J. Mol. Biol. (1977) 117, 33-52
Role of Tubulin-associated Proteins in Microtubule and Elongation
Nucleation
DOUGLAS B. MURPHY?. KENNETH A. JOHNSONJSAND GARY G. BORISY Laboratory
of Molecular Biology, University of Wixomsin Madison, Wise. 53706. IF.S.A.
(Received 13 January
1977, ad
in revixrd .forrrt, II July 1977)
Previous experiments have sllowu that. a fraction of microtubule-associated proteins is essential for the self-assembly of microtubules in vitro. When tubulin was titrated with increasing concentrations of these non-tubulin accessory factors, both the,rate and extent. of polymerization increased in a sigmoidal as opposed to a stoichiometric fashion. The non-tubulin proteins promoted t,he nucleation of microtubules as determined from the analysis of the kinetics of tubulin selfassembly and the examination of the microtubule length distribution following polymerization. The effect, of the non-tubulin factors on microtubule elongation was determined by kinetic experiments in which purified tubulin subunits were added t’o microtubule seeds and the initial rate of polymerization was measured under conditions where spontaneous self-assembly was below detectable levels. flagellar In addition, microtubule growth was also observed when isolated axonemes were incubated with purified tubulin subunits indicating that the non-tubulin factors were not an absolute requirement for elongation. Analysis of the data in terms of the condensation mechanism of microtubule assembly indicated that the non-tubulin proteins stimulat,ed t,he growth of microtubules not by increasing the rate of polymerization but by decreasing the rate of depolymerization. The mechanism b.y which these accessory factors promot,e tubulin assembly may be summarized as follows: under the condit,ions employed, the? are required for tubulin initiation but not for elongation; the factors affect the extent and net rate at which polymer is formed by binding to the polymer, thereby stabilizing the formed microt’ubules and consequently shifting the equilibrium to favor assembly.
1. Introduction The assembly of microtubules ira vitro has been shown to be promoted by accessory proteins in addition to tubulin (Murphy & Borisy. 1975; Weingarten et al., 1975). These non-tubulin accessory proteins have attracted attention recently because of the possibility that they may serve to regulate microtubule assembly in vivo. However, the nature of the accessory factors appears to depend somewhat on the method for isolating microtubule proteins. The principal factor present in microtubule protein prepared according to the procedure of Borisy et al. (1975) is a doublet 7 Present address: Division of Biology, Kansas State Lnivcrsity, Manhattan, Kansas U.S.A. $ Present, address: Department of Biophysics, University of Chicago, Chicago, Ill. 60637, $ In this paper microtubule protein refers to protein cont,aining non-trtbulin accessory and tuhulin as obtained from the reversible assembly procedure, ;i
33
66606, U.S.A.
proteins
34
D. B. MURPHY,
K.
A. JOHNSON
AND
G. G. BORISY
of high molecular weight (HMWt 1 and 2, with 286,000 and 271,000 M,, respectively) that has been identified by electron microscopy as a filamentous projection on the microtubule surface (Dentler et al., 1975; Murphy & Borisy, 1975). In contrast: the principal factor in microtubule protein isolated in the presence of glycerol, according to the method of Shelanski et al. (I 973) as modified by Weingarten et al. (1974), has been referred to as tau factor (Weingarten et al., 1975) and is an asymmetric molecule of 70,000 M, (Penningroth et al., 1976). Both HMW and tau factors appear to be present in both kinds of preparations hut differ in relative abundance in that levels of the HMW species appear to be reduced in the glycerol-isolated protein (Scheele & Borisy, 1976). Tt is not yet known whether the HMW and tau factors are related to each other or are of independent origin. The mechanism by which these fa&ors stimulate polymerization has been under investigation and early results showed that the factors could induce the formation of ring oligomers of tubulin at’ low temperatures and microtubule self-assembly at elevated temperatures (Murphy & Borisy, 1975; Keates & Hall, 1975; Weingarten et al., 1975; Kuriyama, 1975), with increasing concentrations of factor favoring the extent of polymerization (Sloboda ef al., 1976). In our initial report (Murphy & Borisy, 1975) we also found that tubulin subunits were capable of rapid addit’ion ont,o pre-existing fragments and concluded that HMW wa,s not an absolute requirement for elongation of polymer. Hence, we suggested that HMW might serve a role in tubule nucleation rather than elongation, hut we also noted that since HMW associates with microtubules along their entire length, the tubulin-HMW interaction was clearly not restricted to the initiation step. However, Witman et al. (1976) have presented results indicating that tau factor is an absolute requirement for microtubule growth and participates stoichiometrically in the elongation reaction. In order to resolve these apparent inconsistencies and to elucidate the mechanism of stimulation of t,ubulin polymerization by the non-tubulin accessory factors, wt. have investigated t’he effects of the factors on tubule initiation and growth separately, using quantitative methods of kinet’ic analysis as described previously by Johnson & Borisy (1977). The results confirm our previous suggestion that HiNW is required, for tubule initiation but not for elongation under the conditions employed; however, HMW does affect the ext#ent and net rate at which polymer is formed. It does so by binding to the polymer and reducing the dissociation rate constant, thereby stabilizing t,he formed microtubules and shifting the equilibrium t’o favor assembly.
2. Materials and Methods (a) I’reparation
of microtubule protein, microtubule seeds, tubdin, and non-tub&n accessory proteina
subwaitu
Microtubule protein was prepared from porcine brain tissue by 2 cycles of an i,n vitro assembly procedure as described previously (Borisy et al., 1975). Unless otherwise stated, all purifications and experiments were performed in PMG solution (0.1 M-PIPES (pipera1.0 mM-GTP adjusted to pH 6.94 zine-N,N’-bis(2-ethane sulfonic acid)), 0.1 mM-MgSO,, at 23°C with NaOH). Microtubule seeds (average length 1 pm) were prepared by mechanically shearing polymerized microtubules (4 to 7 mg/ml in PMG) by passing the solution through a 22-gauge, 1.5-inch syringe needle. A fresh preparation was used for each experiment; t Abbreviation
usocl : HMW,
high molecular
waight proteins.
STIMULATION
OF MICROTURULE
ASSEMBLY
35
each experiment involving the addition of seeds was completed within 2 11 of the start, of polymerization of the seed protein. Fractions of tubulin and non-tub&n proteins were isolated by chromatography of microtubule protein on DEAE-Sephadex and stepwise elution with salt by a modification of a previously described procedure (Murphy & Borisy, 1975). Briefly, salt was added to a sample of microtubule protein (20 mg/ml) in PMG to a final concn of 0.3 ~-Kc1 and applied to a column containing 4 ml DEAE-Sephadex A50 equilibrated in the same salt solution, but containing 0.1 mM-GTP. Under these conditions, all nori-tubulin components failed to adsorb and eluted together as a fraction of unbound protein. A fraction containing greater than 993 pure tubulin was subsequently elut’ed with t,he same column buff& but containing 0.8 M-KCl. The protein fractions were desalted by chromatography 011 Srphatlex G25 (coarse) equilibrated with PMG solution and generally used within 2 h of preparation. In some instances fractions were also st,ored at - 80°C for up to 1 mont.11 (bvith rct,mtion of SOY/, activity) and thawed immediately before use. The final COIIWIItrations of t,hc desalted non-tubulin and tubulin fractions n-cre usually. 5 and 3 mg/ml. respectively. For one series of experiments (see Results, section (c)). 6 S tubulin was prepared by high-speed centrifugation of microtubule protein iI1 PMG solution at 230,000 g for 90 min at 4°C. The preceding paper (Johnson & Borisy. 1977) has silown that b! analyt)ical centrifugation at polymerizing temperatures (25°C) such preparations exhibit a 6 S tubulin peak and no detectable levels of tubulin oligomers (e.g., 30 S species). Proteill concentrations were determined according to t,hr mrthod of Lowry et al. (1951) using borinrt serum albumin as a standard. (b) .4ssay procedure
for the kinetics
of mic~rotubule
elongation
The kinetics of microtubule elongation were determined by measuring the change irl optical density at 320 nm (Johnson & Borisy, 1977). As described previously, the turbidit,) obscrvcd at this wavelength resulted from the scattering of light by the polymer and was approximately proportional to the concentration of polymer. Typically. a 0.5.ml samplr> of DEAE-purified tubulin at 0°C was placed in a watc+jackcted cuvett’e and warmed to polymerization t,emperature (25.6”C). The half-time for thermal equilibration was 12 s. As reported previously (Murphy & Borisy, 1975). pllrifiod t,uhulitr alone failed to polymerize. After 75 s at 256”C, polymerizatiou was init’iated by t,ltc addition of a small sample (20 ~1) of a solution of microtubule fragments atld the O.D. at) 320 nm was recorded continuously (usually within 3 s after mixing) with a Vary 15 sprctrophotometer. Thp rates of rlonpatiorr was mcasurrd from the initial rat)(x of irlrrc~asc~ in optical derrsit,y.
Ylagellar axollemes were prepared from Chlamydomonas as described previously by. Allen & Borisy (1974). Briefly, cells in 400 ml of culture (3 to 4 x 106 cells/ml) were harvested, the flagella were removed and demcmbranatrtl in 10 rn>x-HEPES (N-2.hydroxyclt,hyl piperazine-A”-2-ethane snlfonio acid) buffer cont,aining O.O36o/b Non-Id& P40 (Shell Chemicals, Ltd, London). and washed in PM(: solution. Thf, axoneme pellet, was tllen resuspended in 0.2 ml PMU sol&on and tllc axoneme suspension was mixed witch tubulin so that a droplet of the mixture placed on AIL calectron microscope grid and negatively stained would contain at least 20 to 30 axonrmes. To test the ability of the isolated axonemes t,o support the addition of tubulin subunits, a sample of axonemes was incubated with a high-speed supernatant of depolymerized microtubule protein and placed at 37°C as described by allen & Borisy (1974) ; the number. of axonenes showing brain tubule addit,ion was usually greater than 95%. To assure maximal activity, it was necessary to prepare the axonemes immediately before use. Plastic tubes and pipettes were used for all stages of isolation to minimize fraying and darnagcl to tht, axotiemes. (d) Electron
microscopy
and carbon, and A 5-~1 sample was placed on a 400-mesh grid coated with Formvar successively displaced with 4 drops of each of the following solutions: 1 mg cytochrome c/ml, distilled water, and 1% aqueous uranyl acetate. The excess stain was removed with
36
D.
B. MURPHY,
K.
A. JOHNSON
AND
G. G. BORISY
a filter paper and the grid was air-dried. Grids were examined with a Philips 300 electron microscope at 80 kV. The length of microtubules was determined by comparison with a calibrated ruling on the phosphor viewing screen of the microscope. (e) I’olyacrylamide
gel electrophoresis
Samples and gels (5%, 0.6 cm x 8.0 cm) were prepared and run according to the procedure of Shapiro et al. (1967). Gels generally containing 3 to 5 rg protein were stained with Coomassie brilliant blue according to the procedure of Fairbanks et al. (1971) and densitometer tracings of the gels were made at 560 nm with a Gilford model 240 spectrophotometer (Gilford Instruments Laboratories, Oberlin, Ohio). Peak areas from the densitometer tracings were quantitated by planimetry. To detect trace levels of possible contaminants in the tubulin preparations gels were overloaded with up to 116 pg protein/ gel.
3. Results (a) Titration
of tubulin
with non-tub&in
proteins
The DEAE-ion exchange procedure used in this study separates microtubule protein into two fractions: one fraction contains tubulin to greater than 99% purity as determined by electrophoresis on sodium dodecyl sulfate gels overloaded with sample (100 pg/gel); the other fraction consists of all other proteins (designated non-tubulin proteins) that are present in microtubule protein purified bp two cycles of assembly and disassembly according to the procedure of Borisy et al. (1975) (Fig. 1). As shown in the Figure, the principal species in the non-tubulin fraction is a doublet of high molecular weight referred to as HMW 1 and 2 (286,000 and 271,000 M,, respectively) but the fraction also contained numerous other minor components including a species of 70,000 M, referred to as tau factor (Weingarten et al.. 1975; Penningroth et al., 1976) and some contaminating tubulin. Previous work has shown that although the HMW components were the principal stimulatory factors in this preparation, tau and possibly other factors also stimulated tubulin polymerization (Murphy et al.. 1977). Therefore we chose to work with this total non-tubulin fraction in order to avoid inadvertently discarding a pertinent factor.
HMW
I and
2
Tau 1
Tubulin 1
J
lb) FIQ. dodecyl
1. Electrophoretic sulfate/polyacrylamide
pattern
of non-tubulin gels.
fraction
(a) and
tubulin
fraction
(b) on sodium
STIMULATION
OF
MICROTUBULE
ASSEMBLY
3:
To examine the effect of the accessory proteins on microtubule assembly, purified tubulin was titrated with the non-tubulin fraction and the extent and rate of assembly were examined by monitoring the change in optical density at 320 nm under polymerizing conditions. The effect of the accessory proteins on t)he kinetics of microtubule polymerization is shown in Figure 2. By polymerizing the protein at 256°C the rate of assembly was sufficiently slow compa,red to the rate at 37°C t’hat the detailed behavior of the assembly kinetics could be observed. As reported previously. purified tubulin without added factors failed to self-assemble (Fig. 2(D)). However, as increasing amounts of the non-t8ubulin fraction were added back to tubulin. both t’he rate and extent of assembly increased (Fig. 2(A). (B) and (C)). In addition, a lag time was ohserved which preceded the onset of t#urbidit,y development (Fig. 2, a,rrows). The effects of the non-tubulin fraction on the rate and extent of assembly over a broad concentration range are shown in Figure 3. In these experiments tubulin at a fixed concentration of 1.3 mg/ml was titrated wit,11 the non-tubulin fraction and polymerization was monitored at a higher temperature (37°C) in order t’o attain equilibrium within a reasonable period of time. As seen in Figure 3, both the extent and rate of polymerization increased in sigmoidal fashion. In both cases. little change in turhidity was detected at concentrations of the non-tubulin fraction below 0.1 mg/ml (7)‘o of the total protein). Above this value. polymerization increased rapidly with half-maximal extent of assembly occurring at 0.35 mg/ml (Sl”,:, of the total protein) and half-maximal rate of assembly at 0.72 mg/ml (350; of t,he total protein). Similar results were obt’ained when polymer formation was monitored b) viscometry and sedimentation and when higher conccmrat8ions of tubulin uere used. However, as will be discussed subsequenbly. t,he fraction of t’ubulin polymerized depended upon the tubulin concentration. With increasing concentrat,ions of tubulin. lower concentrations of non-tubulin fraction were required to oht’ain self-assembly. For example, at 2.7 mg tuhulin/ml. 0.15 mg non-tubulin protc+/rnl (Gob) \l.as sufficient
Time (5) FIQ. 2. Stimulation mg/ml) in PMG buffer following addition of 0.11 mg/ml (curve B), tubulin protein a lag
of self-assembly of purified t,ubulin by non-tubulin protein. Tubulin (1.8 was pre-equilibrated to 26.6”C and turbidity was monitored at 320 nm PMG buffer (curve D), or non-tubulin fraction at 0.08 mg/ml (curve C), and 0.19 mg/ml (curve A) (final concns). At these concentrations of nonperiod was observed prior to assembly (indicated by arrows).
38
D.
B. MURPHY,
K.
A. JOHNSON
AND
G. G. BORISY
Protein concn (mg /ml)
FIa. 3. The effect of non-tubulin protein on the extent (-•-a-) and rate (-o--O--) of tubulin assembly. Polymerization of tubulin (1.3 mg/ml) in PMG buffer was monitored at 320 nm following addition of increasing amounts of non-tubulin fraction (0 to 2.6 mg/ml). Polymerization was performed at 37°C in order to estimate the extent of assembly at equilibrium. Prot,ein concentration indicates the final concentration of non-tubulin protein.
to obtain half-maximal extent of assembly. These data show that the non-tubulin proteins affect the extent of microtubule formation and suggest that the position of the equilibrium for polymerization is determined by the concentration of both tubulin and the accessory factors. In addition, since the amount of factor required to give half-maximal rate of assembly was significantly greater than that required for half-maximal extent of polymerization, it appeared that the factors might have a distinguishable affect on nucleation versus elongation of microtubules. Previous analysis of the kinetics and equilibrium of microtubule assembly (Gaskin et al., 1974; Johnson & Borisy, 1975,1977; Bryan, 1976) had demonstrated that, polymerization occurs as a condensation reaction with distinct phases of nucleation (initiation of new microtubules) and elongation (addition of subunits onto the ends of pre-existing microtubules). However, since the overall rate of self-assembly is a function of both of these phases, it was not possible to distinguish from the overall rate data alone to what extent the factors influenced just one or both of these processes. Experiments were therefore performed to examine the effects of the nontub&n proteins on microtubule nucleation and growth separately. (b) Effect of the non-tub&n
protein
on microtubule
n,ucEea,tioyL
Since an increase in tubule nucleation would result in an increase in the number concentration of microtubules, samples from the titration experiments were examined by electron microscopy to see if an increase in nucleation could be observed directly. With increasing concentrations of the non-tubulin fraction, the total length of tubules per unit area of the grid increased, in agreement with the observed increase in turbidity (Fig. 3). In addition it was observed that few long microtubules formed at low concentrations of factor and that many short microtubules formed at high concentrations of factor. Thus, the non-tubulin fraction appeared to stimulate tubule nucleation. A similar observation has been reported by Sloboda et al. (1976) using microtubule-associated proteins prepared from calf brain.
STIMULATION
OF
MICROTUB17LE
ASSEMBLY
39
It seemed plausible that the long lag periods and low rates of assembly that were observed at low factor concentrations reflected the kinetics of nucleation. In order to explore this possibility further the effects of non-tubulin protein on the kinetics of assembly were examined with respect to the lag time and the maximal rate of assembly. Lag times and maximal rates were extracted from data of the type shown in Figure 2. Lag times were obtained by extrapolating the linear portions of the curves back to the times of zero increase in turbidity. Maximal rates were obtained as the slope? of the initial linear portions of the curves. As shown in Figure 4, at low concentrations of factor, the lag time was extremely long. Over the range of factor concentrations explored (0 to 0.34 mg/ml), the lag decreased from greater than one hour to 20 seconds and the rate of assembly increased exponentially. By plotting the log of t,he rate versus the log of the factor concentration, a st’raight line plot was obtained with a slope of 3.4. This slope is an approximate measure of the order of the microtubule nucleation reaction with respect to the accessory proteins and suggests that’ several accessory protein molecules are involved in the formation of a nucleus (set Discussion). Since it appears that the number of microtubule ends available for growth is larger at higher factor concentrations, the increase in polymerization rate is attributable in part to an increase in microtubule nucleation. However, as shown helow, the accessory proteins also appear to affect the net’ rate of elongation. (0) Effect of the vsom?t&d%n p-otei,n m vnicrot,ubule growth The effect of the accessory proteins on the elongation reaction wras determined using the following procedure. Polymerization was initiated by the addition of a fixed concentration of microbubule seeds and a varying concentration of non-tubulin
Protein concn (mg /ml) Fro. 4. The effect of non-tubulin protein on the lag time preceding tubulin self-assembly (-0 -the initial rat’e of elongation. Accordingly. experiment,s were performed under conditions where t,he non-tubulin fraction alone did not) initiate assembly during the period in which the initial rate, pi of elongat,ion \vt’re bring measured following the addition of seeds. By using these conditions measurtments of initial rate following the addition of seeds measured the change in turbidity due only to microtubuk growth. The effect of the factors on the elongation reaction was then determined by measuring the inital rate of polymerization as :I funct’ion of the concentration of non-tubulin protein. In order to cover a range of possible effects of the non-tubulin protein, the results of t\vo kinds of experiments will he described belo\+,. Ln addition, since previous kinetic experiment)s on elongation were carried out using 6 S monomer preparations obtained by high-speed centrifugation (Johnson & Borisy. 1977) the use of DEAEpurified tubulin and high-speed supernatants as sources of subunits were compared One kind of experiment was performed at, a high tubulin and low seed concentration while the other was performed at a low tubuliu and high seed concentration. In each t,ype of experiment, the highest concentrat,ion of non-tubulin protein which could be investigated was limited by spontaneous nucleation as evidenced by the increase in optical density at 320 nm in the absencca of seeds. Since self-assembly \va.s also favored by increasing tubulin concentrat’ion. a higher concentration of nontubulin protein could be investigated in the experiments involving the lower tubulin concentrat8ions. The determination of the concentration of non-tubulin protein requires comment. Since the seeds were prepared from microtubule protein containing factor as well as t8ubulin, the contribution of unbound fact’or in t’he seed preparabion also had to be estimated. Seed concentrations were used which contributed minimal levels of factor (.
B.
MURPHY,
K.
A.
JOHNSON
ANI>
G.
G.
BORISY
Since the extent as well as the initial rate of polymerization was increased by increasing concentrations of non-tubulin protein, these factors clearly must effect the elongation as well as the initiation reactions. In this regard. our results agree with those obtained by others (Sloboda et al., 1976; Witman et al.. 1976). However, whereas we observed tubule elongation in the absence of added fact,ors, Witman ef al. did not. Their results indicated that purified tubulin did not have the capacity to assemble onto the ends of flagellar microtubules. whereas the addition of small amounts of tau restored the capacity for elongation. From these results as well as turbidometric studies, they concluded that tau is an essential structural component of microtubules. perhaps present in a precise stoichiometric relationship to tuhulin. In contrast, our results showed that DEAE-purified tubulin. when present, in concentrations above the critical concentration. would elongate microtubule seeds derived either from flagellar axonemes or sheared brain microtubules. Although the tubulin fraction alone was sufficient to support microtubule elongation, increasing concentrations of non-tubulin proteins increased bhe net rate of polymerization. Since the question of the absolute requirement, of non-tubulin proteins for microtubule elongation is a fundament’al issue for our understanding of microtubule structure and nssernhl-v. a discussion of the disparity in the two set,s of result’s is warranted. The difference in results does not arise from the source of microtubules used as the seeds since both this study and that of Witman et al. (1976) used flagellar axonemes isolated from the green alga, Chlamydomonas reinhardti, by the method of Allen & Borisy (1974). R’either can our results be explained easily by the suggestion that the tubulin used in our experiments contained small quantities of tau sufficient to produce the observed elongation, The DEAE-purified fraction contained tubulin and no other species were detected on overloaded gels ( >lOO pg/gel). From the densitometric patterns obtained from these gels, a minimum purity of 99% may be established. An independent estimate of the purity of the tubulin was obtained 1j.v its failure to self-assemble detectably at high concent,rations. Experiments at 2.5 mg/ml tubulin showed that as little as 25 pg added non-tub&n protein/ml (l”o of total) would induce self-assembly detectable by turbidity and sedimentation. Thus, since preparations of tubulin at 2.5 mg/ml failed to self-assemble in the absence of added factor. we conclude that our tubulin fraction contained less than l”/ non-tubulin protein. It is difficult to obtain convincing data for levels of purity beyond 99:/, and this question is of particular importance since Witman et al. (1976) showed that as little as 0.5% tau would permit some microtubule elongation onto pre-existing tubules and hence might account for the results reported here. However. we suggest an alternative hypothesis for the observed results. This alternative explanation states that tubulin alone is competent to elongate microtubules and that the concentration of tubulin required to obtain elongation depends on the solution conditions and the concentration of non-tubulin accessory factors present. Although we have not determined the critical concentration for elongation under the conditions employed by Witman et al. (1976). we suggest the possibility that since their isolation method and solution conditions differ from ours, their failure to obtain elongation in the absence of added factors was because the concentration of active tubulin was below the critical concentration.
HTIMULATIOS
OF
MICROTUBULE
ASSEMBLY
49
(c) A model for the effect oj the non-tubulin accessory proteins on the microtubule elongation reaction To determine quantit’atively the effect of the accessory factors on microtubule elongation. the initial rate of polymerization was measured after mixing microtubule seeds and subunits together at low concentrations of non-tubulin protein. These results suggested that the non-tubulin factors were not absolutely required for assembly, but indicated that they did alter the net rate and extent of assembly. Moreover. analysis of the dependence of the initial rate of elongation on tubulin concentration (Fig. 8) demonstrated that the non-tubulin protein increased the net rate of assembly and shifted the equilibrium towards polymer formation by decreasing the rate of depolymerization. In order to describe more fully the effect of the non-tubulin proteins in molecula,r terms and to evaluate whether the decrease in the rate of depolymerization could quantitatively account for the observed st’imulat’ion of assembly. the following model i< presented for the effect of binding of accesdorv proteins on the forward and reverse rates of microtubule assembly. Although. as indicated previously, the nont#ubulin fraction contains a mixture of componerns. the HMW proteins are the major species both in abundance as well as in the ability to stimulate t’ubulin polymerization (Murphy et al., 1977). Therefore, t’his model will be formulated in terms of t,he HMW species: however. the model should he understood to be applicable to the other non-tubulin species as well. Four assumptions form the basis for this analysis. (i) The equilibrium for tubule assembly is assumed to be given by the balance of the net rates of association and dissociation of 6 S tubulin subunits at the end of the microtubule and is defined b? the reaction
(1) wherts M and S represent the end of a microtubule and the 6 8 bubulin subunit. respectively. The net rate of assembly is determined by the difference of the rates of polymerization and depolymerization as follows : d O.U. dS c= - - = k,[MI dt dt
IS] -
k-,lM].
(2,
where k, and k- 1 are the apparent rate constants governingassemblyanddisassembly. respectively, and c is a proportionality constant relating the optical density to the concentration of tubulin subunits polymerized. These postulates were derived from our previous equilibrium and kinetic analyses of microbubule assembly (Johnson & Borisy, 19751977). (ii) The observation that the HMW proteins associated with microtubules to form projections at spacings equal to multiples of a regular interval (Murphy & Borisy, 1975) is taken to indicate that there are periodic binding sites on the tubule lattice not all of which are necessarily occupied. (iii) The HMW-tubule association reaction is assumed to be in a rapid equilibrium with respect to the rate of association and dissociation of tubulin subunits at the end of the tubule. The basis for this assumption is twofold: (a) the HMW proteins altered the initial rate of assembly when mixed simultaneously with seeds and 6 S tubulin; and (b) analysis of the temperature dependence of assembly suggested that the HMW association reaction was in a rapid equilibrium (Johnson, K. A. & Borisy, G. G., unpublished
5)
I).
H.
MURPHY,
I> 1, most of the sites are occupied and k-,,,,, = k’,. This case accounts for the plateau in Figure 5 as the net rate of assembly approaches a maximum of of HMW shifted the rate of k,[M][S] - k’_,[M]. Since a saturating concentration depolymerization (Fig. 8) to approximately zero, kl, is presumed to be very small and in the subsequent analysis is tentat,ively assigned a value of zero. (ii) When the concentration of non-tubulin protein is zero, all of the binding sites are free and the rate constant for disassembly becomes Icy 1. For this case, the net rate of assembly is given by k.JM] [S] - kl,[M]. Thus, the question of whether or not assembly
STIMULATION
OF MICROTUBU
LE
ilSSEMBLP
51
occurs in the absence of non-tubulin proteins depends upon whether or not k,[S J k:, is positive or negative for a given set of conditions. The two experiments described in Figure 5 demonstrate this point. In the experiment at the high tubulin concentration, the data indicate a positive rate of elongation while in the experiment at the lower tubulin concentration, a net negat’ive rwt,e was obtained by extrapolation to zero non-tubulin protein concentration. The smooth curves drawn in Figure 5 were calculated according to equation (6) on thtb assumption that the stimulatory activity was solely a function of HMW. The curve fitting was done as follows. First, on t’he basis of quantitative titration data (Peloquin, J. & Borisy, G. G., unpublished result’s), it was assumed that’ one HMW binding site was defined by 12 tubulin dimers polymerized in the seed preparation. This ratio is also in agreement with t’he HM3V superlattice as described l)y Amos (1977). From the number of tubulin dimers per HMW binding site. the molecular weight of the dimer (110,000) and the roncentrat,ion of polymer (mg/ml). the concentration of HMW binding sites was calculated. From the concentration of total HMW, the concentration of binding sites, and assuming identical and noninteracting sites, and assuming a value for K, the association constant for HMW binding, the fract’ion of bound sites, 0, was calculated. Setting lc: 1 == 0 and assuming a value of kL,.k-,,,,, was calculated. The molar concentration of microtubule seeds ]M], was calculated from the mass concentration, the average length of t)he seeds (1 pm) and the mass per unit length of tubule as defined by the microtubule lattice parameters of Erickson (1974). Knowing the subunit concentration LX] and values of the constants, lc, and c from previous studies (Johnson & Borisy, 1977), the initial rate of elongation. do.n./dt was calculated. The procedure was then reiterat,ed for varying values of K and kl r. The best fit for both experiments (Fig. 5) using kl, = 0 se1 for the was obtained with kl, = 19 s-l and K = 7~ lo6 M-l, reasons described above and k, = 2 x lo6 1\1-lssl as ha,s been previously measured (Johnson & Borisy, 1977). The values of these constants are constrained within relatively narrow limits (1200:) by the requirement of fitting the data obtained from two experiments performed under markedly different sets of conditions. How ever, t’he actual values will need to be measured experimentally. From the rat)e paramct’ers derived above, the critical concentration in the absence of factors may be found by setting k,[S] - lill = 0 and solving for IS 1. The value obtained is 1 mg/ml. Thus, the proposed model quantitatively account,s for t’he available data in terms of reasonable rate and association constants. The model presented here differs in a number of essential features from the model of Witman et al. (1976). In their model, tau is viewed as an integral component of the microtubule structure and elongation is directly dependent upon a stoichiom&ic incorporation of tau along with tubuhn. In contrast, the model presented here views thcx accessory proteins as not affecting at all the elongation reaction per SC. The facbors are neither required for the addition of a tubulin subunit at the end of the microtubule nor do they affect the rate constant for the addition reaction. Rather. the, factors bind at sites made available after polymerization has occurred and the) reduce the dissociation rate constant,, hence stabilizing the formed microtubules and shifting the equilibrium towards polymerization. It is intriguing to consider t,hat regulation of microtubule assembly in &vo may be cont’rolled by reactions that> alter the affinity of the accessory proteins for their binding sites on microtubules.
.52
D. B. MURPHY,
K.
A. ,JC)HNSON
ANI)
G. G. BORISY
This study was supported by National lrrstitutes of Health grant GM-21963 to one of 11s (G. G. B.); anothrr author (D. B. M.) was an N.I.H. Postdoctoral Bellow supported I)y grant CA-00923; tltc> third allt,llor (K. A. .J.) \vas sllpport.cd hy H~I N. 1.H. Predoctoral ‘I‘minrc&ip. HI1’FE:RENClGS 1 Allen, C. & Borisy, G. G. (lY74). J. Mol. Hiol. 90, 381-402. Amos, L. A. (1977). J. Cell. Biol. 72, 642- 654. Bloodgood, R. A. & Rosenbaum, J. L. (1976). J. Cell Biol. 71, 322-331. Borisy. G. G., Marcum, J. M., Olmsted. .J. B., Murphy, D. B. BE Johnson, K. A. (1975). Ann. X. Y. Acad. Sci. 253, 107- 132. Bryan, *J. (1976). J. Cell. Biol. 71, 749~~76i. Dentler, W. L., Granott, S. & Rosenbaum, J. L. (1975). J. Cell. Biol. 65, 237-241. Dentler, F$:. L., Granrtt, S., Witmarr, G. B. & Roscnbaum, J. L. (1974). 1%~. Nat. AC&. Sci.. U.S.A. 71, 1710~1714. Erickson, H. I’. (1974). .7. Cell Biol. 60, I53- 167. Erickson, H. P. & Vot,er, W. A. (1!)76). f’roc. &at. Acad. Sci., U.S.A. 73, 2813-2817. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971). Biochemistry, 10, 2606-2617. Gaskin, F., Cantor, C. K. & Shelanski, M. L. (1974). J. Mol. Biol. 89, 737.~758. Herzog, W. & Weber, K. (1977). I’roc. A’&. Acad. Sk., U.S.A. 74, 1860-~1864. Himes, R. H., Burt,on, P. H., Kprsry, Ft. N. & Pierson, G. B. (1976). Proc. X’at. Acad. Sci., U.S.A. 73,4397-439Y. ,Johnson, K. A. & Borisy, G. G. (l!J75). It1 Molecules and Cell Movement (InouB, S. Ur Stephens, It. E., ads), pp. I 1% 139, Raven Press, New York. Johnson, K. A. & Borisy, G. G. (1977). J. Mol. Biol. 117, 1-31. Keates, R. A. & Hall, I%. H. (1975). A’ature (Lomlon), 257, 418- 421. Kuriyama, R. (1975). J. Biochem. 77, 23. 31. Lee, J. C. & Timasheff, S. N. (1975). Biochemistry, 14, 5183-5187. Lowry, 0. A., Rosebrough. N. J., Farr, A. L. B Randall. R. .J. (1951). .I. Biol. Chem. 193, 265-275. Murplly, D. B. & Borisy, G. G. (lY75). 1%~. iVat. Acad. Sk., U.S.A. 72, 2696-2700. Murphy, D. B., Valler, R. B. & Borisy, G. G. (1977). Riochemistry, 16, 2598-2605. Penningroth, S. M., Cleveland, D. W. & Kirschner, M. W. (1976). In Cell Motility (GoldT. & R,osenbaum. .I . . eds), pp. 1233-1257, Cold Spring Harbor man, R., Pollard, Laboratory, New York. Scheele, K. B. & Borisy, G. G. (1976). Biochem. Biophys. Res. Cornmurk. 70, l--7. Biophys. Res. Commun. Shapiro, A. L., Vinuela, E. & Maizel, .r. V. (1967). Biochem. 28, 815-820. Shelanski, M. L.. Gaskin, F. & Cantor, C. R. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 765-768. Sloboda, R. D., Dentler, W. L. & Rosenbaum, J. L. (1976). Biochemistry, 15, 4497-4505. Tanford, C. (1961). Physical Chemistry of Macromolecules, pp. 532-535, John Wiley and Sons, New York. Weingarten, M. D., Suter, M. M., Littman, D. R. & Kirschner, M. W. (1974). Biochemistry, 13, 5529-5537. Weingarten, M. D., Lockwood, A. H.. Hwo, S. & Kirschner, M. W. (1975). Proc. iVat. Acad. Sci., U.S.A. 72, 1858-1862. Witman, G. B., Cleveland, D. W., Weingarten, M. D. & Kirschner, M. W. (1976). Prop. Nat. Acad. Sci.. [J.S.A. 73, 4070-4074.
Role of Tubulin-associated Proteins in Microtubule and Elongation
Nucleation
DOUGLAS B. MURPHY?. KENNETH A. JOHNSONJSAND GARY G. BORISY Laboratory
of Molecular Biology, University of Wixomsin Madison, Wise. 53706. IF.S.A.
(Received 13 January
1977, ad
in revixrd .forrrt, II July 1977)
Previous experiments have sllowu that. a fraction of microtubule-associated proteins is essential for the self-assembly of microtubules in vitro. When tubulin was titrated with increasing concentrations of these non-tubulin accessory factors, both the,rate and extent. of polymerization increased in a sigmoidal as opposed to a stoichiometric fashion. The non-tubulin proteins promoted t,he nucleation of microtubules as determined from the analysis of the kinetics of tubulin selfassembly and the examination of the microtubule length distribution following polymerization. The effect, of the non-tubulin factors on microtubule elongation was determined by kinetic experiments in which purified tubulin subunits were added t’o microtubule seeds and the initial rate of polymerization was measured under conditions where spontaneous self-assembly was below detectable levels. flagellar In addition, microtubule growth was also observed when isolated axonemes were incubated with purified tubulin subunits indicating that the non-tubulin factors were not an absolute requirement for elongation. Analysis of the data in terms of the condensation mechanism of microtubule assembly indicated that the non-tubulin proteins stimulat,ed t,he growth of microtubules not by increasing the rate of polymerization but by decreasing the rate of depolymerization. The mechanism b.y which these accessory factors promot,e tubulin assembly may be summarized as follows: under the condit,ions employed, the? are required for tubulin initiation but not for elongation; the factors affect the extent and net rate at which polymer is formed by binding to the polymer, thereby stabilizing the formed microt’ubules and consequently shifting the equilibrium to favor assembly.
1. Introduction The assembly of microtubules ira vitro has been shown to be promoted by accessory proteins in addition to tubulin (Murphy & Borisy. 1975; Weingarten et al., 1975). These non-tubulin accessory proteins have attracted attention recently because of the possibility that they may serve to regulate microtubule assembly in vivo. However, the nature of the accessory factors appears to depend somewhat on the method for isolating microtubule proteins. The principal factor present in microtubule protein prepared according to the procedure of Borisy et al. (1975) is a doublet 7 Present address: Division of Biology, Kansas State Lnivcrsity, Manhattan, Kansas U.S.A. $ Present, address: Department of Biophysics, University of Chicago, Chicago, Ill. 60637, $ In this paper microtubule protein refers to protein cont,aining non-trtbulin accessory and tuhulin as obtained from the reversible assembly procedure, ;i
33
66606, U.S.A.
proteins
34
D. B. MURPHY,
K.
A. JOHNSON
AND
G. G. BORISY
of high molecular weight (HMWt 1 and 2, with 286,000 and 271,000 M,, respectively) that has been identified by electron microscopy as a filamentous projection on the microtubule surface (Dentler et al., 1975; Murphy & Borisy, 1975). In contrast: the principal factor in microtubule protein isolated in the presence of glycerol, according to the method of Shelanski et al. (I 973) as modified by Weingarten et al. (1974), has been referred to as tau factor (Weingarten et al., 1975) and is an asymmetric molecule of 70,000 M, (Penningroth et al., 1976). Both HMW and tau factors appear to be present in both kinds of preparations hut differ in relative abundance in that levels of the HMW species appear to be reduced in the glycerol-isolated protein (Scheele & Borisy, 1976). Tt is not yet known whether the HMW and tau factors are related to each other or are of independent origin. The mechanism by which these fa&ors stimulate polymerization has been under investigation and early results showed that the factors could induce the formation of ring oligomers of tubulin at’ low temperatures and microtubule self-assembly at elevated temperatures (Murphy & Borisy, 1975; Keates & Hall, 1975; Weingarten et al., 1975; Kuriyama, 1975), with increasing concentrations of factor favoring the extent of polymerization (Sloboda ef al., 1976). In our initial report (Murphy & Borisy, 1975) we also found that tubulin subunits were capable of rapid addit’ion ont,o pre-existing fragments and concluded that HMW wa,s not an absolute requirement for elongation of polymer. Hence, we suggested that HMW might serve a role in tubule nucleation rather than elongation, hut we also noted that since HMW associates with microtubules along their entire length, the tubulin-HMW interaction was clearly not restricted to the initiation step. However, Witman et al. (1976) have presented results indicating that tau factor is an absolute requirement for microtubule growth and participates stoichiometrically in the elongation reaction. In order to resolve these apparent inconsistencies and to elucidate the mechanism of stimulation of t,ubulin polymerization by the non-tubulin accessory factors, wt. have investigated t’he effects of the factors on tubule initiation and growth separately, using quantitative methods of kinet’ic analysis as described previously by Johnson & Borisy (1977). The results confirm our previous suggestion that HiNW is required, for tubule initiation but not for elongation under the conditions employed; however, HMW does affect the ext#ent and net rate at which polymer is formed. It does so by binding to the polymer and reducing the dissociation rate constant, thereby stabilizing t,he formed microtubules and shifting the equilibrium t’o favor assembly.
2. Materials and Methods (a) I’reparation
of microtubule protein, microtubule seeds, tubdin, and non-tub&n accessory proteina
subwaitu
Microtubule protein was prepared from porcine brain tissue by 2 cycles of an i,n vitro assembly procedure as described previously (Borisy et al., 1975). Unless otherwise stated, all purifications and experiments were performed in PMG solution (0.1 M-PIPES (pipera1.0 mM-GTP adjusted to pH 6.94 zine-N,N’-bis(2-ethane sulfonic acid)), 0.1 mM-MgSO,, at 23°C with NaOH). Microtubule seeds (average length 1 pm) were prepared by mechanically shearing polymerized microtubules (4 to 7 mg/ml in PMG) by passing the solution through a 22-gauge, 1.5-inch syringe needle. A fresh preparation was used for each experiment; t Abbreviation
usocl : HMW,
high molecular
waight proteins.
STIMULATION
OF MICROTURULE
ASSEMBLY
35
each experiment involving the addition of seeds was completed within 2 11 of the start, of polymerization of the seed protein. Fractions of tubulin and non-tub&n proteins were isolated by chromatography of microtubule protein on DEAE-Sephadex and stepwise elution with salt by a modification of a previously described procedure (Murphy & Borisy, 1975). Briefly, salt was added to a sample of microtubule protein (20 mg/ml) in PMG to a final concn of 0.3 ~-Kc1 and applied to a column containing 4 ml DEAE-Sephadex A50 equilibrated in the same salt solution, but containing 0.1 mM-GTP. Under these conditions, all nori-tubulin components failed to adsorb and eluted together as a fraction of unbound protein. A fraction containing greater than 993 pure tubulin was subsequently elut’ed with t,he same column buff& but containing 0.8 M-KCl. The protein fractions were desalted by chromatography 011 Srphatlex G25 (coarse) equilibrated with PMG solution and generally used within 2 h of preparation. In some instances fractions were also st,ored at - 80°C for up to 1 mont.11 (bvith rct,mtion of SOY/, activity) and thawed immediately before use. The final COIIWIItrations of t,hc desalted non-tubulin and tubulin fractions n-cre usually. 5 and 3 mg/ml. respectively. For one series of experiments (see Results, section (c)). 6 S tubulin was prepared by high-speed centrifugation of microtubule protein iI1 PMG solution at 230,000 g for 90 min at 4°C. The preceding paper (Johnson & Borisy. 1977) has silown that b! analyt)ical centrifugation at polymerizing temperatures (25°C) such preparations exhibit a 6 S tubulin peak and no detectable levels of tubulin oligomers (e.g., 30 S species). Proteill concentrations were determined according to t,hr mrthod of Lowry et al. (1951) using borinrt serum albumin as a standard. (b) .4ssay procedure
for the kinetics
of mic~rotubule
elongation
The kinetics of microtubule elongation were determined by measuring the change irl optical density at 320 nm (Johnson & Borisy, 1977). As described previously, the turbidit,) obscrvcd at this wavelength resulted from the scattering of light by the polymer and was approximately proportional to the concentration of polymer. Typically. a 0.5.ml samplr> of DEAE-purified tubulin at 0°C was placed in a watc+jackcted cuvett’e and warmed to polymerization t,emperature (25.6”C). The half-time for thermal equilibration was 12 s. As reported previously (Murphy & Borisy, 1975). pllrifiod t,uhulitr alone failed to polymerize. After 75 s at 256”C, polymerizatiou was init’iated by t,ltc addition of a small sample (20 ~1) of a solution of microtubule fragments atld the O.D. at) 320 nm was recorded continuously (usually within 3 s after mixing) with a Vary 15 sprctrophotometer. Thp rates of rlonpatiorr was mcasurrd from the initial rat)(x of irlrrc~asc~ in optical derrsit,y.
Ylagellar axollemes were prepared from Chlamydomonas as described previously by. Allen & Borisy (1974). Briefly, cells in 400 ml of culture (3 to 4 x 106 cells/ml) were harvested, the flagella were removed and demcmbranatrtl in 10 rn>x-HEPES (N-2.hydroxyclt,hyl piperazine-A”-2-ethane snlfonio acid) buffer cont,aining O.O36o/b Non-Id& P40 (Shell Chemicals, Ltd, London). and washed in PM(: solution. Thf, axoneme pellet, was tllen resuspended in 0.2 ml PMU sol&on and tllc axoneme suspension was mixed witch tubulin so that a droplet of the mixture placed on AIL calectron microscope grid and negatively stained would contain at least 20 to 30 axonrmes. To test the ability of the isolated axonemes t,o support the addition of tubulin subunits, a sample of axonemes was incubated with a high-speed supernatant of depolymerized microtubule protein and placed at 37°C as described by allen & Borisy (1974) ; the number. of axonenes showing brain tubule addit,ion was usually greater than 95%. To assure maximal activity, it was necessary to prepare the axonemes immediately before use. Plastic tubes and pipettes were used for all stages of isolation to minimize fraying and darnagcl to tht, axotiemes. (d) Electron
microscopy
and carbon, and A 5-~1 sample was placed on a 400-mesh grid coated with Formvar successively displaced with 4 drops of each of the following solutions: 1 mg cytochrome c/ml, distilled water, and 1% aqueous uranyl acetate. The excess stain was removed with
36
D.
B. MURPHY,
K.
A. JOHNSON
AND
G. G. BORISY
a filter paper and the grid was air-dried. Grids were examined with a Philips 300 electron microscope at 80 kV. The length of microtubules was determined by comparison with a calibrated ruling on the phosphor viewing screen of the microscope. (e) I’olyacrylamide
gel electrophoresis
Samples and gels (5%, 0.6 cm x 8.0 cm) were prepared and run according to the procedure of Shapiro et al. (1967). Gels generally containing 3 to 5 rg protein were stained with Coomassie brilliant blue according to the procedure of Fairbanks et al. (1971) and densitometer tracings of the gels were made at 560 nm with a Gilford model 240 spectrophotometer (Gilford Instruments Laboratories, Oberlin, Ohio). Peak areas from the densitometer tracings were quantitated by planimetry. To detect trace levels of possible contaminants in the tubulin preparations gels were overloaded with up to 116 pg protein/ gel.
3. Results (a) Titration
of tubulin
with non-tub&in
proteins
The DEAE-ion exchange procedure used in this study separates microtubule protein into two fractions: one fraction contains tubulin to greater than 99% purity as determined by electrophoresis on sodium dodecyl sulfate gels overloaded with sample (100 pg/gel); the other fraction consists of all other proteins (designated non-tubulin proteins) that are present in microtubule protein purified bp two cycles of assembly and disassembly according to the procedure of Borisy et al. (1975) (Fig. 1). As shown in the Figure, the principal species in the non-tubulin fraction is a doublet of high molecular weight referred to as HMW 1 and 2 (286,000 and 271,000 M,, respectively) but the fraction also contained numerous other minor components including a species of 70,000 M, referred to as tau factor (Weingarten et al.. 1975; Penningroth et al., 1976) and some contaminating tubulin. Previous work has shown that although the HMW components were the principal stimulatory factors in this preparation, tau and possibly other factors also stimulated tubulin polymerization (Murphy et al.. 1977). Therefore we chose to work with this total non-tubulin fraction in order to avoid inadvertently discarding a pertinent factor.
HMW
I and
2
Tau 1
Tubulin 1
J
lb) FIQ. dodecyl
1. Electrophoretic sulfate/polyacrylamide
pattern
of non-tubulin gels.
fraction
(a) and
tubulin
fraction
(b) on sodium
STIMULATION
OF
MICROTUBULE
ASSEMBLY
3:
To examine the effect of the accessory proteins on microtubule assembly, purified tubulin was titrated with the non-tubulin fraction and the extent and rate of assembly were examined by monitoring the change in optical density at 320 nm under polymerizing conditions. The effect of the accessory proteins on t)he kinetics of microtubule polymerization is shown in Figure 2. By polymerizing the protein at 256°C the rate of assembly was sufficiently slow compa,red to the rate at 37°C t’hat the detailed behavior of the assembly kinetics could be observed. As reported previously. purified tubulin without added factors failed to self-assemble (Fig. 2(D)). However, as increasing amounts of the non-t8ubulin fraction were added back to tubulin. both t’he rate and extent of assembly increased (Fig. 2(A). (B) and (C)). In addition, a lag time was ohserved which preceded the onset of t#urbidit,y development (Fig. 2, a,rrows). The effects of the non-tubulin fraction on the rate and extent of assembly over a broad concentration range are shown in Figure 3. In these experiments tubulin at a fixed concentration of 1.3 mg/ml was titrated wit,11 the non-tubulin fraction and polymerization was monitored at a higher temperature (37°C) in order t’o attain equilibrium within a reasonable period of time. As seen in Figure 3, both the extent and rate of polymerization increased in sigmoidal fashion. In both cases. little change in turhidity was detected at concentrations of the non-tubulin fraction below 0.1 mg/ml (7)‘o of the total protein). Above this value. polymerization increased rapidly with half-maximal extent of assembly occurring at 0.35 mg/ml (Sl”,:, of the total protein) and half-maximal rate of assembly at 0.72 mg/ml (350; of t,he total protein). Similar results were obt’ained when polymer formation was monitored b) viscometry and sedimentation and when higher conccmrat8ions of tubulin uere used. However, as will be discussed subsequenbly. t,he fraction of t’ubulin polymerized depended upon the tubulin concentration. With increasing concentrat,ions of tubulin. lower concentrations of non-tubulin fraction were required to oht’ain self-assembly. For example, at 2.7 mg tuhulin/ml. 0.15 mg non-tubulin protc+/rnl (Gob) \l.as sufficient
Time (5) FIQ. 2. Stimulation mg/ml) in PMG buffer following addition of 0.11 mg/ml (curve B), tubulin protein a lag
of self-assembly of purified t,ubulin by non-tubulin protein. Tubulin (1.8 was pre-equilibrated to 26.6”C and turbidity was monitored at 320 nm PMG buffer (curve D), or non-tubulin fraction at 0.08 mg/ml (curve C), and 0.19 mg/ml (curve A) (final concns). At these concentrations of nonperiod was observed prior to assembly (indicated by arrows).
38
D.
B. MURPHY,
K.
A. JOHNSON
AND
G. G. BORISY
Protein concn (mg /ml)
FIa. 3. The effect of non-tubulin protein on the extent (-•-a-) and rate (-o--O--) of tubulin assembly. Polymerization of tubulin (1.3 mg/ml) in PMG buffer was monitored at 320 nm following addition of increasing amounts of non-tubulin fraction (0 to 2.6 mg/ml). Polymerization was performed at 37°C in order to estimate the extent of assembly at equilibrium. Prot,ein concentration indicates the final concentration of non-tubulin protein.
to obtain half-maximal extent of assembly. These data show that the non-tubulin proteins affect the extent of microtubule formation and suggest that the position of the equilibrium for polymerization is determined by the concentration of both tubulin and the accessory factors. In addition, since the amount of factor required to give half-maximal rate of assembly was significantly greater than that required for half-maximal extent of polymerization, it appeared that the factors might have a distinguishable affect on nucleation versus elongation of microtubules. Previous analysis of the kinetics and equilibrium of microtubule assembly (Gaskin et al., 1974; Johnson & Borisy, 1975,1977; Bryan, 1976) had demonstrated that, polymerization occurs as a condensation reaction with distinct phases of nucleation (initiation of new microtubules) and elongation (addition of subunits onto the ends of pre-existing microtubules). However, since the overall rate of self-assembly is a function of both of these phases, it was not possible to distinguish from the overall rate data alone to what extent the factors influenced just one or both of these processes. Experiments were therefore performed to examine the effects of the nontub&n proteins on microtubule nucleation and growth separately. (b) Effect of the non-tub&n
protein
on microtubule
n,ucEea,tioyL
Since an increase in tubule nucleation would result in an increase in the number concentration of microtubules, samples from the titration experiments were examined by electron microscopy to see if an increase in nucleation could be observed directly. With increasing concentrations of the non-tubulin fraction, the total length of tubules per unit area of the grid increased, in agreement with the observed increase in turbidity (Fig. 3). In addition it was observed that few long microtubules formed at low concentrations of factor and that many short microtubules formed at high concentrations of factor. Thus, the non-tubulin fraction appeared to stimulate tubule nucleation. A similar observation has been reported by Sloboda et al. (1976) using microtubule-associated proteins prepared from calf brain.
STIMULATION
OF
MICROTUB17LE
ASSEMBLY
39
It seemed plausible that the long lag periods and low rates of assembly that were observed at low factor concentrations reflected the kinetics of nucleation. In order to explore this possibility further the effects of non-tubulin protein on the kinetics of assembly were examined with respect to the lag time and the maximal rate of assembly. Lag times and maximal rates were extracted from data of the type shown in Figure 2. Lag times were obtained by extrapolating the linear portions of the curves back to the times of zero increase in turbidity. Maximal rates were obtained as the slope? of the initial linear portions of the curves. As shown in Figure 4, at low concentrations of factor, the lag time was extremely long. Over the range of factor concentrations explored (0 to 0.34 mg/ml), the lag decreased from greater than one hour to 20 seconds and the rate of assembly increased exponentially. By plotting the log of t,he rate versus the log of the factor concentration, a st’raight line plot was obtained with a slope of 3.4. This slope is an approximate measure of the order of the microtubule nucleation reaction with respect to the accessory proteins and suggests that’ several accessory protein molecules are involved in the formation of a nucleus (set Discussion). Since it appears that the number of microtubule ends available for growth is larger at higher factor concentrations, the increase in polymerization rate is attributable in part to an increase in microtubule nucleation. However, as shown helow, the accessory proteins also appear to affect the net’ rate of elongation. (0) Effect of the vsom?t&d%n p-otei,n m vnicrot,ubule growth The effect of the accessory proteins on the elongation reaction wras determined using the following procedure. Polymerization was initiated by the addition of a fixed concentration of microbubule seeds and a varying concentration of non-tubulin
Protein concn (mg /ml) Fro. 4. The effect of non-tubulin protein on the lag time preceding tubulin self-assembly (-0 -the initial rat’e of elongation. Accordingly. experiment,s were performed under conditions where t,he non-tubulin fraction alone did not) initiate assembly during the period in which the initial rate, pi of elongat,ion \vt’re bring measured following the addition of seeds. By using these conditions measurtments of initial rate following the addition of seeds measured the change in turbidity due only to microtubuk growth. The effect of the factors on the elongation reaction was then determined by measuring the inital rate of polymerization as :I funct’ion of the concentration of non-tubulin protein. In order to cover a range of possible effects of the non-tubulin protein, the results of t\vo kinds of experiments will he described belo\+,. Ln addition, since previous kinetic experiment)s on elongation were carried out using 6 S monomer preparations obtained by high-speed centrifugation (Johnson & Borisy. 1977) the use of DEAEpurified tubulin and high-speed supernatants as sources of subunits were compared One kind of experiment was performed at, a high tubulin and low seed concentration while the other was performed at a low tubuliu and high seed concentration. In each t,ype of experiment, the highest concentrat,ion of non-tubulin protein which could be investigated was limited by spontaneous nucleation as evidenced by the increase in optical density at 320 nm in the absencca of seeds. Since self-assembly \va.s also favored by increasing tubulin concentrat’ion. a higher concentration of nontubulin protein could be investigated in the experiments involving the lower tubulin concentrat8ions. The determination of the concentration of non-tubulin protein requires comment. Since the seeds were prepared from microtubule protein containing factor as well as t8ubulin, the contribution of unbound fact’or in t’he seed preparabion also had to be estimated. Seed concentrations were used which contributed minimal levels of factor (.
B.
MURPHY,
K.
A.
JOHNSON
ANI>
G.
G.
BORISY
Since the extent as well as the initial rate of polymerization was increased by increasing concentrations of non-tubulin protein, these factors clearly must effect the elongation as well as the initiation reactions. In this regard. our results agree with those obtained by others (Sloboda et al., 1976; Witman et al.. 1976). However, whereas we observed tubule elongation in the absence of added fact,ors, Witman ef al. did not. Their results indicated that purified tubulin did not have the capacity to assemble onto the ends of flagellar microtubules. whereas the addition of small amounts of tau restored the capacity for elongation. From these results as well as turbidometric studies, they concluded that tau is an essential structural component of microtubules. perhaps present in a precise stoichiometric relationship to tuhulin. In contrast, our results showed that DEAE-purified tubulin. when present, in concentrations above the critical concentration. would elongate microtubule seeds derived either from flagellar axonemes or sheared brain microtubules. Although the tubulin fraction alone was sufficient to support microtubule elongation, increasing concentrations of non-tubulin proteins increased bhe net rate of polymerization. Since the question of the absolute requirement, of non-tubulin proteins for microtubule elongation is a fundament’al issue for our understanding of microtubule structure and nssernhl-v. a discussion of the disparity in the two set,s of result’s is warranted. The difference in results does not arise from the source of microtubules used as the seeds since both this study and that of Witman et al. (1976) used flagellar axonemes isolated from the green alga, Chlamydomonas reinhardti, by the method of Allen & Borisy (1974). R’either can our results be explained easily by the suggestion that the tubulin used in our experiments contained small quantities of tau sufficient to produce the observed elongation, The DEAE-purified fraction contained tubulin and no other species were detected on overloaded gels ( >lOO pg/gel). From the densitometric patterns obtained from these gels, a minimum purity of 99% may be established. An independent estimate of the purity of the tubulin was obtained 1j.v its failure to self-assemble detectably at high concent,rations. Experiments at 2.5 mg/ml tubulin showed that as little as 25 pg added non-tub&n protein/ml (l”o of total) would induce self-assembly detectable by turbidity and sedimentation. Thus, since preparations of tubulin at 2.5 mg/ml failed to self-assemble in the absence of added factor. we conclude that our tubulin fraction contained less than l”/ non-tubulin protein. It is difficult to obtain convincing data for levels of purity beyond 99:/, and this question is of particular importance since Witman et al. (1976) showed that as little as 0.5% tau would permit some microtubule elongation onto pre-existing tubules and hence might account for the results reported here. However. we suggest an alternative hypothesis for the observed results. This alternative explanation states that tubulin alone is competent to elongate microtubules and that the concentration of tubulin required to obtain elongation depends on the solution conditions and the concentration of non-tubulin accessory factors present. Although we have not determined the critical concentration for elongation under the conditions employed by Witman et al. (1976). we suggest the possibility that since their isolation method and solution conditions differ from ours, their failure to obtain elongation in the absence of added factors was because the concentration of active tubulin was below the critical concentration.
HTIMULATIOS
OF
MICROTUBULE
ASSEMBLY
49
(c) A model for the effect oj the non-tubulin accessory proteins on the microtubule elongation reaction To determine quantit’atively the effect of the accessory factors on microtubule elongation. the initial rate of polymerization was measured after mixing microtubule seeds and subunits together at low concentrations of non-tubulin protein. These results suggested that the non-tubulin factors were not absolutely required for assembly, but indicated that they did alter the net rate and extent of assembly. Moreover. analysis of the dependence of the initial rate of elongation on tubulin concentration (Fig. 8) demonstrated that the non-tubulin protein increased the net rate of assembly and shifted the equilibrium towards polymer formation by decreasing the rate of depolymerization. In order to describe more fully the effect of the non-tubulin proteins in molecula,r terms and to evaluate whether the decrease in the rate of depolymerization could quantitatively account for the observed st’imulat’ion of assembly. the following model i< presented for the effect of binding of accesdorv proteins on the forward and reverse rates of microtubule assembly. Although. as indicated previously, the nont#ubulin fraction contains a mixture of componerns. the HMW proteins are the major species both in abundance as well as in the ability to stimulate t’ubulin polymerization (Murphy et al., 1977). Therefore, t’his model will be formulated in terms of t,he HMW species: however. the model should he understood to be applicable to the other non-tubulin species as well. Four assumptions form the basis for this analysis. (i) The equilibrium for tubule assembly is assumed to be given by the balance of the net rates of association and dissociation of 6 S tubulin subunits at the end of the microtubule and is defined b? the reaction
(1) wherts M and S represent the end of a microtubule and the 6 8 bubulin subunit. respectively. The net rate of assembly is determined by the difference of the rates of polymerization and depolymerization as follows : d O.U. dS c= - - = k,[MI dt dt
IS] -
k-,lM].
(2,
where k, and k- 1 are the apparent rate constants governingassemblyanddisassembly. respectively, and c is a proportionality constant relating the optical density to the concentration of tubulin subunits polymerized. These postulates were derived from our previous equilibrium and kinetic analyses of microbubule assembly (Johnson & Borisy, 19751977). (ii) The observation that the HMW proteins associated with microtubules to form projections at spacings equal to multiples of a regular interval (Murphy & Borisy, 1975) is taken to indicate that there are periodic binding sites on the tubule lattice not all of which are necessarily occupied. (iii) The HMW-tubule association reaction is assumed to be in a rapid equilibrium with respect to the rate of association and dissociation of tubulin subunits at the end of the tubule. The basis for this assumption is twofold: (a) the HMW proteins altered the initial rate of assembly when mixed simultaneously with seeds and 6 S tubulin; and (b) analysis of the temperature dependence of assembly suggested that the HMW association reaction was in a rapid equilibrium (Johnson, K. A. & Borisy, G. G., unpublished
5)
I).
H.
MURPHY,
I> 1, most of the sites are occupied and k-,,,,, = k’,. This case accounts for the plateau in Figure 5 as the net rate of assembly approaches a maximum of of HMW shifted the rate of k,[M][S] - k’_,[M]. Since a saturating concentration depolymerization (Fig. 8) to approximately zero, kl, is presumed to be very small and in the subsequent analysis is tentat,ively assigned a value of zero. (ii) When the concentration of non-tubulin protein is zero, all of the binding sites are free and the rate constant for disassembly becomes Icy 1. For this case, the net rate of assembly is given by k.JM] [S] - kl,[M]. Thus, the question of whether or not assembly
STIMULATION
OF MICROTUBU
LE
ilSSEMBLP
51
occurs in the absence of non-tubulin proteins depends upon whether or not k,[S J k:, is positive or negative for a given set of conditions. The two experiments described in Figure 5 demonstrate this point. In the experiment at the high tubulin concentration, the data indicate a positive rate of elongation while in the experiment at the lower tubulin concentration, a net negat’ive rwt,e was obtained by extrapolation to zero non-tubulin protein concentration. The smooth curves drawn in Figure 5 were calculated according to equation (6) on thtb assumption that the stimulatory activity was solely a function of HMW. The curve fitting was done as follows. First, on t’he basis of quantitative titration data (Peloquin, J. & Borisy, G. G., unpublished result’s), it was assumed that’ one HMW binding site was defined by 12 tubulin dimers polymerized in the seed preparation. This ratio is also in agreement with t’he HM3V superlattice as described l)y Amos (1977). From the number of tubulin dimers per HMW binding site. the molecular weight of the dimer (110,000) and the roncentrat,ion of polymer (mg/ml). the concentration of HMW binding sites was calculated. From the concentration of total HMW, the concentration of binding sites, and assuming identical and noninteracting sites, and assuming a value for K, the association constant for HMW binding, the fract’ion of bound sites, 0, was calculated. Setting lc: 1 == 0 and assuming a value of kL,.k-,,,,, was calculated. The molar concentration of microtubule seeds ]M], was calculated from the mass concentration, the average length of t)he seeds (1 pm) and the mass per unit length of tubule as defined by the microtubule lattice parameters of Erickson (1974). Knowing the subunit concentration LX] and values of the constants, lc, and c from previous studies (Johnson & Borisy, 1977), the initial rate of elongation. do.n./dt was calculated. The procedure was then reiterat,ed for varying values of K and kl r. The best fit for both experiments (Fig. 5) using kl, = 0 se1 for the was obtained with kl, = 19 s-l and K = 7~ lo6 M-l, reasons described above and k, = 2 x lo6 1\1-lssl as ha,s been previously measured (Johnson & Borisy, 1977). The values of these constants are constrained within relatively narrow limits (1200:) by the requirement of fitting the data obtained from two experiments performed under markedly different sets of conditions. How ever, t’he actual values will need to be measured experimentally. From the rat)e paramct’ers derived above, the critical concentration in the absence of factors may be found by setting k,[S] - lill = 0 and solving for IS 1. The value obtained is 1 mg/ml. Thus, the proposed model quantitatively account,s for t’he available data in terms of reasonable rate and association constants. The model presented here differs in a number of essential features from the model of Witman et al. (1976). In their model, tau is viewed as an integral component of the microtubule structure and elongation is directly dependent upon a stoichiom&ic incorporation of tau along with tubuhn. In contrast, the model presented here views thcx accessory proteins as not affecting at all the elongation reaction per SC. The facbors are neither required for the addition of a tubulin subunit at the end of the microtubule nor do they affect the rate constant for the addition reaction. Rather. the, factors bind at sites made available after polymerization has occurred and the) reduce the dissociation rate constant,, hence stabilizing the formed microtubules and shifting the equilibrium towards polymerization. It is intriguing to consider t,hat regulation of microtubule assembly in &vo may be cont’rolled by reactions that> alter the affinity of the accessory proteins for their binding sites on microtubules.
.52
D. B. MURPHY,
K.
A. ,JC)HNSON
ANI)
G. G. BORISY
This study was supported by National lrrstitutes of Health grant GM-21963 to one of 11s (G. G. B.); anothrr author (D. B. M.) was an N.I.H. Postdoctoral Bellow supported I)y grant CA-00923; tltc> third allt,llor (K. A. .J.) \vas sllpport.cd hy H~I N. 1.H. Predoctoral ‘I‘minrc&ip. HI1’FE:RENClGS 1 Allen, C. & Borisy, G. G. (lY74). J. Mol. Hiol. 90, 381-402. Amos, L. A. (1977). J. Cell. Biol. 72, 642- 654. Bloodgood, R. A. & Rosenbaum, J. L. (1976). J. Cell Biol. 71, 322-331. Borisy. G. G., Marcum, J. M., Olmsted. .J. B., Murphy, D. B. BE Johnson, K. A. (1975). Ann. X. Y. Acad. Sci. 253, 107- 132. Bryan, *J. (1976). J. Cell. Biol. 71, 749~~76i. Dentler, W. L., Granott, S. & Rosenbaum, J. L. (1975). J. Cell. Biol. 65, 237-241. Dentler, F$:. L., Granrtt, S., Witmarr, G. B. & Roscnbaum, J. L. (1974). 1%~. Nat. AC&. Sci.. U.S.A. 71, 1710~1714. Erickson, H. I’. (1974). .7. Cell Biol. 60, I53- 167. Erickson, H. P. & Vot,er, W. A. (1!)76). f’roc. &at. Acad. Sci., U.S.A. 73, 2813-2817. Fairbanks, G., Steck, T. L. & Wallach, D. F. H. (1971). Biochemistry, 10, 2606-2617. Gaskin, F., Cantor, C. K. & Shelanski, M. L. (1974). J. Mol. Biol. 89, 737.~758. Herzog, W. & Weber, K. (1977). I’roc. A’&. Acad. Sk., U.S.A. 74, 1860-~1864. Himes, R. H., Burt,on, P. H., Kprsry, Ft. N. & Pierson, G. B. (1976). Proc. X’at. Acad. Sci., U.S.A. 73,4397-439Y. ,Johnson, K. A. & Borisy, G. G. (l!J75). It1 Molecules and Cell Movement (InouB, S. Ur Stephens, It. E., ads), pp. I 1% 139, Raven Press, New York. Johnson, K. A. & Borisy, G. G. (1977). J. Mol. Biol. 117, 1-31. Keates, R. A. & Hall, I%. H. (1975). A’ature (Lomlon), 257, 418- 421. Kuriyama, R. (1975). J. Biochem. 77, 23. 31. Lee, J. C. & Timasheff, S. N. (1975). Biochemistry, 14, 5183-5187. Lowry, 0. A., Rosebrough. N. J., Farr, A. L. B Randall. R. .J. (1951). .I. Biol. Chem. 193, 265-275. Murplly, D. B. & Borisy, G. G. (lY75). 1%~. iVat. Acad. Sk., U.S.A. 72, 2696-2700. Murphy, D. B., Valler, R. B. & Borisy, G. G. (1977). Riochemistry, 16, 2598-2605. Penningroth, S. M., Cleveland, D. W. & Kirschner, M. W. (1976). In Cell Motility (GoldT. & R,osenbaum. .I . . eds), pp. 1233-1257, Cold Spring Harbor man, R., Pollard, Laboratory, New York. Scheele, K. B. & Borisy, G. G. (1976). Biochem. Biophys. Res. Cornmurk. 70, l--7. Biophys. Res. Commun. Shapiro, A. L., Vinuela, E. & Maizel, .r. V. (1967). Biochem. 28, 815-820. Shelanski, M. L.. Gaskin, F. & Cantor, C. R. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 765-768. Sloboda, R. D., Dentler, W. L. & Rosenbaum, J. L. (1976). Biochemistry, 15, 4497-4505. Tanford, C. (1961). Physical Chemistry of Macromolecules, pp. 532-535, John Wiley and Sons, New York. Weingarten, M. D., Suter, M. M., Littman, D. R. & Kirschner, M. W. (1974). Biochemistry, 13, 5529-5537. Weingarten, M. D., Lockwood, A. H.. Hwo, S. & Kirschner, M. W. (1975). Proc. iVat. Acad. Sci., U.S.A. 72, 1858-1862. Witman, G. B., Cleveland, D. W., Weingarten, M. D. & Kirschner, M. W. (1976). Prop. Nat. Acad. Sci.. [J.S.A. 73, 4070-4074.
