A reassessment of the factors affecting microtubule assembly and disassembly in vitro

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics.     

The sequence of a region of bacteriophage phiX174 DNA coding for parts of genes A and B

The kinetics of microtubule assembly were investigated by monitoring changes in turbidity which result from the scattering of incident light by the polymer. These studies indicated that assembly occurred by a pathway involving a nucleation phase, followed by an elongation phase as evidenced by a lag in the polymerization kinetics, followed by a psuedo-first-order exponential increase in turbidity. Analytical ultracentrifugation of solutions polymerized to equilibrium showed that 6 S tubulin was the only species detectable in equilibrium with microtubules. Investigation of the elongation reaction in mixtures of 6 S tubulin and microtubule fragments demonstrated that: (1) the net rate of assembly was the sum of the rates of polymerization and depolymerization; (2) the rate of polymerization was proportional to the product of the microtubule number concentration and the 6 S tubulin concentration; and (3) the rate of depolymerization was proportional to the number concentration of microtubules. These results demonstrate that microtubule assembly occurs by a condensation polymerization mechanism consisting of distinct nucleation and elongation steps. Microtubules are initiated in a series of protein association reactions in a pathway that has not been fully elucidated. Elongation proceeds by the consecutive association of 6 S tubulin subunits onto the ends of existing microtubules. Similarly, depolymerization occurs by dissociation of 6 S subunits from the ends of microtubules. The rate constants measured for polymerization and depolymerization at 30 °C were 4 × 10⁶m^?1 s^?1 and 7 s^?1, respectively.     

Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed.     

GDP-Tubulin Incorporation into Growing Microtubules Modulates Polymer Stability

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule “structural plasticity.”     

Colchicine inhibition of microtubule assembly via copolymer formation.

Colchicine.tubulin complex (CD) inhibits microtubule assembly. We examined this inhibition under conditions where spontaneous nucleation was suppressed and assembly was restricted to an elongation polymerization. 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 proposed 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; (b) these copolymers can have appreciable numbers of incorporated CDs which are, most likely, randomly distributed in the copolymers; (c) CD-tubulin copolymers have assembly-competent ends with association and dissociation rate constants which decrease as the CD/tubulin ratio in the copolymers, (CD/T)MT, increases; and (d) the critical tubulin concentrations required for microtubule assembly increase in the presence of CD, indicating that copolymer affinity for tubulin decreases as (CD/T)MT increases.     

A Metastable Intermediate State of Microtubule Dynamic Instability That Differs Significantly between Plus and Minus Ends

The current two-state GTP cap model of microtubule dynamic instability proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation while loss of this GTP-tubulin cap converts the microtubule end to shortening. However, when this model was directly tested by using a UV microbeam to sever axoneme-nucleated microtubules and thereby remove the microtubule's GTP cap, severed plus ends rapidly shortened, but severed minus ends immediately resumed elongation (Walker, R.A., S. Inoué, and E.D. Salmon. 1989. J. Cell Biol. 108: 931–937).

To determine if these previous results were dependent on the use of axonemes as seeds or were due to UV damage, or if they instead indicate an intermediate state in cap dynamics, we performed UV cutting of self-assembled microtubules and mechanical cutting of axoneme-nucleated microtubules. These independent methods yielded results consistent with the original work: a significant percentage of severed minus ends are stable after cutting. In additional experiments, we found that the stability of both severed plus and minus ends could be increased by increasing the free tubulin concentration, the solution GTP concentration, or by assembling microtubules with guanylyl-(α,β)-methylene-diphosphonate (GMPCPP).

Our results show that stability of severed ends, particularly minus ends, is not an artifact, but instead reveals the existence of a metastable kinetic intermediate state between the elongation and shortening states of dynamic instability. The kinetic properties of this intermediate state differ between plus and minus ends. We propose a three-state conformational cap model of dynamic instability, which has three structural states and four transition rate constants, and which uses the asymmetry of the tubulin heterodimer to explain many of the differences in dynamic instability at plus and minus ends.

     

Equilibrium and kinetic analysis of microtubule assembly in the presence of guanosine diphosphate

In this paper we expand upon a previously reported observation of the effects of GDP on microtubule assembly. A ratio of GDP to GTP of ten (1 mm-GDP and 0.1 mm-GTP) is generally sufficient to completely block microtubule assembly, but only limited depolymerization is induced if GDP is added after assembly has reached a plateau in the presence of GTP. When added during polymerization, GDP arrests further elongation, and greater steady-state levels of assembly are obtained the later the time of addition of GDP. To explain this behavior we examined the rates of assembly and disassembly and the apparent critical concentration (C₀) of tubulin in the presence of GDP. GDP-tubulin polymerizes very slowly as compared to GTP-tubulin, while depolymerization rates, as determined by dilution, are nearly identical in GTP and GDP. The C₀ value calculated from the assembly and disassembly rates in GTP is within experimental error of the C₀ value at steady-state determined directly. In the presence of GDP, however, the C₀ value calculated from rate measurements is at least 60 times greater than that determined by equilibrium analysis. Our results indicate that the net assembly rate in GDP is not a valid measure of the reaction occurring at steady-state. A limited amount of depolymerization may occur upon addition of GDP to microtubules, and this appears to be due to a decrease in the fraction of protein able to participate in the polymerization reaction. The amount of tubulin “inactivated” by GDP is increased by the removal of microtubule-associated proteins. GDP-tubulin will stabilize existing microtubules, even when its polymerization cannot be demonstrated. These results are inconsistent with present models of microtubule assembly, and a new model involving co-operative interaction of microtubule-associated protein-tubulin oligomers at microtubule ends is proposed.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

Interaction of vinblastine with steady-state microtubules in vitro

At low concentrations, vinblastine binds rapidly and reversibly to a very limited number of high affinity sites on steady-state bovine brain microtubules (mean K_d, 1.9 × 10^?6m; 16.8 ± 4.3 vinblastine binding sites per microtubule) which appear to be located at one or both ends of the microtubules. At high concentrations, vinblastine binds to a high binding capacity class of sites of undetermined affinity, located on helical strands of protofilaments which form at the ends of depolymerizing microtubules, and/or along the surface of the microtubules. Substoichiometric inhibition of microtubule assembly, which occurs at low vinblastine concentrations, appears to be due to the binding of vinblastine to the high affinity class of sites. Fifty per cent inhibition of tubulin addition to the net assembly ends of steady-state microtubules occurred at 1.38 × 10^?7m-drug, and at this concentration, 1.16 ± 0.27 molecules of vinblastine were bound to the high affinity class of sites. Vinblastine appeared to bind directly to the microtubule ends, and our results indicate that vinblastine inhibits the assembly of steady-state bovine brain microtubules by binding rapidly and with high affinity to one or two molecules of tubulin at the net assembly ends. Splaying and peeling of protofilaments at microtubule ends and the active depolymerization of microtubules occurred only at vinblastine concentrations greater than 1 × 10^?6 to 2 × 10^?6m. This action of vinblastine is associated with and may be due to the binding of vinblastine to the high capacity class of sites. Both actions of vinblastine may be due to the binding of vinblastine to the same binding sites on the tubulin molecule, with the sites exhibiting either a high or low affinity depending upon the location in the microtubule.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

Arabidopsis AUGMIN Subunit8 Is a Microtubule Plus-End Binding Protein That Promotes Microtubule Reorientation in Hypocotyls

In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis.     

Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.     

Microtubule elongation and guanosine 5'-triphosphate hydrolysis. Role of guanine nucleotides in microtubule dynamics

The tubulin concentration dependence of the rates of microtubule elongation and accompanying GTP hydrolysis has been studied over a large range of tubulin concentration. GTP hydrolysis followed the elongation process closely at low tubulin concentration and became gradually uncoupled at higher concentrations, reaching a limiting rate of 35-40 s-1. The kinetic parameters for microtubule growth were different at low and high tubulin concentrations. Elongation of microtubules has also been studied in solutions containing GDP and GTP in variable proportions. Only traces of GTP present in GDP were necessary to confer a high stability (low critical concentration) to microtubules. Pure GDP-tubulin was found unable to elongate microtubules in the absence of GTP but blocked microtubule ends with an equilibrium dissociation constant of 5-6 microM. These data were accounted for by a model within which, in the presence of GTP-tubulin at high concentration, microtubules grow at a fast rate with a large GTP cap; the GTP cap may be quite short in the region of the critical concentration; microtubule stability is linked to the strong interaction between GTP and GDP subunits at the elongating site; dimeric GDP-tubulin does not have the appropriate conformation to undergo reversible polymerization. These results are discussed with regard to possible role of GDP and GTP and of GTP hydrolysis in microtubule dynamics.     

Role of tubulin-associated proteins in microtubule nucleation and elongation

Previous experiments have shown 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 the 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 to microtubule seeds and the initial rate of polymerization was measured under conditions where spontaneous self-assembly was below detectable levels. In addition, microtubule growth was also observed when isolated flagellar 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 stimulated the growth of microtubules not by increasing the rate of polymerization but by decreasing the rate of depolyerization. The mechanism by which these accessory factors promote tubulin assembly may be summarized as follows: under the conditions employed, they 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 microtubules and consequently shifting the equilibrium to favor assembly.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

The determinants that govern microtubule assembly from the atomic structure of GTP-tubulin

Tubulin alternates between a soluble curved structure and a microtubule straight conformation. GTP binding to αβ-tubulin is required for microtubule assembly, but whether this triggers conversion into a straighter structure is still debated. This is due, at least in part, to the lack of structural data for GTP-tubulin before assembly. Here, we report atomic-resolution crystal structures of soluble tubulin in the GDP and GTP nucleotide states in a complex with a stathmin-like domain. The structures differ locally in the neighborhood of the nucleotide. A loop movement in GTP-bound tubulin favors its recruitment to the ends of growing microtubules and facilitates its curved-to-straight transition, but this conversion has not proceeded yet. The data therefore argue for the conformational change toward the straight structure occurring as microtubule-specific contacts are established. They also suggest a model for the way the tubulin structure is modified in relation to microtubule assembly.     

Theory for modeling the copolymerization of tubulin and tubulin-colchicine complex

Substoichiometric concentrations of tubulin-colchicine complex (TC) inhibits microtubule assembly through a copolymerization reaction between tubulin and TC. We have determined the rates and extent of TC incorporation into bovine brain microtubules and developed a theory that models copolymerization. Our analysis suggests that while the apparent association rate constants for tubulin and TC are similar, the apparent dissociation rate constants for TC are a factor of five or more larger than those of tubulin. Copolymer composition showed only slight changes during assembly despite changes in the solution phase and showed little dependence at high TC upon the initial tubulin concentration. The theory was based on coupled Oosawa-Kasai equations that allow for the co-assembly of two components, tubulin and TC. An expression was derived that relates copolymer composition to reaction mixture composition and to the affinity of microtubule ends for tubulin and TC. This expression predicts copolymer composition at TC concentrations less than 10 microM and correlates composition with assembly inhibition. We perceive copolymerization as a facilitated incorporation of TC requiring the presence of tubulin. TC incorporation was dependent on the ratio of total tubulin to the dissociation constant for TC bound to microtubule ends. The copolymerization reaction is thus characterized by an interplay of two effects (a) where tubulin facilitates the incorporation of TC into the microtubule, and (b) where TC inhibits the assembly of tubulin into microtubules.