Magnesium ion effects on microtubule nucleation in vitro

Much interest has currently been attached to the length distribution of microtubules polymerized in vitro and the related question of their possible 'dynamic instability'. Fundamental to this question is the mechanism of microtubule nucleation, which controls the rates of assembly and disassembly of microtubule protein in vitro. These kinetics are affected by a number of factors, including both the guanine nucleotides, GTP and GDP, and magnesium ion. Mg2+ exerts complex effects, as indicated by the existence of an optimal Mg2+ concentration for the maximum assembly rate of microtubule protein, and we investigate these effects in this report. At [Mg2+] greater than 0.5 mM, the characteristic lag-phase is substantially increased and the rate of assembly is greatly reduced without affecting the critical concentration significantly. We show that increasing [Mg2+] has two effects on the assembly process: nucleation is less efficient and the intrinsic rate constant for the elongation reaction is reduced. Lowering [Mg2+] (less than 0.5 mM) also inhibits nucleation. These effects of varying [Mg2+] can be explained predominantly in terms of enhanced stability of the microtubule-associated protein-containing oligomeric species present in the microtubule protein preparation. [Mg2+] is thus found to be a further important factor in microtubule nucleation, and hence, in determining length distributions in assembling microtubules.     

Control of nucleation in microtubule self-assembly

The inhibition of the rate and amplitude of assembly of microtubule protein at low GTP concentration is shown by measurement of microtubule length distributions to be due to the suppression of microtubule nucleation. This inhibitory effect is enhanced by GDP added before assembly, but can be overcome by a number of molecules such as pyrophosphate or ADP. The selective inhibition of nucleation by GDP in vitro, which occurs in addition to inhibition of elongation, could provide a mechanism for the control of spontaneous microtubule nucleation in vivo.     

Microtubules and nucleoside diphosphate kinase. Comparison of kinetics of GTP- and CTP-induced assembly.

The kinetics of assembly of MAP2-tubulin microtubule protein were examined as a function of the GTP concentration in order to test the hypothesis that CTP-induced assembly results from the generation of GTP by nucleoside diphosphate kinase. These studies show that (a) there is no assembly below a minimum GTP concentration and that this represents a nucleation requirement, (b) the rate of elongation is inconsistent with a single assembly-species, and (c) the elongation rate increases markedly as the GTP concentration is raised, although GTP is not absolutely required for elongation. These assembly kinetics have been compared with those with increasing CTP concentrations, by using microtubule protein containing a very low nucleoside diphosphate kinase activity of known substrate specificity. Neither nucleation nor the observed rates of elongation can be attributed to the formation of GTP, either (a) in terms of the generation of free GTP and subsequent binding to tubulin or (b) by the direct charging of GDP bound to the tubulin exchangeable site. The results show that nucleoside diphosphate kinase is not required for CTP-induced microtubule assembly, and suggest that CTP directly effects microtubule assembly.     

Assembly of microtubules with ATP: evidence that only a fraction of the protein is assembly-competent

Chick brain microtubule protein can be assembled in vitro with ATP, although the extent of assembly is less than that with GTP. The ATP-induced assembly is not the result of generation of GTP by the co-purifying nucleoside diphosphate kinase. Neither an observed increase in the critical concentration nor the phosphorylation of MAP2 can account for the decreased extent of assembly. However, whereas microtubules are formed with both ATP and GTP, incubation with ATP yields additional filaments and polymorphic aggregates. The results demonstrate that of the total protein which can be assembled into microtubules by GTP, about 25-35% is assembled into other structural forms in the presence of ATP.     

Taxol assembles tubulin in the absence of exogenous guanosine 5'-triphosphate or microtubule-associated proteins

Taxol increases the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells [Schiff, P. B., Fant, J., & Horwitz, S. B. (1979) Nature (London) 277, 665-667; Schiff, P. B., & Horwitz, S. B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1561-1565]. We report herein that taxol has the ability to promote microtubule assembly in the absence of microtubule-associated proteins, rings, and added guanosine 5'-triphosphate (GTP or organic buffer. The drug enhances additional microtubule assembly when added to microtubules at apparent steady state. This additional assembly can be attributed to both elongation of existing microtubules and spontaneous nucleation of new microtubules. Taxol-treated microtubules have depressed dissociation reactions as determined by dilution experiments. The drug does not inhibit the binding of GTP or the hydrolysis of GTP or guanosine 5'-diphosphate (GDP) in our microtubule protein preparations. Taxol does not competitively inhibit the binding of colchicine to tubulin.     

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.     

The Balance Between τ Protein's Microtubule Growth and Nucleation Activities: Implications for the Formation of Axonal Microtubules

Abstract: The microtubule-associated protein τ is found primarily in neuronal tissues and is highly enriched in the axon. It promotes microtubule assembly in vitro and stabilizes microtubules in cells. To study how τ protein might be involved in the unique features of axonal microtubules, we have analyzed the effect of E. coli -synthesized τ protein using an in vitro centrosome-mediated microtubule regrowth assay over a wide range of τ/tubulin ratios. We report that microtubule assembly promoted by τ protein exhibits characteristic changes dependent on the τ/tubulin ratio. Above a threshold level, nucleation of new microtubules is favored over growth of existing ones, τ isoform variation does not change this phase transition in microtubule assembly. We discuss how τ might participate in the elaboration of axonal morphology based on our results and present evidence that the phase transition from microtubule growth to nucleation is critical for axonal development.     

An oscillatory mode for microtubule assembly.

Depending upon the conditions under which polymerization takes place, pure tubulin can assemble into microtubules following either the usual monotonic kinetics or a more complex oscillatory mechanism. When present, these oscillations involve large cyclic changes in the extent of polymer formed before a steady-state is reached. Analysis of the microtubules formed at different times shows that these oscillations involve marked redistribution in both the length and number of microtubules. No significant difference is found between two populations of microtubules corresponding to the same level of assembly, one for which the extent of polymerization will remain stable with time and one for which it will decrease by as much as 90% in the next oscillation. The amplitude of these oscillations is sensitive to changes in the concentrations of protein, nucleotide (GTP, GDP or GMPpNp), magnesium ion or GTP regenerating system. A complete shift from an oscillatory to a monotonic polymerization can be induced by a minor increase in the concentration of free nucleotide, GTP or GDP.     

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.     

Dynamics of the microtubule oscillator: role of nucleotides and tubulin-MAP interactions.

Microtubules can be induced to perform synchronous and periodic cycles of assembly and disassembly at constant temperature. The process depends on GTP hydrolysis. Time-resolved X-ray scattering using synchrotron radiation shows a cyclic interconversion of tubulin subunits, microtubules and oligomers (= short protofilament fragments). Oscillations are correlated with conditions that stabilize polymers and destabilize oligomers, and others of opposite effect. Microtubule stabilizers include GTP, Mg2+ or microtubule-associated proteins (MAPs), destabilizers include GDP or elevated ionic strength. K+ at intracellular concentrations noticeably increases the stability of tubulin-MAP oligomers, in contrast to Na+. ATP and the non-hydrolyzable analogue AMP-PNP enhance oscillations by mechanisms that are not directly linked to the role of nucleotide hydrolysis in assembly. We propose a mechanism of oscillations that include oligomers as microtubule disassembly products which transiently lock the protein in an unpolymerizable state; this may point to a role of oligomers in controlling microtubule assembly cycles in cells.     

Monotonic versus oscillating microtubule assembly: a cryo-electron microscope study

Depending on the free GTP concentration, microtubules can assemble following either a monotonic or an oscillatory mode. We have used cryoelectron microscopy to compare the tubulin assemblies characteristic of each polymerization pathway. We focus on the first assembly peak. At this particular time point, despite their strikingly different subsequent evolution, both systems are similar with regard to the extent of tubulin polymerization and to the microtubule length distribution. The present study shows that whilst the observed microtubule structures are the same in both systems, the oscillatory system shows quantities of closed ring-like tubulin oligomers, far in excess of those observed in the monotonic system. Furthermore, the conversion of the oscillating system to a monotonic one by GTP addition during the first oscillation is accompanied by a marked decrease in the number of rings. Based on these results we propose that the GTP dependent step which governs microtubule oscillations is the opening of inactive tubulin oligomers.     

Effect of cibacron blue on tubulin assembly in the absence and presence of microtubule-associated proteins

Cibacron blue was found to inhibit assembly and increase the critical concentration of microtubule proteins. In the presence of 4 mol Cibacron blue/mol tubulin, assembly was completely inhibited and pre-formed microtubules disassembled. Addition of 8% (v/v) dimethylsulfoxide to Cibacron blue-inhibited samples induced assembly of normal microtubules in addition to sheets of protofilaments. Disassembly was induced upon addition of 1 mM colchicine or 2mM Ca²⁺. No obvious difference was seen in the protein composition of these microtubules compared with controls. GTP exchange was not affected by the presence of Cibacron blue nor was GTP able to counteract its effect. This indicates that the exchangeable GTP site is not involved. The extent of assembly of phosphocellulose purified tubulin in the presence of 8% (v/v) dimethylsulfoxide was only slightly less in the presence of Cibacron blue, although the assembly rate was decreased. These results suggest that Cibacron blue might alter the binding of one or more of the associated proteins stimulating assembly.     

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)     

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.     

Tubulin oligomers and microtubule oscillations. Antagonistic role of microtubule stabilizers and destabilizers

Several types of non-equilibrium phenomena have been observed in microtubule polymerization, including dynamic instability, assembly overshoot and oscillations. They can be interpreted in terms of interactions between tubulin subunits (= alpha, beta heterodimers), microtubules, and a third state, oligomers, which represent intermediates between microtubule disassembly and the regeneration of assembly-competent subunits by GTP. Here we examine the role of oligomers by varying conditions that stabilize or destabilize microtubules and/or oligomers. By varying their ratio one can drive tubulin assembly either into steady-state microtubules or oligomers. These regimens of assembly conditions are separated by a region where microtubules oscillate. The oscillations can be simulated by computer modelling, based on a reaction scheme involving the three states of tubulin and nucleotide exchange on tubulin subunits, but not on microtubules or oligomers.     

Microtubule assembly in rat brain extracts. Further characterization of the effects of zinc on assembly and cold stability

Microtubule assembly has been studied turbidometrically in supernatant fluids prepared from rat brain by high-speed centrifugation. It was confirmed that assembly occurred in the absence of added GTP. Zinc ions (500 microM, but in the presence of 1 mM EGTA) stimulated assembly under these conditions. Zinc-stimulated assembly produced microtubules with normal characteristics, as judged by electron microscopy, SDS-polyacrylamide gel electrophoresis and inhibition of assembly by fructose-6-phosphate or colchicine. However, microtubules formed in the presence of such zinc concentrations were more stable to cold than controls, although the rate constant for the disassembly reaction was unchanged. Neither the stimulation of assembly by zinc nor the effect on cold stability was affected by trifluoperazine suggesting that a calmodulin-related mechanism is not involved. Microtubule "seeds" had little effect in the presence of zinc, suggesting that it may be acting on the nucleation phase of the assembly reaction. This was supported by the findings that zinc reduced the critical concentration of brain supernatant necessary for assembly and that zinc did not affect the rate constant for assembly. The results suggest zinc can in some way stabilize microtubules; possible mechanisms are discussed.     

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.     

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.     

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.     

Tubulin-nucleotide interactions. Effects of removal of exchangeable guanine nucleotide on protein conformation and microtubule assembly.

The removal of tightly bound GDP from the exchangeable nucleotide-binding site of tubulin has been performed with alkaline phosphatase under conditions which essentially retain the assembly properties of the protein. When microtubule protein is treated with alkaline phosphatase, nucleotide is selectively removed from tubulin dimer rather than from MAP (microtubule-associated protein)-containing oligomeric species. Tubulin devoid of E-site (the exchangeable nucleotide-binding site of the tubulin dimer) nucleotide shows enhanced proteolytic susceptibility of the beta-subunit to thermolysin and decreased protein stability, consistent with nucleotide removal causing changes in protein tertiary structure. Pyrophosphate ion (3 mM) is able to promote formation of normal microtubules in the complete absence of GTP by incubation at 37 degrees C either with nucleotide-depleted microtubule protein or with nucleotide-depleted tubulin dimer to which MAPs have been added. The resulting microtubules contain up to 80% of tubulin lacking E-site nucleotide. In addition to its effects on nucleation, pyrophosphate competes weakly with GDP bound at the E-site. It is deduced that binding of pyrophosphate at a vacant E-site can promote microtubule assembly. The minimum structural requirement for ligands to induce tubulin assembly apparently involves charge neutralization at the E-site by bidentate ligation, which stabilizes protein domains in a favourable orientation for promoting the supramolecular protein-protein interactions involved in microtubule formation.