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.     

Enthalpy changes in microtubule assembly from pure tubulin

The enthalpy changes that occur in the self-assembly of tubulin into microtubules were examined by adiabatic differential heat capacity microcalorimetry and by isothermal batch microcalorimetry. Tubulin solutions at concentrations between 7 and 17 mg/mL were heated from 0 to 40 degrees C at heating rates of 1 or 2 deg/min in pH 6.8 or 7.0 assembly buffers containing 20 mM MES, 100 mM glutamic acid, 5 mM MgCl2, 3.4 M glycerol, and either 0.5 mM GMP-PCP or 1 mM GTP. The assembly reaction in the presence of GTP was characterized by a complex heat-uptake pattern consisting of a broad endotherm with a sharper exotherm superimposed on it, similar to assembly in a GTP phosphate buffer [Hinz, H.-J., Gorbunoff, M.J., Price, B., & Timasheff, S.N. (1979) Biochemistry 18,3084]. Replacement of GTP by the nonhydrolyzable analogue resulted in a pattern typical for an endothermic reaction only. These results have permitted the assignment of the endothermic process to microtubule assembly and of the exothermic process to the resultant GTP hydrolysis. In these studies equilibration was found to be slow, several hours of cooling being required for the system to return to its original state. Turbidity scans also revealed hysteresis between consecutive scans and a displacement of the depolymerization transition midpoint to a lower temperature than that of assembly. The disassembly of microtubules was examined in batch calorimetry experiments in pH 7.0 phosphate, 1 mM GTP, 16 mM MgCl2, and 3.4 M glycerol, in which tubulin assembled into microtubules was diluted to below the critical concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.     

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.     

Energetics of the cooperative assembly of cell division protein FtsZ and the nucleotide hydrolysis switch

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 +/- 100 cal mol-1 K-1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 degrees C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol-1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.     

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.     

Interaction of tubulin and cellular microtubules with the new antitumor drug MDL 27048. A powerful and reversible microtubule inhibitor

We have characterized the binding of trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2- propen- 1-one (MDL 27048) to purified procine brain tubulin, and the inhibition of microtubule assembly by this compound in vitro and using cultured cells. Binding measurements were performed by difference absorption and fluorescence spectroscopy. MDL 27048 binds to one site/tubulin heterodimer with an apparent equilibrium constant Kb = (2.8 +/- 0.8) X 10(6) M-1 (50 mM 2-(N-morpholino)ethanesulfonic acid, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.5 mM MgCl2, 0.1 mM GTP buffer, pH 6.7, at 25 degrees C). Podophyllotoxin displaced the binding of MDL 27048, suggesting an overlap with the colchicine-binding site. Assembly of purified tubulin into microtubules was inhibited by substoichiometric concentrations of MDL 27048, which also induced a slow depolymerization of preassembled microtubules. The cytoplasmic microtubules of PtK2 cells were disrupted in a concentration and time-dependent manner by MDL 27048, as observed by indirect immunofluorescence microscopy. Maximal depolymerization took place with 2 X 10(-6) M MDL 27048 in 3 h. When the inhibitor was washed off from the cells, fast microtubule assembly (approximately 8 min) and complete reorganization of the cytoplasmic microtubule network (15-30 min) were observed. MDL 27048 also induced mitotic arrest in SV40-3T3 cell cultures. Due to all these properties, this anti-tumor drug constitutes a new and potent microtubule inhibitor, characterized by its specificity and reversibility.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselectivity of the guanyl-exchangeable nucleotide-binding site of tubulin probed by guanosine 5'-O-(2-thiotriphosphate) diastereoisomers

The active site of the exchangeable nucleotide-binding site of tubulin was studied by using diastereoisomers A (Sp) and B (Rp) of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) where the phosphorus atom to which sulfur is attached is chiral. Turbidimetric measurements were used to follow kinetics, and electron microscopy was used to evaluate polymeric forms. Both isomers at 0.5 mM promoted the assembly of tubulin in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid, 30% glycerol, 3 mM MgCl2, and 1 mM EGTA, pH 6.6, 23-37 degrees C. GTP beta S(A) promoted assembly into microtubules, although a few bundles were also found by electron microscopy. However, GTP beta S(B) induced assembly of tubulin into bundles of sheets and microtubules. As expected, 0.5 mM GTP induced tubulin to assemble into microtubules, thin sheets, and a few bundles. Both GTP and GTP beta S(A) were hydrolyzed in the tubulin polymers. However, more than 95% of the bound GTP beta S(B) was not hydrolyzed. Higher concentrations of GTP beta S(B), i.e., 1 mM, also induced bundles of sheets and microtubules, with 86% of the thionucleotide bound as the triphosphate. The GTP beta S(B)-induced polymers were considerably more cold stable than the GTB beta S(A)-induced microtubules, which were more cold stable than GTP-induced polymers. Mg(II) (2-5 mM) had minimal effects on the structures induced by GTP beta S(A) or -(B) isomers in the tubulin assembly system. However, at 1 mM Mg(II), no assembly was found with GTP beta S(A) and tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions

We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end.     

A synchrotron X-ray scattering characterization of purified tubulin and of its expansion induced by mild detergent binding

This report presents a synchrotron radiation X-ray scattering characterization of calf brain tubulin purified by the modified Weisenberg procedure. The results show that under nonassembly conditions (i.e., in 10 mM sodium phosphate and 0.1 mM GTP, pH 7, buffer) these preparations consist of a uniform population of molecules with a radius of gyration of 3.1 +/- 0.1 nm, which can be interpreted as arising from the native alpha-beta heterodimer. The uniformity in the population persists even at unusually high concentrations of protein. Binding of colchicine or substitution of GTP by GDP does not induce, within the statistical accuracy and resolution range of our measurements, any significant structural modification in soluble tubulin. In assembly buffer [i.e., 10 mM sodium phosphate, 6 mM magnesium chloride, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 1 mM GTP, and 3.4 M glycerol, pH 6.5], these preparations readily assemble into microtubules upon increasing the temperature from 4 to 37 degrees C. Binding of nondenaturing amphiphiles to soluble tubulin provides a simplified model for tubulin-membrane interactions. The X-ray scattering data show that the radius of gyration of tubulin progressively increases upon binding of the mild detergent sodium deoxycholate, reaching a maximum value of 4.3 +/- 0.1 nm at detergent saturation. The relative increase in the radius of gyration coincides within experimental error with the previously determined relative increase in the frictional coefficient [Andreu, J.M., & Mu?oz, J.A. (1986) Biochemistry 25, 5220-5230]. Analysis of these observations suggests that the effect of detergent binding is to induce an isotropic swelling of the protein structure.     

Direct incorporation of microtubule oligomers at high GTP concentrations

Chick brain microtubule protein consists primarily of a mixture of MAP2:tubulin oligomers and dimeric tubulin. The assembly of this protein is described by a single pseudofirst-order reaction at 20 microM GTP, but by the summation of two pseudofirst-order reactions at 1 mM GTP. The protein contains two GTP-binding species, corresponding to the tubulin dimers and the oligomers, and conditions which alter the dimer: oligomer equilibrium, affect the kinetics of microtubule assembly. The results indicate that the oligomers are only direct assembly intermediates at high GTP concentrations.     

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.     

Microtubular protein reaction with nucleotides.

The dissociation constants for GTP and GDP with tubulin were determined to be equal to 1.1 ± 0.4 × 10^?7 M and 1.5 ± .6 × 10^?7 (4°), respectively. A lower limit for the dissociation constant for ATP was established as equal to 6 × 10^?4 M. The equivalent binding of GTP and GDP is not readily consistent with a mechanism in which the role of GTP in microtubule assembly is to bind to the protein to induce a conformation which is able to polymerize. An ATP-induced polymerization of tubulin apparently involves a transphosphorylation reaction in which GTP is formed and mediates the assembly. For this reaction to occur with desalted tubulin trace amounts of GDP are required; in the reaction of 0.1 mM ATP with 22.0 μM tubulin, 0.1 μM GDP induces about 80% as much tubule formation as is seen with 0.1 mM GTP alone.     

Guanosine 5'-O-(3-thiotriphosphate), a potent nucleotide inhibitor of microtubule assembly

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the two diastereoisomers of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) were prepared enzymatically, and their interactions with tubulin and microtubule-associated proteins (MAPs) in 0.1 M 2-(N-morpholino)ethanesulfonate, 0.5 mM MgCl2 were examined. GTP gamma S did not support microtubule assembly but instead inhibited the reaction. This analog was 1.5-2 times more potent than GDP in inhibiting both tubulin polymerization and GTP hydrolysis under conditions in which these reactions were dependent on MAPs. In contrast to the analog's inhibitory effects on polymerization and hydrolysis, however, radiolabeled GTP gamma S was only feebly bound by purified tubulin at 0 degrees C relative to the binding of GDP and GTP. There was a marked increase in the amount of GTP gamma S bound when the reaction temperature was raised to 37 degrees C or when MAPs were included in the reaction mixture. Only when both MAPs were present and the higher reaction temperature was used did the binding of GTP gamma S exceed that of GDP. Since substitution of sulfur for oxygen in a molecule should decrease its hydrophilic properties, these findings suggest that the exchangeable nucleotide binding site of tubulin becomes more hydrophobic at higher temperatures and in the presence of MAPs. The two isomers of GTP beta S were able to support MAP-dependent polymerization, although a 50-100-fold higher concentration of the analogs as compared to GTP was required. Neither isomer of GTP beta S had a significant inhibitory effect on GTP hydrolysis dependent on tubulin + MAPs.     

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.     

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.     

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.