady state, preferentially suppressing dynamics at plus ends while
destabilizing minus ends (38). Unlike Taxol. vinblastine stabilizes
microtubule dynamics at plus ends through an interaction directly at
the microtubule end (35, 38, 52). The preferential suppression of
dynamics at plus ends by vinblastine could be due to higher-affinity
binding sites at microtubule plus ends than at minus ends (38). The
synergy of the antitumor effects of vinblastine and Taxol (53) likely
results from their similar suppressive actions on microtubule dynam
ics brought about by binding to different sites on microtubules.
Mechanism of Mitotic Block by Taxol in Cells. The most potent
mechanism of action of Taxol in HeLa cells and many other mam
malian cells is mitotic block at the transition from metaphase to
anaphase (14). In cells, Taxol-induced spindle abnormalities and
mitotic block occurred in the absence of microtubule bundling or
increases in polymer mass suggesting, together with the known effects
of Taxol on plus-end dynamics (27), that Taxol induces mitotic block
by stabilizing dynamics (14). Recent demonstrations that Taxol sup
presses dynamics at plus ends of individual microtubules in human
cancer cells at the same concentrations that block mitosis have
strongly supported this hypothesis.3 Taxol suppresses growth and
shortening at plus ends of kinetochore fibers in mitotic spindles, as
indicated by inhibition of the normal oscillations of mitotic chromo
somes as they congress to the metaphase plate in the presence of low
concentrations of Taxol (6). In addition. Wilson and Forer (11)
recently observed that low concentrations of Taxol reduced the length
of nonacetylated microtubules near the kinetochore-associated plus
ends of microtubules in crane fly spermatocyte spindles, further
suggesting that Taxol suppressed plus-end dynamics.
However, although the action of Taxol on microtubule dynamics at
low Taxol concentrations is restricted to the plus ends, it seems certain
that Taxol exerts profound effects on the interactions of spindle
components with the minus ends of microtubules, which are located at
the poles of the mitotic spindle. Centrosomal components that are
normally located at the minus ends of microtubules in the mitotic
spindle often become disorganized in the presence of Taxol. Centro
somal integrity is lost. At high Taxol concentrations (10-20 /J.M),
some pericentriolar proteins, such as the NuMa (SPN) antigen, dis
sociate from the centrioles. and microtubules are no longer focused
primarily around centriole-containing centrosomes (28. 29, 38, 39).
The microtubule-dependent motor protein CENP-E accumulates at the
spindle poles following incubation of HeLa cells with 7.5 nM Taxol
(54). These effects may result from the potent inhibition of spindle
microtubule flux in cells incubated with Taxol: thus, although minusend depolymerization continues to some degree, plus-end dynamics
are blocked after a brief period of net plus-end lengthening. Flux or
treadmilling must be inhibited in the partially "frozen" spindle, al
lowing acetylation (a marker of stable microtubules) to "catch up" to
the kinetochore, as observed by Wilson and Forer (11). As would be
predicted by these findings, concentrations of Taxol that block mitosis
result in shorter kinetochore microtubules and shorter spindles rather
than in lengthened spindles (7, 11, 14. 30). Thus, it is clear that Taxol
can potently suppress dynamics at plus ends of spindle microtubules,
whereas its low potency at minus ends permits continued microtubule
depolymerization at the spindle poles. It is unnecessary to postulate a
special microtubule-severing or microtubule-depolymerizing
protein
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LOW POTENCY OF TAXOL AT MICROTUBULE MINUS ENDS
acting at minus ends to explain the continued minus-end depolymerization in spindles in the presence of Taxol (7). The differential effects
of Taxol by itself at opposite microtubule ends may suffice.
Plus-end stabilization and continued minus-end depolymerization
or destabilization resulting from an increased catastrophe frequency
may lead to dissociation of important mitotic regulators from the
centrosome, thus enhancing the Taxol-induced perturbation of mito
sis. The suppression of normal tubulin flux or treadmilling in kinetochore microtubules may reduce tension on kinetochores and their
associated proteins and block the transition from metaphase to anaphase (55). Reduced poleward rate of tubulin flow in the spindle
and/or the decreased tension at the kinetochores may disrupt the flow
of critical signals from the kinetochore regions of the spindle to the
pole required for normal spindle function.
How Does the Effect of Taxol at Low Concentrations on Mi
crotubule Dynamics Relate to the Chemotherapeutic Mechanism
of Taxol in Humans? The effects of Taxol at its lowest effective
concentrations on microtubule dynamics represent the most potent
known effect of the drug (14, 27, 37). Important for Taxol's chemotherapeutic action is the finding that mitotic block by low concentra
tions leads to apoptosis in human tumor cells (33). Comparisons of the
Taxol concentrations that block microtubule dynamics (10 nM-1 JUIM)
with the effective plasma concentrations during chemotherapy have
raised the question of the relevance of suppression of microtubule
dynamics and induction of mitotic block to the anticancer drug mech
anism. Taxol concentrations in plasma lie in the submicromolar to low
micromolar range (56-60 M). As much as 95% of Taxol in plasma
binds to plasma proteins and. thus, may be unavailable to enter cells
(61). If Taxol and its metabolites are present in patients for hours or
days with time- and metabolism-dependent variations in concentra
tions of Taxol, the relevant therapeutic concentration may be the
long-term available Taxol concentration rather than the peak plasma
concentration. Little information is available concerning the effective
concentrations of Taxol in cells during chemotherapy. Taxol accumu
lates in cultured HeLa cells under conditions of mitotic block to
transient peak intracellular concentrations of ~5 JLIM(14). However.
Taxol may localize to compartments in cells other than microtubules
(62) and. thus, may be partially unavailable to bind to microtubules.
In addition, microtubule-binding proteins and alterations in tubulin
isotype or posttranslational modifications of tubulin may alter the
affinity of cellular microtubules for Taxol, as compared with the
affinity of purified bovine brain tubulin (used for studies of the effects
of Taxol on microtubule dynamics). Given these variables and the
importance to the cell of its most drug-sensitive functions, the evi
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trations may play a major role in Taxol chemotherapy.
We are only beginning to probe the importance of differential
effects at opposite microtubule ends of chemotherapeutically impor
tant antimitotic drugs like Taxol and vinblastine in blocking mitosis
and inducing cell death. With increased knowledge of how such
agents work, they become more valuable as tools to probe mitotic and
cell cycle mechanisms and may lead to improved design of chemotherapeutic agents.
ACKNOWLEDGMENTS
We are indebted to Herbert P. Miller for purification of tubulin and to Drs.
Jacob Israelachvili. Dulal Panda. Richard Himes. and James Flynn and Jennifer
Gregory for stimulating and insightful discussions.
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Low Potency of Taxol at Microtubule Minus Ends: Implications
for its Antimitotic and Therapeutic Mechanism
W. Brent Derry, Leslie Wilson and Mary Ann Jordan
Cancer Res 1998;58:1177-1184.
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