THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254,,No 20, Issue of October 25, pp. 10540-10550, 1979 Prrnted m U.S.A.

Colchicine Inhibition of Microtubule Assembly via Copolymer Formation*

(Received for publication, February 5, 1979, and in revised form, May 25, 1979)

Himan Sternlichtt and Israel Ringel

From the Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106

Colchicine l tubulin complex (CD) inhibits microtu- bule assembly. We examined this inhibition under con- ditions where spontaneous nucleation was suppressed and assembly was restricted to an elongation polym- erization. We found that CD inhibited assembly by a mechanism which preserved the ability of microtubule ends to add tubulin. This observation is inconsistent with the end-poisoning model which recently was pro- posed as a general mechanism for assembly inhibition by CD. Our data are consistent with the following model: (a) microtubules formed in the presence of CD are CD-tubulin copolymers; @) these copolymers can have appreciable numbers of incorporated CDs which are, most likely, randomly distributed in the copoly- mers; (c) CD-tubulin copolymers have assembly-com- petent ends with association and dissociation rate con- stants which decrease as the CD/tubulin ratio in the copolymers, (CD/!&T, increases; and (d) the critical tubulin concentrations required for microtubule assem- bly increase in the presence of CD, indicating that copolymer affinity for tubulin decreases as (CD/T)MT increases.

Colchicine is a potent drug that is extensively used to probe microtubule-dependent processes (1, 2). CD’ is a 1:l complex of colchicine with tubulin, the dimeric subunit protein of the microtubule polymer (3,4). This complex is believed to be the direct inhibitor of microtubule assembly (5). Substoichio- metric amounts of colchicine relative to tubulin prevent po- lymerization (1, 6, 7). Other potent antimicrotubule drugs, both competitive and noncompetitive inhibitors of colchicine binding, also inhibit assembly substoichiometrically. Margolis and Wilson (5, 8) recently proposed a general mechanism of drug action to explain substoichiometric inhibition of micro- tubule assembly. They suggested that microtubule ends are poisoned by acquiring a number of bound CDs that block further tubulin addition. This conclusion was supported by

* This work was supported in part by American Cancer Society Grant 0-99 to H. S. A preliminary report of several aspects of this study was presented at the June 1978 meeting of the American Society of Biological Chemists (27) and at the November 1978 meeting of the American Society for Cell Biology (28). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

4 To whom reprint requests should be addressed. ’ The abbreviations used are: CD, tubulin dimer containing a bound

colchicine; D,,,a~, total tubulin including active and inactive compo- nents; MAP, microtubule-associated protein; MTF, microtubule frag- ments obtained from CD-free, spontaneously assembled microtubules, and used as seeds for elongation polymerization; PB-2.5 M, a micro- tubule-stabilizing buffer (pH 6.7? consisting of 0.1 M 2-(N-morpho- 1ino)ethanesulfonic acid (Mes), 2 mM ethylenebis(oxyethyleneni- trilo)tetraacetate (EGTA), 0.1 mM EDTA, 2 mM 2-mercaptoethanol, 0.5 rnM MgC12, and 2.5 M glycerol; AU, absorbance unit.

the results of experiments involving CD addition to sponta- neously assembled microtubules at or near steady state (5,8). Their pioneering proposal stimulated our investigation of CD inhibition of microtubule assembly.

Earlier studies of microtubule assembly in the absence of CD have shown that tubulin polymerization occurs as a re- action with distinct phases of nucleation (initiation of new microtubule) and elongation (addition of subunits onto the ends of pre-existing microtubules) (9-13). The nucleation phase can be suppressed by a variety of techniques, e.g. with polyanions, and assembly ceases to be spontaneous (10, 13, 14). In such cases, polymerization can be induced by adding microtubule fragments (MTF). Assembly proceeds almost exclusively by dimeric protein addition to the MTF, which elongate into microtubules; microtubule mass increases during this elongation reaction, but the number of microtubules remains constant and equal to the number of MTF added, a

property which significantly simplifies interpretation of as- sembly kinetics (10, 14). We report below the results of our kinetic study of microtubules assembled in the presence of CD. Heparin, a sugar polyanion with four ionized acid groups/ sugar subunit, was used to suppress the nucleation phase and limit assembly to an elongation reaction.

Analysis of the inhibition process was facilitated by earlier studies of elongation reactions (10, 14). The rate of assembly during an elongation reaction can be expressed as the differ- ence between the rates of polymerization and depolymeriza- tion. Polymerization involves stepwise addition of dimer sub- units to microtubule ends and has been theoretically analyzed in the absence of CD as a “bimolecular” association reaction

with rates, (rate),, proportional to the product of [D], the concentration of free tubulin, and [m*], the concentration of

assembly-competent microtubule ends. Depolymerization has been theoretically analyzed as a “unimolecular” dissociation reaction with rates, (rate)d, proportional to [m*]. Thus,

(rate), = K, [D][m*]; (l-4)

(rate)d = km [m*]. (1B)

K, is an apparent second order association rate constant, whereas h- is an apparent first order dissociation rate con- stant. Other components in the assembly process, such as GTP, polyanions, etc., affect K+ and h-, but were not explicitly considered in the kinetic analysis, as their concentrations were assumed to be approximately constant during assembly (10, 12,14). When [n*] is constant during assembly, the expression for the association rate (Equation 1A) reduces to a pseudo- first order rate expression (Equation lC), whereas the expres- sion for the dissociation rate (Equation 1B) reduces to a constant term (Equation 1D):

(rate), = k [D]; UC)

(rate)d = km [m*] = constant, (10

where 12 = k+ [m*]. U-0

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Colchicine Inhibition of Microtubule Assembly 10541

Elongation reactions in the absence of CD have been suc-

cessfully analyzed in terms of pseudo-fast order association reactions (Equation 1C) and constant rates of dissociation

(Equation lD), consistent with [m*] being constant during

assembly and equal to m, the number concentration” of added

MTF. To a very good approximation, assembly proceeded

exponentially to an equilibrium steady state with rate con-

stants, k, linearly dependent on m, whereas the mass concen- trations of microtubule polymer formed were independent of

m but related to k,, k-, and tubulin concentrations in the

manner predicted by Equations 1C to 1E (10, 13, 14).

If CD inhibits microtubule assembly by an end-poisoning

mechanism, one would expect [m*] to decrease with time

during an elongation reaction as CDs accumulate at the

microtubule ends and block assembly. The condition for

pseudo-fast order kinetics, i.e. constant [m*], would no longer

be satisfied, and as a result a nonexponential approach to

steady state would be predicted. The results of our elongation

reaction study are not consistent with this prediction. Al- though we could readily confirm that CD inhibits microtubule

assembly substoichiometrically (5), we were unable to detect

any deviations from exponential kinetics during assembly that

could be ascribed to CD-induced changes in [m*]. In addition,

when we studied the initiating, i.e. seeding, competence of

microtubules assembled to steady state in the presence of CD,

we found that they had ends capable of initiating elongation

reactions. Furthermore, we found that the degree to which CD inhibited assembly did not correlate with the number of

CDs incorporated per microtubule, but did correlate with the

relative amounts of incorporated CD and tubulin, i.e. with the

composition of the microtubule polymer. We interpret our

data as indicating that CD substoichiometrically inhibits elon-

gation reactions by a mechanism other than end poisoning.

MATERIALS AND METHODS

The procedures for preparing tubulin and CD, together with the methods used to determine (a) CD/tubulin ratios in the microtubules; (b) microtubule lengths; (c) a, the fraction of active tubulin; and (d) characterize the assembly process are presented in the miniprint supplement.”

Absorbance Measurements-Microtubule assembly at 37’C was monitored spectrophotometrically (9) by use of a Gilford 2400-2, a multichambered spectrophotometer capable of measuring the absorb- antes in four samples simultaneously. Changes in the absorbances at 350 nm with time, A(t), were recorded and analyzed as described below. Previous studies of the light-scattering properties of microtu- bule solutions have shown that absorbance changes are proportionally related to the mass concentration of microtubule present (15). In this study we used the relationship microtubule (mg/ml) = 6.5 x A (Table IS).

MTF As Initiators of Elongation Polymerization-MTF stocks containing -23 pM tubulin (D toti) in PB-2.5 M buffer were prepared by sonicating l-ml aliquots of spontaneously assembled microtubule preparations (-0.3 AU) for 20 s at 40 W with a Heat System model W185 sonicator equipped with a microtip. Small volumes, V,, of stock were used immediately to initiate assembly in l-ml aliquots of hepa- rin-inhibited tubulin solutions containing 1 mM GTP and varying amounts of CD (0 to 1.2 PM). Small volumes of stock were also removed immediately for length determinations by electron micros- copy. The reproducibility of the sonication procedure was tested by

’ The number concentration, expressed in molar units, is (100O/N0) x (number of MTF/ml), where NO is Avogadro’s number, 6.023 X 10”‘.

’ Portions of this paper (including Figs. IS to 3S and Table 1s) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda. Md. 20014. Reauest Document No. 79M-235. cite author(s), and include a check or money order for $1.20 per set of photocopies.

seeding identically prepared tubulin solutions (23 PM &,f, 0.13 mM heparin) with constant volumes of MTF from 8 different stock prep- arations and comparing initial assembly rates. Initial assembly rates were determined from the initial slopes observed in absorbance versus time plots. Initial slope values were identical to within approximately 30% and gave a standard deviation of approximately f16% when averaged. When MTF from a common stock were used to initiate polymerization in identical samples and assembly rates compared, initial slope values were identical to within &5%.

Electron microscopy examinations of the MTF stocks indicated that the fragment lengths varied from -0.4 to 1.2 pm and had average lengths of -0.7 + 0.15 am. MTF number concentrations in the stocks, M,s, were determined by a process described earlier by Bryan (14). M, = 9 X 10” fragments/ml = 1.5 x lo-” M, based on an average fragment length of 0.7 pm. In this study, m, the MTF number concentration present in the heparin-inhibited solutions at the start of assembly, was estimated from the number concentration of the stock and the dilution factor: m - rno = M,V, (1 + V,)-‘.

In the majority of cases, kinetic comparisons were made between samples which differed in CD concentrations but which were initiated with MTF from a common stock. As a result, relative differences in assembly rates could be determined accurately. In these cases we set m = mo. The results from repeat runs, using different MTF stocks but analyzed similarly (m assumed equal to mo), were often averaged for greater statistical accuracy. In other cases (see initial rate studies below) a series of runs each containing a different but constant CD concentration were initiated with different MTF stocks, and com- pared. In order to facilitate the study, a CD-free “internal reference” sample was also included in each run as a control. Initial assembly rates of the internal reference samples were compared with that from the identically prepared internal reference sample of the CD-free run in order to derive scaling factors, F,, and effective m values, m = F, x ml,, which compensated for sonication differences between runs. This procedure facilitated the kinetic analysis and obviated the need to average over large numbers of samples for statistical accuracy.

Tub&in Activity Analysis-Analysis of the kinetic data below required knowing a, the fraction of active tubulin available for assem- bly. Values for (Y were determined using CD-free solutions for conven- ience, noting that active tubulin in solution is in equilibrium with tubulin in the microtubule phase at steady state. The solution con- centration of active tubulin at steady state was assumed to have a constant value, D,“, independent of DH.I., the tubulin concentration in the microtubule phase at steady state, and &,I, the total protein concentration, as required by Oosawa’s condensation theory (14, 16, 17). D,” corresponds to the critical tubulin concentration required for assembly and varies with polyanion concentration (13, 14). Values for LY and D,” were determined from a mass balance based on active tubulin concentrations:

Di,,.l. + DC0 = a Dtota,; (2)

D,..,.I. = a Dt,td - D,‘.

The experimental procedures and results for spontaneous and hepa- rin-inhibited assembly are discussed in the miniprint.

Data Analysis-The steady state absorbances, A,, were observed to decrease slowly with time in the heparin-inhibited solutions both in the presence and absence of CD: A, = AT(t) (Fig. 3S, also below). Generally, A(t) reached a maximum absorbance change and then slowly decreased. Adding an additional 1 mu GTP after the maximum was reached, to compensate for GTP hydrolysis during assembly, had little or no effect on A,(t). The observed decrease in A,(t) with time is interpreted as arising from a spontaneous net depolymerization of microtubules and, presumably, is related to thermal denaturation of free tubulin, although other inactivation mechanisms may also be contributing.

It was desirable to have an analysis procedure which would com- pensate for thermal denaturation and background interference, thus facilitating comparisons of assembly parameters. The following pro- cedure was used: Am(t) values were linearly extrapolated from the time range where assembly was complete into the time range where assembly was occurring (Fig. 3s). This procedure enabled us to estimate A,“, the A, value at zero time. A,’ values were assumed equal to the steady state absorbance values which would have been obtained in the absence of denaturation. A linear extrapolation was equivalent to fitting A,(t) to an exponential with a small, negative time constant. When a single value for A, was needed for purposes of comparing assembly behavior, it was A,” which was often used. Thus the assembly parameter “per cent inhibition,” which compares assem-

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10542 Colchicine Inhibition of Microtubule Assembly

bly in the presence of CD with assembly in a CD-free reference of identical tubulin and heparin concentrations, was defined as:

per cent inhibition = A,” (no CD) - A,” (with CD)

A,” (no CD) x 100%. (3)

Per cent inhibition is based on microtubule yields corrected for protein denaturation:

&r(mg/ml) = 6.5 A,“.

In a similar manner, when A, was plotted against 01 Dw,I, A,” values were used for A,. However, when microtubule lengths were calculated and compared with observed values, the absorbances at the time of sample removal were used (Equation IS).

Assembly rate constants, k, were obtained in the presence and absence of CD, by fitting least square lines to plots of In 8 uer.sus time:

In ([A,(t) - A(t)]/&(t)) = -kt = In 8. (4A)

A,(t) and A(t) denote, respectively, the extrapolated A,(t) and the observed A(t) values corrected for a background contribution, Aback(t) - 10-4t (min), and for the turbidity contribution introduced with the seeds which causes an abrupt jump in absorbance at zero time:

A,(t) = Am(t) - A,,mp - IO-% (4B)

/i(t) = A(t) - A ,u,n,, - lo-?. (4C)

Aback(t) appeared to arise in part from a nonspecific aggregation (denatured tubulin?) and, for about the fist 3 h, was approximated well as a linear function of time, Aback(t) -. 1 x 10m4t. After approxi- mately 3 h, Aback(t) in these studies became nonlinear and increased rapidly with time (see below). Ahack is discussed in more detail in the miniprint supplement (Fig. 3s). A,(t) used in the kinetic simulation studies below (Figs. 1 to 3) had the form: A,(t) = A,” - A,,,,,,, - 1.8 f 0.2 x 10 9.

Eq. 4 can be rewritten as:

In ([A,(t) - A(t)]/[A&) - A,,,,,,,, - 10e4t]) = -kt. (5)

Equation 5 was used previously for assembly studies in the absence of CD (13,14). In these earlier studies, background contributions were neglected, and A,(t) was approximated by a constant.

Equation 3A can also be expressed as:

A(t) = A,(t)(l - emK’). (6)

This equation was used successfully below to simulate assembly kinetics in the presence and absence of CD.

Initial rates of assembly were determined from the initial slopes, dA/dt, of absorbances uersus time plots, and were studied below as a function of varying &,A at fixed CD values. Initial slope values were corrected for background interference by subtracting dAhack(t)/ dt - 1 X 10m4, and corrected for sonication variability by multiplying the remainder by a resealing factor, F,. F, was set equal to 1 in the CD-free runs (see “Materials and Methods”). The corrected slopes were plotted against (01 &,,,I),, the active tubulin concentration in solution at the start of assembly reaction, and analyzed in accord with

II

06

I I I I I I I I 1 I I II 30 60 90

Johnson and Borisy’s procedure (10):

dA/dt = (h+ m/C)(a D,<,,,Jl - (hm m/C),

where C is the conversion factor, 59 PM/AU (Equation 4s).

(7)

RESULTS

Figs. 1 and 2 show A(t) results obtained from heparin- inhibited tubulin solutions that underwent elongation polym- erization at 37°C in the presence and absence of CD. In one study (Fig. l), CD concentrations were varied while concen- trations of tubulin, heparin, and MTF were held constant. A large m (80 ~1 of MTF) was used to induce rapid polymeriza- tion and minimize the effects of protein denaturation on assembly kinetics. Progressively reduced assembly rates and steady state yields were observed with increasing CD concen- trations (Fig. lA), confirming that CD inhibits microtubule assembly (5). In all cases, the observed kinetics were simulated successfully as exponential approaches to steady states, A,(t), in accord with Eq. 6, with rate constants, 12, which decrease with increasing CD (Fig. 1A). The reduction in k with increas- ing CD was evident at the start of the assembly reaction. The k values used in the simulation were least square values derived from semilog plots of the approach to steady state (Equation 4A, Fig. 1B). Although some deviations from lin- earity were observed in the semilog plots, the extent of these deviations, measured by comparing initial and least square- determined rates (Table I), were small and independent of CD concentration. We conclude from the above results that elongation kinetics in the presence and absence of CD are approximated well, and to the same degree of fitness, by pseudo-fist order kinetics. The kinetic results are summarized in Table I. In the absence of CD, polymer yield and k+, the association rate constant, agreed well with previously reported values obtained with polyadenylic acid (14). This confirmed earlier observations that polyanions at equivalent charge con- centrations inhibit microtubule assembly similarly (14). CD/ tubulin ratios in the microtubules ((CD/T)MT) were deter- mined at steady state (Fig. 1, Table I). (CD/T)MT values increased with increasing CD and were less than 0.01, confiim- ing earlier observations that substoichiometric amounts of incorporated CD inhibit assembly (5).

In a subsequent study, MTF concentrations were varied while concentrations of tubulin, heparin, and CD were held constant (Fig. 2). In this example, [CD] = 0.24 PM. The kinetic behavior observed was qualitatively identical to the behavior observed in the absence of CD (13,14). A common absorbance, A,(t), is reached that is independent of MTF concentration (Fig. 2A). A,(t) was observed to decrease slowly with time:

A,(t) = A,” - 6.5 x 10 “t,

FIG. 1. Microtubule assembly as a function of CD concentration. One- milliliter aliquots containing 23 pM tu- bulin (D,,,,L), 0.13 mM heparin, 1 mM GTP, and 0.0 (O), 0.17 (0). 0.34 (A), and 0.68 (a) PM CD were incubated at 37°C for approximately I5 min prior to the addition of 80 al of MTF (m = 11 X lo-“’ M). Assembly kinetics were simulated (solid curues, A) in accord with Equation 6 using assembly rate constants, k, de- rived from least square linear plots of In 6’ uersus time (solid lines, B). Aliquots were removed approximately I’% h after assembly began, and the CD to tubulin ratios in the microtubules were deter- mined.

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Colchicine Inhibition of Microtubule Assembly 10543

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min 2 6 IO 14

fJ (~lo-‘” M )

FIG. 2. Microtubule assembly as a function of MTF addition. MTF from a common stock (80 (O), 40 (O), 20 (A), and 10 (A) ~1) were added to l-ml ali- quots containing 23 PM tubulin, 0.13 mM heparin, 1 mM GTP, and 0.24 PM CD. m varied from -1.4 X lo-“’ M (for lo-~1 addition) to 11 x lo-” M (for 80-~1 ad- dition). A control sample (- - -) lacking CD, but otherwise of identical composi- tion, was initiated with MTF, and polym- erized in the manner shown (A). The control had an A,” value of approxi- mately 0.108. Assembly kinetics were simulated (solid curues, A) in accord with Equation 6, using rate constants, k, derived from B and assuming a com- mon A&) equal to 0.079 f 0.003 - 6.5 x 10m5 t. The rate constants were linearly dependent on m: k = k+ m + k” (C).

TABLE I

Assembly as a function of CD

Assembly was initiated by adding 80 1.11 of MTF (Fig. 1). D,,,,l = 23 FM, heparin = 0.13 mM, a - 0.57, m = 11 x lo-“’ M.

Assembly rate constants

CD Microtubule yield R inhibi-

m.vr”) tion Least square* ki” Incorporation ratio Initial (CD/TIM.,

(k) (k/j

PM PM x lo-‘min-’ x lo-‘min-’ X 10”mine’Me’ x lo-”

0.0 6.2 0 7.7 8.8 0.70 0 0.17 5.2 16 5.7 6.5 0.52 4.1 0.34 4.3 31 4.5 5.0 0.41 6.0 0.68 3.1 50 4.1 4.5 0.37 8.3

o DM.,, is expressed in tubulin equivalence (9.1 pM = 1 mg/ml). DWI. (PM) = 59 A,” (Equation 4S), and denotes microtubule yield corrected for protein denaturation during assembly.

’ Based on semilog data covering approximately 2 time constants (-85% of the reaction). ’ Based on semilog data covering approximately 0.3 to 0.4 time constants (-35% of the reaction). ” Apparent association rate constant (h+ = h/m).

with t in minutes and A,” - 0.079 f 0.003. A,’ was approxi- mately 27% less than the value observed in the absence of CD (Fig. 2A, control). Semilog plots of the approach to steady state were linear to a good approximation at all m values, and gave k values which increased with m (Fig. 2B) and which varied from approximately 0.6 to 4.6 X 10m2 min-‘. The cal- culated A(t) based on these k values (solid lines, Fig. 2A) agreed well with the observed A(t). A linear relationship between k and m was noted, k = k+ m + k0 (Fig. 2C), with k, = 0.41 + 0.02 X 10’ mini’ Mm’ for an &fold variation in m in this particular example (23 pM D,,,,I, 0.24 FM CD, 0.13 mM heparin). k” was -1 x 10e3 min-‘, a value consistent with the small residual spontaneous assembly reaction evident when m

= 0 (Fig. 3s). We interpret k+ to be a second order association rate constant for assembly in the presence of CD (see “Dis- cussion”).

The kinetic results obtained in the presence of CD-expo- nential relaxation to a steady state absorbance A,(t), with rate constants, k, proportional to m (Figs. 1 and 2)-suggest that microtubule ends remain assembly-competent during polymerization.

If the microtubules formed in the presence of CD have assembly-competent ends at steady state, it should be possible to use these microtubules instead of MTF to initiate polym- erization in heparin-inhibited solutions. Results from experi- ments similar to that displayed in Fig. 3 confirmed this pre- diction. MTF were added to a l-ml aliquot from a heparin- inhibited tubulin solution (Solution IO), which contained 0.24 pM CD, and initiated an elongation polymerization. When the polymerizing solution, Solution I (Fig. 3), reached steady state, it was mixed 1:l with an aliquot from Solution 10 and initiated

FIG. 3. Microtubules assembled in the presence of CD have assembly-competent ends. Forty microliters of MTF were added to l-ml aliquots of solution containing 20 PM tubulin (D,,,,J, 0.24 pM

CD, 0.13 mM heparin, and 1 mM GTP (solution I,,), and initiated polymerization (0, A and B). A control sample containing no CD but otherwise of identical composition, was initiated with MTF as indi- cated (A). After approximately 120 min, an aliquot from Solution I at steady state was mixed 1:l with solution Zo, and initiated polymeri- zation (0, A and B). Assembly kinetics were simulated in accord with Eq. 6 (solid curves, A) using assembly rate constants derived from B, and assuming A,” values of 0.055 and 0.051 for Solutions I and II, respectively. The A,” differences presumably represent denaturation effects. Background interferences, apparent after approximately 180 min, were responsible for the divergences between calculated and observed absorbances which developed at late times. The k value estimated for Solution II assembly was derived from data limited to the initial 60 min of Solution II assembly (B). Similar background interferences were often observed in these studies after long times (CL Fig. 2) and occurred both in the presence and absence of CD.

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10544 Colchicine Inhibition of Microtubule Assembly

polymerization (Solution II, Fig. 3). The number concentra- tion of microtubule seeds in the 1:l mixture was half the number concentration of MTF used to initiate polymerization in the earlier & aliquot, and the 1:l mixture, Solution II, underwent an elongation polymerization characterized by an assembly rate constant, k, which was half that observed for Solution I. The results of the kinetic analysis are summarized in Table II. K, values calculated for Solutions I and II assem- bly agree well with each other (Table II) and compare well with the k, value of 0.41 x 10’ min-’ M-' obtained earlier for samples of the same composition which were initiated with MTF (Fig. 2C). The number of CDs incorporated per micro- tubule of average length from Solutions I and II were esti- mated at steady state from the absorbances, A,, and the measured CD/tubulin ratios for the microtubules, (CD/T)MT, and had values of approximately 17 ? 2 and 27 & 2, respec- tively (Table II, Equation 3S with A, - 0.045). (CD/T)MT values for the microtubules from Solutions I and II were similar and indicated that substoichiometric amounts of in- corporated CD inhibit assembly (Fig. 3, Table II).

A varying MTF experiment analogous to that shown in Fig. 2 was done in order to investigate CD incorporation under conditions of constant per cent inhibition (Fig. 4). A lowered heparin concentration facilitated the study. Per cent inhibi- tion and k, were observed to be constant, to a very good approximation, over an &fold variation in m. The observation that (CD/T)Mr was also constant as m was varied (Table III) suggested to us that it is the relative numbers of the incor- porated CDs and incorporated tubulins, i.e. polymer compo- sition, which determines inhibition, rather than the absolute number of CDs per microtubule. This hypothesis was sup- ported by a determination of N,, the CD incorporation num- ber, using measured (CD/T)MT and average microtubule lengths at steady state (LMT) (Table III, Equation 2s). LMT values as a function of m were determined from microtubule length distributions (Fig. 5) obtained by dark field microscopy (18). The sum of the observed microtubule lengths in 1 ml at steady state is equal to 6.0 x 10’” x m x LMT, and had an average value of 1.0 -t 0.4 X lOI* pm rn-’ over the g-fold variation in m. This average value agrees well with the theo- retical value of 1.05 + 0.04 x 10” pm ml-’ predicted for an elongation polymerization based on the common A, value of -0.048 -t 0.002 at the time the samples were removed (Fig. 4, Equation IS). The observed average microtubule lengths (Fig. 5) and their predicted values at steady state (Equation 1s) are in good agreement, with discrepancies between observed and predicted values generally no greater than about 35% (Table III). N, varied from approximately 25 to approximately

FIG. 4. A, Varying volumes of MTF were added to l-ml aliquots containing approximately 14 PM tubulin (&,,.I), 0.34 pM CD, 38 FM

heparin, and 1 mM GTP, and induced assembly to a common A,” (A,” = 0.048 * 0.002). After steady state was reached, aliquots were removed for microtubule length and (CD/T)M.I. determinations. B, assembly rates, k, were estimated from initial slopes, dA/dt, (-. -) and were linearly dependent on m: k = k+ m + k” with k+ = 1.1 x 10’ min-’ M-’ and k” - 2 x 10m3 mini’ (k = dA/dt/A,“).

TABLE III

Per cent inhibition is independent of the number of incorporated CDs when microtubule composition is constant

Varying MTF experiment done under conditions of constant Dt,,,,l (14 PM), CD (0.34 FM), and heparin (38 PM).

Average length of mi- Number of crotubules CDs incorpo-

m % inhibi- rated/micro-

tiorf (CD/T)M.I Observed” Pre- tubule ofav-

.&I dieted

LF.41 erage lengthd

(NJ

x 10-10 M x 10-3 Pm P

11 t t 2.3 + 0.11 1.7 26 -+ 2 5.5 Constant Constant 3.2 + 0.15 3.2 36 +- 2 2.8 (30 + 2%) (7.1 -c 0.3) 4.8 + 0.2 6.4 55 -+ 3

1.4 1 1 7.9 -c 0.35 12.2 90 -t 5

“ Per cent inhibition values were calculated from A,” data (Fig. 4 and Eq. 3).

’ Length distributions based on sample sizes of -75 to 100 micro- tubules. Lengths were determined using dark field microscopy (Fig. 5). Errors are S.E.M.

’ Based on m, number concentration of seeds, and the absorbances at time of sample removal (Fig. 4 and Equation 1s).

” Calculated from the observed average microtubule lengths, LWI., and (CD/T)MT values (equation 2s).

90 as m varied over a factor of 8, while per cent inhibition and (CD/T)Mr remained constant. This finding suggests that, if it were practical to repeat the experiment with a set of very small m values so as to obtain very large length microtubules,

TABLE II

Microtubules assembled in the presence of CD as initiators of elongation polymerization

D loral = 20 pM, CD = 0.24 pM, heparin = 0.13 mM.

Number concentration Average length of Number of CDs incorporated Seeds of seeds in polymeriz-

Assembly rate constants” (k) k, = k/m (CD/T)W~ microtubules’ into microtubules of average

ing solution (m) LR, lengthd (N,)

x 10-‘O M X 10e2 mine’ X 10’ min-’ hi -’ x 1o-3 w

MTF’ Solution I 5 2.0 + 0.06 0.40 + 0.02 3.3 -c 0.3 3.3 17 + 2

MT’ Solution II 2.5 0.94 + 0.05 0.38 k 0.02 2.6 f 0.3 6.5 27 + 2

” From Fig. 3. ’ Determined as described under “Materials and Methods” in the miniprint supplement. ’ Calculated from the absorbances at steady state (Fig. 3) and from the seed concentrations, m (Equation 1s). The calculated lengths were

consistent with estimates based on visual examinations of electron photomicrographs. “Calculated from LR,r and (CD/T)M.I. values (Equation 3s). ’ Forty microliters of MTF were added to l-ml aliquots from solution ZO and initiated polymerization (Fig. 3, Solution I curve). ‘MT = Solution I microtubules which were assembled to steady state in the presence of 0.24 pM CD. These microtubules were added in a

1:l volume ratio to a l-ml aliquot from solution ZO, and initiated polymerization (Fig. 3, Solution II curve).

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Colchicine Inhibition of Microtubule Assembly 10545

FIG. 5. Microtubule length determinations. Assembly was in- duced by adding varying MTF concentrations to heparin-inhibited tubulin solutions containing 0.34 PM CD (Fig. 4). After steady state was reached, aliquota were removed for microtubule length determi- nations. A, a representative dark field photomicrograph of glutaral- dehyde-fixed microtubules which were assembled by the addition of

very large values of N, indeed would be obtained while (CD/ T)MT and per cent inhibition would remain unchanged. We conclude that, at constant (CD/T)MT, assembly inhibition is independent of N,.

Although per cent inhibition does not correlate with the number of CDs per microtubule, it does correlate with (CD/ T)MT, i.e. with polymer composition. (CD/T)MT ratios in the microtubule polymer increases with increasing CDtotal for con- stant &,I (Table I). When (CD/T)M~ increases, per cent inhibition increases (Table I); when (CD/T)MT remains con- stant, per cent inhibition remains constant (Table III). In the example chosen (23 pM &,,,,I, 0.13 mu heparin), a 4-fold increase in CDtow results in approximately a 2-fold increase in (CD/T)Mr, and approximately 3-fold increase in the relative per cent inhibition (Table I).

Microtubule assembly in the presence of heparin and CD was studied as a function of Dtotsl. Representative data are shown in Fig. 6, A and B. The data were analyzed to obtain steady state absorbances, A,‘, and initial rates of assembly (Fig. 6, C and D). When A,’ values are plotted against a Dto,l for various fixed CD concentrations, a series of parallel lines are obtained (Fig. 6C) whose abscissa intercepts correspond to the critical, i.e. minimum tubulin concentrations (D,) re- quired for assembly. The concentrations of active free tub&n in the solution phase at steady state were observed to be independent of microtubule yield and DIOtal, but to be depend- ent on CD, and were observed to equal the critical concentra- tions, D, (Fig. 6C, inset). Previous studies done in the absence

15 - _ B

L ova. P

7.9

4 6 8 IO 12 14

pm, MT LENGTH

10 pl of MTF (m = 1.4 X IO-‘” M). The field displayed corresponds to an area of approximately 2.4 X lo3 pm*. Four to five such fields, containing approximately 75 to 100 microtubules, were scored to obtain average microtubule lengths &MT). Horizontal bar f-) corresponds to a length of 10 pm. B, microtubule length distributions as a function of m derived from dark field microscopy measurements.

of CD have indicated that a necessary condition for this equality to hold is that the solution and microtubule phases be in equilibrium (16, 17). Polymerization and depolymeriza- tion rates are equal at equilibrium, with k- = k+ D, (cf. Equations IA and 1B). (D,)-’ has been interpreted as the affinity constant for tubulin addition to microtubule in the absence of CD (19). The results shown in Fig. SC, supported by the results of the other experiments described in this section, suggest to us that the same interpretation of D,-’ and the same relationship between k-, k+, and D, hold when assembly occurs in the presence of CD. D, increases with increasing CD. We therefore infer that the affinity of the microtubule for tubulin decreases with increasing CD, i.e. increasing (CD/T)MT. Noting that k+ decreases with increas- ing CD (Table I), we estimate that k- remains approximately constant, or decreases slightly with increasing CD over the CD range studied (CD d 1 PM). In the absence of CD we estimate k- to have a value of approximately 5 x lo* mm-‘, similar to the value reported by Johnson and Borisy (10) for their CD-free system depleted of microtubule-associated pro- teins (MAPS).

Initial rates of assembly in the presence of CD were studied by plotting initial values for dA/dt against (a DtotaJ,, the active tubulin concentration in solution at the start of the assembly reaction. Straight line plots were obtained (Fig. 6D). As CD increased, the slopes of these lines decreased, whereas the abscissa and ordinate intercepts both increased. Following Johnson and Borisy (Ref. 10, Eq. 7), we estimated k,, D,, and

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10546 Colchicine Inhibition of Microtubule Assembly

00’ I I I

30 60 90

.08

0. a

04

TOTAL ACTIVE TUBULIN, pM

min.

8(

x C Y a u-4(

-81

60 90

k- by setting slopes equal to k+ m/C, abscissa intercepts to D,., and ordinate intercepts to (-km m/C). C is the conversion factor, 59 PM/AU (Eq. 4s). This analysis assumes that initial assembly rates are equal to the difference between initial rates of polymerization and depolymerization (10). The estimates of k,, D,, and k- from the initial rate analysis are in good agreement with the values in Table I for k+ and Fig. 6C for D,, and with the values for k- as estimated from the product k+D,. (Table I, Fig. 6C). This agreement between two inde- pendent methods of analysis supports our perception that assembly in the presence of CD proceeds with constant [m*]

but with altered (CD-dependent) k, and k- values. In this example, where CD increased from 0.0 to 1.2 pM (Fig. 6D), k, decreased from approximately 0.67 to 0.35 x 10’ min-’ M-l,

whereas k- decreased from approximately 5 to 4 x 10’ min-‘, as D, increased from approximately 7 to 11 pM.

DISCUSSION

The earlier observation that CD inhibits microtubule as- sembly substoichiometrically was suggestive that CD poisons microtubule ends during assembly (5). However, we interpret our data as indicating that microtubules assembled in the presence of CD are inhibited by a different mechanism in which the ability of microtubule ends to add dimers is pre- served.

Elongation kinetics in the presence of CD was monitored by absorbance measurements at 350 nm. The approach to steady state had a functional dependence on time which was similar to that reported for assembly in the absence of CD (10, 13), and consistent with pseudo-fust order kinttics. The

FIG. 6. Assembly as a function of D total. Assembly in l-ml aliquot samples were initiated with 80 ~1 of MTF. In A and B, the solutions contained varying tubulin concentrations (D,,,,,), 0.13 mM heparin, 1 rnM GTP, and the indicated CD concentrations (0.0 or 0.68 FM). Dt,til values were: A, 17.3; 0, 19.1; A, 20.9; n , 21.8; l ,22.8; and Q24.6 m&r. Each of the runs was initiated with MTF from differ- ent stocks. A CD-free control sample (0) was therefore included with each run. The initial slopes (*) of these con- trols were compared, and resealing fac- tors were derived to correct for sonica- tion variability as discused above. Steady state absorbances A,” and initial rates, dA/dt, were obtained from data similar to that shown in A and B, and were plotted against total active tubulin ((Y &,,,I), and corrected for MTF addition (C and D). CD concentrations were: 0, 0.0; A, 0.17; 0, 0.68; and A, 1.2 pM; and (Y - 0.60. Critical tubulin concentrations, D,, were set equal to the abscissa inter- cept values in C and were observed to equal a &,,,I - DP.w, the concentrations of active free tubulin in the solution phase at steady state (C, inset).

observed kinetics were approximated well by the expression:

A(t) = A&)(1 - e-&*),

= A&)(1 - e-‘*“O. (6)

Assembly rate constants, k, were proportionally dependent on m, while A,“, steady state absorbances corrected for protein denaturation, were independent of m (Figs. 2 and 4). k”, the contribution to k from a small spontaneous nucleation com- ponent, is neglected in Equation 6. The similar kinetic be- havior observed in the absence and presence of CD suggests to us that CD incorporation affects k, and A,“, but does not cause changes in [m*], the number concentration of assembly- competent ends. For a constant Dtotal, k, and A,” values were observed to decrease with increasing (CD/T)MT (Table I, Fig. 6C). On the basis of these findings, we perceive microtubule assembly in the presence of CD and heparin to be a copo- lymerization reaction involving tubulin and CD in which [m*] remains constant during assembly whereas k, and A,’ depend on the composition of the CD-tubulin copolymer formed. We view k, as a composite which corresponds to an appropriately weighed average of tub&n and CD association rate constants. The major contribution to k+ presumably comes from tubulin, since it is the major component in the microtubule.

This interpretation of the kinetic results is supported by the following observations.

1. We were able to show that microtubules assembled in the presence of CD have appreciable numbers of incorporated CDs at steady state (e.g. Table II). Nevertheless, these micro- tubules have assembly-competent ends at steady state capable

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Colchicine Inhibition of Microtubule Assembly 10547

of initiating elongation polymerization reactions in heparin- inhibited solutions that proceed with assembly rates consist- ent with [n*] = m (the number concentration of added microtubules). Thus, in all cases, irrespective of whether seeds were derived from microtubules assembled in the presence of CD or whether MTF were used, we found that k (observed) = k, m, with m denoting the number concentration of added seed, and k, being a common constant (Fig. 3, Table II).

2. I f an end-poisoning mechanism was operative with bound CDs confined primarily to the assembly end of the microtu- bule (5), it seems reasonable to expect the degree of inhibition to correlate with the number of CDs at the end, i.e. with the number of CDs bound per microtubule. In this study, N,, the number of incorporated CDs per microtubule of average length at steady state, was used as a measure of CD incorpo- ration. Inhibition at constant (CD/T)MT was independent of N,,, even though N,, in one instance, was approximately as large as 90 (Table III). However, inhibition did correlate with microtubule composition, i.e. with (CD/T)MT (Table I).

3. Assembly rates are progressively reduced in the presence of CD relative to the CD-free control (Fig. 1, Table I). In all cases, the reduction is evident at the start of the assembly reaction, while assembly can be simulated well by an expo- nential approach to steady state. This finding is difficult to understand if inhibition occurs by an end-poisoning mecha- nism. If an end-poisoning mechanism were operative, one would expect initial assembly rates to be similar, if not iden- tical, to the CD-free control, since the ends of the elongating microtubules would initially be free of CD. As ends accumu- late CD, the apparent rate constants would be expected to decrease with time. The experimental results, however, can be understood if CD and tubulin copolymerize while copoly- mer ends remain assembly-competent. The copolymerization reactions are presumed to be characterized by assembly rates dependent on copolymer composition. An exponential ap- proach to steady state would be expected if copolymer com- positions remained essentially constant during assembly. Co- polymer composition, in turn, should depend on CD and tubulin concentrations in the solution phase during assembly. In this study, per cent changes in solution phase composition during assembly were small.

The inhibition mechanism we propose for assembly in the presence of CD is similar in several respects to the way in which MAPS and nucleotide enhance assembly (19, 20). Both the inhibition and enhancement processes are sensitive to substoichiometric amounts of a secondary species; both proc- esses appear to involve copolymerization reactions which form copolymers whose assembly kinetics and affinity constants for tubulin depend upon composition. In the case of MAPS, affinity constants for tubulin increases with increasing (MAP/ T)MT (19,21). In the case of CD, affinity constants for tub&n, as measured by D,-’ (Fig. 6C), appear to decrease as (CD/ T)wI. increases. MAPS stabilize microtubules by decreasing k-, whereas k, is relatively unaffected (19). However, CD in the presence of heparin appears to destabilize microtubules by decreasing k+, whereas k- is relatively unaffected. These results were obtained under MAP-depleted conditions with low concentrations of CD (~1.2 PM), and appear to be inde- pendent of glycerol and the particular polyanion used.4

We suspect that incorporated CDs are randomly distributed in the copolymer and that the composition of the copolymers remains nearly constant during assembly. A random distri- bution of CD, averaged over the large number of microtubules present, should lead to a macroscopically uniform copolymer- ization process. As a result, exponential elongation polymeri- zation kinetics would be expected for a constant number of

’ H. Sternlicht and I. Ringel, unpublished results.

assembly competent ends, as was observed. A random distri- bution would also be consistent with our finding that inhibi- tion correlates with (CD/T)M,~ values, i.e. with the relative amounts of incorporated CD and tubulin (Tables I and III). Substoichiometric amounts of incorporated CD were capable of significantly altering copolymer affinity for tubulin. The molecular basis for this remains to be determined. We pre- sume that (CD/T)w, ratios measured correspond to CDs directly incorporated into the microtubule lattice, and that it is these incorporated CDs which perturb assembly kinetics. We regard this interpretation as the most simple and plausible. CD differs conformationally from tubulin (22). As a result, CDs may incorporate differently than tubulin into the micro- tubule and affect assembly kinetics by perturbing short and long range organization. These perturbations could be signif- icant at substoichiometric levels of CD and would be expected to correlate with (CD/T)MT ratios. Recent electron micros- copy and optical diffraction studies suggest that specific inter- actions between tubulin subunits exist which give rise to long range structural features in the microtubules. Microtubules show large (96 nm) axial periodicities (23, 24). These perio- dicities have been discussed in connection with MAP-micro- tubule associations (23, 24) and appear to be related to the periodic change in tubulin subunit orientation along the mi- crotubule protofiiaments observed under MAP free conditions (25, 26). We suggest that an investigation of the effects incor- porated CDs have on microtubule lattice periodicities may help clarify the inhibition mechanism.

We regard as less likely alternate interpretations which may assume that (CD/T),,, ratios contain significant contributions from CD aggregates or CDs adventitiously attached to micro- tubules, or which may assume that the observed decrease in affinity constant for tubulin in the presence of CD reflects a MAP depletion process caused, for example, by MAP. CD complex formation. (CD/T)MT values were observed to be reproducible from brain preparation to brain preparation, and the values obtained were confirmed using the sucrose-layering technique.4 The latter centrifugation technique was used by Margolis and Wilson (8) in their “endcapping” studies of spontaneously assembled microtubules, which indicated con- siderably smaller numbers of incorporated CDs per microtu- bule under conditions of considerably larger concentrations of colchicine and CD relative to the present study. Gel electro- phoresis studies of microtubules assembled in the presence of 0.13 mM heparin indicated that microtubules were essentially devoid of high molecular weight MAPS because of MAP complexation with heparin, a result consistent with previous polyanion studies (13, 14). Furthermore, we observed no dif- ferences between gel electrophoresis patterns when microtu- bules were obtained by assembly in the presence of 0.13 mM

heparin, or by assembly in the presence of 0.13 mM heparin and ?l PM CD. Assembly kinetics were not characteristic of a MAP depletion process. As discussed above, we observed that k, decreased, whereas k- remained essentially constant as CD increased (Table I, Fig. 6D). However, Murphy et at. observed, in a study done in the absence of CD, that k, remains essentially constant, whereas k- increases, as MAPS are removed (19). We have also studied spontaneous assembly in the presence of CD (no heparin (Refs. 27 and 28)).” D,. was observed to increase with increasing CD in a manner similar to that reported in this heparin study (Fig. 6C). Gel electro- phoresis studies of microtubules formed in the presence of CD concentrations as high as 5 pM indicated significant amounts of MAPS, and showed no significant differences in MAP content and electrophoresis pattern when compared with microtubules assembled in the absence of CD.” These findings

’ H. Sternlicht and I. Ringel, manuscript in preparation.

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10548 Colchicine Inhibition of Microtubule Assembly

support our interpretation that the decrease in copolymer affinity for tubulin with increasing CD, as indicated by the increase in D, (Fig. 6C), arise from CDs directly incorporated into the microtubule lattice.

We envisage microtubule assembly in the presence of CD to be a copolymerization reaction involving tubulin, CD, and MAPS as “monomer” components with yields and assembly rates dependent on monomer ratios and copolymer composi- tion. Although this interpretation differs from the prevailing view of end poisoning, we suggest that the two mechanisms are in a general sense consistent with each other. Differences in interpretation may reflect the different approaches to the study. We examined microtubule assembly in the presence of colchicine under conditions where tubulin was entirely in the solution phase when assembly was initiated, and we observed that an equilibrium state was reached dependent on copoly- mer composition. Previous investigators, who proposed the end-poisoning model, were concerned with understanding the effects of colchicine added at steady state, where the major fraction of the tubulin was in the microtubule phase, and they observed a different kind of behavior (5, 8). One expects, according to the copolymerization model, that when CDs are added to microtubules at steady state, microtubule mass would be lost as the system relaxes from the original equilib- rium state, determined by total tubulin and MAP concentra- tions, to the new equilibrium state determined by total tubu- lin, MAP, and CD concentrations. However, net disassembly does not occur to the extent expected (5,8). We suspect that, under the experimental conditions of the earlier investiga- tions, short lengths of copolymer form at the microtubule ends which mask the ends, and which prevent the system from reaching the expected equilibrium state in a reasonable length of time. A definitive reconciliation of the two mecha- nisms will most likely require a better understanding of the microtubule assembly process, and a better characterization of the copolymer caps which form in the presence of CD under conditions where a significant or major fraction of total tubulin is initially in the microtubule phase.

Acknowledgments-We thank R. Bast, J. Bryan, J. Mieyal, S. Rudolph and L. Wilson for several stimulating discussions. We are particularly grateful to L. Wilson for clarifying a number of experi- mental procedures. We also thank B. TandIer for assistance with electron microscopy measurements, and D. Tishko for assistance with dark field microscopy measurements. J. Szasz provided excellent technical support for many aspects of this study.

REFERENCES

1. Taylor, E. W. (1965) J. Cell Biol. 25, 145-160 2. Dustin, P. (1978) Microtubules, pp. 167-184, Springer-Verlag.

New York 3. Bryan, J. (1972) Biochemistry 11,2611-2616 4. McClure, W. O., and Paulson, J. C. (1977) Mol. Pharmacol. 13,

560-575 5. Margolis, R., and Wilson, L. (1977) Proc. Natl. Acad. Sci. U. S.

A. 74,3466-3470 6. Olmsted, J. B., and Borisy, G. G. (1973) Biochemistry 12, 4282-

4289 7. Wilson, L., Anderson, K., Grisham, L., and Chin, D. (1975) in

Microtubules and Microtubule Inhibitors (Borgers, M., and De Brabander, M., eds) pp. 103-113, American Elsevier Publishers, New York

8. Margolis, R. L., and Wilson, L. (1978) Cell 13, 1-8 9. Gaskin, F., Cantor, C. R., and Shelanski, M. L. (1974) J. Mol.

Biol. 89, 737-755 10. Johnson, K. A., and Borisy, G. G. (1977) J. Mol. Biol. 117, 1-31 11. Dentler, W. L., Granett, S., Witman, G. B., and Rosenbaum, J.

(1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1710-1714 12. Lee, J. C., Tweedy, N., and Timasheff, S. N. (1978) Biochemistry

17,2783-2790 13. Nagle, B., and Bryan, J. (1976) in Cell Motility (Goldman, R.,

Pollard, T., and Rosenbaum, J., eds) pp. 1213-1231, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

14. Bryan, J. (1976) J. Cell Biol. 71, 749-767 15. Berne, B. J. (1974) J. Mol. Biol. 89, 755-758 16. Oosawa, F., and Kasai, M. (1962) J. Mol. Biol. 4, lo-21 17. Johnson, K. A., and Borisy, G. G. (1975) in Molecules and Cell

Movement (Inoue, S., and Stephens, R. E., eds) pp. 119-141, Raven Press, New York

18. Kuriyama, R., and Miki-Noumura, T. (1975) J. Cell Sci. 19, 607- 620

19. Murphy, D. B., Johnson, K. A., and Borisy, G. G. (1977) J. Mol. Biol. 117,33-52

20. Penningroth, S. M., and Kirschner, M. W. (1978) Biochemistry 17,734-740

21. Sloboda, R. D., and Rosenbaum, J. L. (1979) Biochemistry 18, 48-55

22. Garland, D. L. (1978) Biochemistry 17,4266-4272 23. Amos, L. A. (1977) J. Cell Biol. 72,642-654 24. Kim, H., Binder, L. I., and Rosenbaum, J. L. (1979) J. Cell Biol.

80,266-276 25. Langford, M. (1978) J. Cell Biol. 79, 289a 26. Mandelkow, E., Thomas, J., and Cohen, C. (1977) Proc. Natl.

Acad. Sci. U. S. A. 74, 3370-3374 27. Sternhcht, H., and Ringel, I. (1978) Fed. Proc. 37, 1791a 28. Sternhcht, H., and Ringel, I. (1978) J. Cell Biol. 79, 302a Additional references are found on p. 12549.

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