Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Structural changes accompanying GTP hydrolysis in microtubules: information from a slowly hydrolyzable analogue guanylyl-(alpha,beta)- methylene-diphosphonate

We have used cryoelectron microscopy to try to understand the structural basis for the role of GTP hydrolysis in destabilizing the microtubule lattice. We have measured a structural difference introduced into microtubules by replacing GTP with guanylyl- (alpha,beta)-methylene-diphosphonate (GMPCPP). In a stable GMPCPP microtubule lattice, the moire patterns change and the tubulin subunits increase in size by 1.5 A. This information provides a clue to the role of hydrolysis in inducing the structural change at the end of a microtubule during the transition from a growing to a shrinking phase.     

Microtubule structure at improved resolution.

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.     

The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice [published erratum appears in J Cell Biol 1995 Apr;129(2):549]

The standard free energy for hydrolysis of the GTP analogue guanylyl- (a,b)-methylene-diphosphonate (GMPCPP), which is -5.18 kcal in solution, was found to be -3.79 kcal in tubulin dimers, and only -0.90 kcal in tubulin subunits in microtubules. The near-zero change in standard free energy for GMPCPP hydrolysis in the microtubule indicates that the majority of the free energy potentially available from this reaction is stored in the microtubule lattice; this energy is available to do work, as in chromosome movement. The equilibrium constants described here were obtained from video microscopy measurements of the kinetics of assembly and disassembly of GMPCPP-microtubules and GMPCP- microtubules. It was possible to study GMPCPP-microtubules since GMPCPP is not hydrolyzed during assembly. Microtubules containing GMPCP were obtained by assembly of high concentrations of tubulin-GMPCP subunits, as well as by treating tubulin-GMPCPP-microtubules in sodium (but not potassium) Pipes buffer with glycerol, which reduced the half-time for GMPCPP hydrolysis from > 10 h to approximately 10 min. The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s-1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.     

Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules.

Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly(catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCpP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 to 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of the rate constant for dissociation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microtubules, now found to be 0.118 s-1, has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as previously reported.     

Diffusion inside microtubules

Recent high-resolution analysis of tubulin's structure has led to the prediction that the taxol binding site and a tubulin acetylation site are on the interior of microtubules, suggesting that diffusion inside microtubules is potentially a biologically and clinically important process. To assess the rates of transport inside microtubules, predictions of diffusion time scales and concentration profiles were made using a model for diffusion with parameters estimated from experiments reported in the literature. Three specific cases were considered: 1) diffusion of αβ-tubulin dimer, 2) diffusion/binding of taxol, and 3) diffusion/binding of an antibody specific for an epitope on the microtubule's interior surface. In the first case tubulin is predicted to require only ∼1 min to reach half the equilibrium concentration in the center of a 40 μm microtubule open at both ends. This relatively rapid transport occurs because of a lack of appreciable affinity between tubulin and the microtubule inner surface and occurs in spite of a three-fold reduction in diffusivity due to hindrance. By contrast the transport of taxol is much slower, requiring days (at nm concentrations) to reach half the equilibrium concentration in the center of a 40 μm microtubule having both ends open. This slow transport is the result of fast, reversible taxol binding to the microtubule's interior surface and the large capacity for taxol (∼12 mm based on interior volume of the microtubule). An antibody directed toward an epitope in the microtubule's interior is predicted to require years to approach equilibrium. These results are difficult to reconcile with previous experimental results where substantial taxol and antibody binding is achieved in minutes, suggesting that these binding sites are on the microtubule exterior. The slow transport rates also suggest that microtubules might be able to serve as vehicles for controlled-release of drugs. Received: 2 March 1998 / Revised version: 3 May 1998 / Accepted: 3 May 1998     

Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.

Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly.     

Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization

A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.     

Magnesium requirements for guanosine 5'-O-(3-thiotriphosphate) induced assembly of microtubule protein and tubulin

Two conflicting interpretations on the role of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in microtubule protein and tubulin assembly have been previously reported. One study finds that GTP gamma S promotes assembly while another study reports that GTP gamma S is a potent inhibitor of microtubule assembly. We have examined the potential role of Mg2+ to learn if the conflicting interpretations are due to a metal effect. Turbidity, electron microscopy, and nucleotide binding and hydrolysis were used to analyze the effect of the Mg2+ concentration on GTP gamma S-induced assembly of microtubule protein (tubulin + microtubule-associated proteins) in the presence of buffer +/- 30% glycerol and in buffer with GTP added before or after GTP gamma S. GTP gamma S substantially lowers the Mg2+ concentration required to induce cross-linked or clustered rings of tubulin. These cross-linked rings do not assemble well into microtubules, and GTP only partially restores microtubule assembly. However, taxol will promote GTP gamma S-induced cross-linked rings of microtubule protein to assemble into microtubules. The effect of GTP gamma S on microtubule protein assembly in the presence of Zn2+ with and without added Mg2+ suggests that GTP gamma S also effects the formation of Zn2+-induced sheet aggregates. Purified tubulin was used in assembly experiments with Mg2+, Zn2+, and taxol to better understand GTP gamma S interactions with tubulin. The optimal Mg2+ concentration for assembly of tubulin is lower with GTP gamma S than with GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taxol binds to cellular microtubules

Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. B. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [3H]taxol and the formation of microtubule bundles. [3H]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubules in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [3H]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaN3 prevents the characteristic taxol-induced bundle formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there formation. The binding of [3H]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.     

Equilibrium studies of a fluorescent paclitaxel derivative binding to microtubules

A fluorescent derivative of paclitaxel, 3'-N-m-aminobenzamido-3'-N-debenzamidopaclitaxel (N-AB-PT), has been prepared in order to probe paclitaxel-microtubule interactions. Fluorescence spectroscopy was used to quantitatively assess the association of N-AB-PT with microtubules. N-AB-PT was found equipotent with paclitaxel in promoting microtubule polymerization. Paclitaxel and N-AB-PT underwent rapid exchange with each other on microtubules assembled from GTP-, GDP-, and GMPCPP-tubulin. The equilibrium binding parameters for N-AB-PT to microtubules assembled from GTP-tubulin were derived through fluorescence titration. N-AB-PT bound to two types of sites on microtubules (K(d1) = 61 +/- 7.0 nM and K(d2) = 3.3 +/- 0.54 microM). The stoichiometry of each site was less than one ligand per tubulin dimer in the microtubule (n(1) = 0.81 +/- 0.03 and n(2) = 0.44 +/- 0.02). The binding experiments were repeated after exchanging the GTP for GDP or for GMPCPP. It was found that N-AB-PT bound to a single site on microtubules assembled from GDP-tubulin with a dissociation constant of 2.5 +/- 0.29 microM, and that N-AB-PT bound to a single site on microtubules assembled from GMPCPP-tubulin with a dissociation constant of 15 +/- 4.0 nM. It therefore appears that microtubules contain two types of binding sites for paclitaxel and that the binding site affinity for paclitaxel depends on the nucleotide content of tubulin. It has been established that paclitaxel binding does not inhibit GTP hydrolysis and microtubules assembled from GTP-tubulin in the presence of paclitaxel contain almost exclusively GDP at the E-site. We propose that although all the subunits of the microtubule at steady state are the same "GDP-tubulin-paclitaxel", they are formed through two paths: paclitaxel binding to a tubulin subunit before its E-site GTP hydrolysis is of high affinity, and paclitaxel binding to a tubulin subunit containing hydrolyzed GDP at its E-site is of low affinity.     

The kinetic mechanism of kinesin

The chemical kinetic mechanism of kinesin (K) is considered by using a consensus scheme incorporating biochemically defined open, closed and trapped states. In the absence of microtubules, the dominant species is a trapped K*ADP state, which is defined by its ultra-slow release of ADP (off rate, k(off) approximately 0.002 s(-1)) and weak microtubule binding (dissociation constant, K(d) approximately 10-20 microM). Once bound, this trapped state equilibrates with a strongly binding open state that rapidly releases ADP (k(off) approximately 300 s(-1)). After ADP release, Mg*ATP binds (on rate, k(on) approximately 2 microM(-1)s(-1)) driving formation of a closed state that is defined by hydrolysis competence and by strong binding to microtubules. Hydrolysis (k(hyd) approximately 100-300 s(-1)) and phosphate release (k(off)>100 s(-1)) both occur in this microtubule-bound closed state. Phosphate release acts as a gate that controls reversion to the trapped K*ADP state, which detaches from the microtubule, completing the cycle.     

Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5′-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.     

In vivo and in vitro studies on the role of HMW-MAPs in taxol-induced microtubule bundling

The involvement of high molecular weight microtubule-associated proteins (HMW-MAPs) in the process of taxol-induced microtubule bundling has been studied using immunofluorescence and electron microscopy. Immunofluorescence microscopy shows that HMW-MAPs are released from microtubules in granulosa cells which have been extracted in a Triton X-100 microtubule-stabilizing buffer (T-MTSB), unless the cells are pretreated with taxol. 1.0 microM taxol treatment for 48 h results in microtubule bundle formation and the retention of HMW-MAPs in these cells upon extraction with T-MTSB. Electron microscopy demonstrates that microtubules in control cytoskeletons are devoid of surface structures whereas the microtubules in taxol-treated cytoskeletons are decorated by globular particles of a mean diameter of 19.5 nm. The assembly of 3 X cycled whole microtubule protein (tubulin plus associated proteins) in vitro in the presence of 1.0 microM taxol, results in the formation of closely packed microtubules decorated with irregularly spaced globular particles, similar in size to those observed in cytoskeletons of taxol-treated granulosa cells. Microtubules assembled in vitro in the absence of taxol display prominent filamentous extensions from the microtubule surface and center-to-center spacings greater than that observed for microtubules assembled in the presence of taxol. Brain microtubule protein was purified into 6 s and HMW-MAP-enriched fractions, and the effects of taxol on the assembly and morphology of these fractions, separately or in combination, were examined. Microtubules assembled from 6 s tubulin alone or 6 s tubulin plus taxol (without HMW-MAPs) were short, free structures whereas those formed in the presence of taxol from 6 s tubulin and a HMW-MAP-enriched fraction were extensively crosslinked into aggregates. These data suggest that taxol induces microtubule bundling by stabilizing the association of HMW-MAPs with the microtubule surface which promotes lateral aggregation.     

Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.     

The minimum GTP cap required to stabilize microtubules

Background: Microtubules polymerized from pure tubulin show the unusual property of dynamic instability, in which both growing and shrinking polymers coexist at steady state. Shortly after its addition to a microtubule end, a tubulin subunit hydrolyzes its bound GTP. Studies with non-hydrolyzable analogs have shown that GTP hydrolysis is not required for microtubule assembly, but is essential for generating a dynamic polymer, in which the subunits at the growing tip have bound GTP and those in the bulk of the polymer have bound GDP. It has been suggested that loss of the ‘GTP cap’ through dissociation or hydrolysis exposes the unstable GDP core, leading to rapid depolymerization. However, evidence for a stabilizing cap has been very difficult to obtain.Results We developed an assay to determine the minimum GTP cap necessary to stabilize a microtubule from shrinking. Assembly of a small number of subunits containing a slowly hydrolyzed GTP analog (GMPCPP) onto the end of dynamic microtubules stabilized the polymer to dilution. By labeling the subunits with rhodamine, we measured the size of the cap and found that as few as 40 subunits were sufficient to stabilize a microtubule.Conclusion On the basis of statistical arguments, in which the proportion of stabilized microtubules is compared to the probability that when 40 GMPCPP-tubulin subunits have polymerized onto a microtubule end, all protofilaments have added at least one GMPCPP-tubulin subunit, our measurements of cap size support a model in which a single GTP subunit at the end of each of the 13 protofilaments of a microtubule is sufficient for stabilization. Depolymerization of a microtubule may be initiated by an exposed tubulin–GDP subunit at even a single position. These results have implications for the structure of microtubules and their means of regulation.     

Mechanism of the microtubule GTPase reaction

The rate of GTP hydrolysis by microtubules has been measured at tubulin subunit concentrations where microtubules undergo net disassembly. This was made possible by using microtubules stabilized against disassembly by reaction with ethylene glycol bis-(succinimidylsuccinate) (EGS) as sites for the addition of tubulin-GTP subunits. The tubulin subunit concentration was varied from 25 to 90% of the steady state concentration, and there was no net elongation of stabilized microtubule seeds. The GTPase rate with EGS microtubules was linearly proportional to the tubulin-GTP subunit concentration when this concentration was varied by dilution and by using GDP to compete with GTP for the tubulin E-site. The linear dependence of the rate is consistent with a GTP mechanism in which hydrolysis is coupled to the tubulin-GTP subunit addition to microtubule ends. It is inconsistent with reaction schemes in which: microtubules are capped by a single tubulin-GTP subunit, which hydrolyzes GTP when a tubulin-GTP subunit adds to the end; hydrolysis occurs primarily in subunits at the interface of a tubulin-GTP cap and the tubulin-GDP microtubule core; hydrolysis is not coupled to subunit addition and occurs randomly in subunits in a tubulin-GTP cap. It was also found that GDP inhibition of the microtubule GTPase rate results from GDP competition for GTP at the tubulin subunit E-site. There is no additional effect of GDP on the GTPase rate resulting from exchange into tubulin subunits at microtubule ends.     

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.     

X‐ray and Cryo‐EM structures reveal mutual conformational changes of Kinesin and GTP‐state microtubules upon binding

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin‐1/KIF5C preferentially binds to the GTP‐state microtubules over GDP‐state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide‐free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide‐free KIF5C and the cryo‐electron microscopic structure of nucleotide‐free KIF5C complexed with the GTP‐state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP‐state microtubule and KIF5C. KIF5C acquires the ‘rigor conformation’, where mobile switches I and II are stabilized through L11 and the initial portion of the neck‐linker, facilitating effective ADP release and the weak‐to‐strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP‐state microtubule in a similar manner to GDP‐taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP‐state microtubules.