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.     

Kinetics and mechanism of microtubule length changes by dynamic instability

Microtubules at steady state were found to undergo dramatic changes in length, with only very little change in number concentration and mean length. This result is accounted for by a mechanism in which microtubules are capped at ends by tubulin-GTP subunits; loss of the tubulin-GTP cap at one end results in disassembly of all the tubulin-GDP subunits, so that the medial edge of the distal tubulin-GTP cap is exposed; the exposed tubulin-GTP cap is sufficiently stable, so that microtubule regrowth from the cap rather than loss of the cap occurs. This mechanism predicts that a bell-shaped length distribution of sheared microtubules will be transiently bimodal, with peaks of short and moderate length microtubules, in rearranging to an exponential length distribution. We have observed the predicted transient bimodal length distribution experimentally and in a Monte Carlo simulation. Dynamic instability has recently been accounted for by assuming that microtubule ends are capped with only a single tubulin-GTP subunit at each end of the five helices that serve as elongation sites. Such a minimal tubulin-GTP cap is apparently ruled out by our observations, which require that the remnant tubulin-GTP cap generated from disassembly be able to serve as nucleating site; we do not expect that a stable nucleating site can be generated from five tubulin-GTP subunits, oriented as the five helices that serve as elongation sites.     

Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling

The length dynamics both of microtubule-associated protein (MAP)-rich and MAP-depleted bovine brain microtubules were examined at polymer mass steady state. In both preparations, the microtubules exhibited length redistributions shortly after polymer mass steady state was attained. With time, however, both populations relaxed to a state in which no further changes in length distributions could be detected. Shearing the microtubules or diluting the microtubule suspensions transiently increased the extent to which microtubule length redistributions occurred, but again the microtubules relaxed to a state in which changes in the polymer length distributions were not detected. Under steady-state conditions of constant polymer mass and stable microtubule length distribution, both MAP-rich and MAP-depleted microtubules exhibited behavior consistent with treadmilling. MAPs strongly suppressed the magnitude of length redistributions and the steady-state treadmilling rates. These data indicate that the inherent tendency of microtubules in vitro is to relax to a steady state in which net changes in the microtubule length distributions are zero. If the basis of the observed length redistributions is the spontaneous loss and regain of GTP-tubulin ("GTP caps") at microtubule ends, then in order to account for stable length distributions the microtubule ends must reside in the capped state far longer than in the uncapped state, and uncapped microtubule ends must be rapidly recapped. The data suggest that microtubules in cells may have an inherent tendency to remain in the polymerized state, and that microtubule disassembly must be induced actively.     

Regeneration of mirror symmetrical limbs in the axolotl

Measurements of tubulin exchange into and from bovine brain microtubules at steady state in vitro were made with 3H-GTP as a marker for tubulin addition to or loss from microtubules. Tubulin has an exchangeable GTP binding site that becomes nonexchangeable in the microtubule. We found that tubulin addition to and loss from microtubules under steady state conditions occurred at equivalent rates, that loss and gain were linear, and that exchange rates (percentage of total tubulin in microtubules lost or gained per hour) were dependent upon microtubule length. Furthermore, we found that podophyllotoxin blocked steady state assembly, but did not alter the rate of steady state tubulin loss. When the assembling microtubule end was pulsed with 3H-GTP at steady state, the label was almost completely retained during a subsequent chase. We conclude that the microtubule assembly-disassembly "equilibrium" is a steady state summation of two different reactions which occur at opposite ends of the microtubule, and that assembly and disassembly occur predominantly and perhaps exclusively at the opposite ends under steady state conditions in vitro.     

Phosphate release during microtubule assembly: what stabilizes growing microtubules?

The molecular mechanism underlying microtubule dynamic instability depends on the relationship between the addition of tubulin-GTP to a growing microtubule and its hydrolysis in the microtubule lattice to tubulin-GDP, with release of inorganic phosphate (Pi). Since this relationship remains controversial, we have re-examined the release of Pi upon microtubule assembly using a fluorometric assay for Pi, based on the phosphate-binding protein of Escherichia coli [Brune M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994) Biochemistry 33, 8262-8271]. Microtubule assembly and Pi release were monitored simultaneously in a standard fluorimeter as an increase in the turbidity and fluorescence, respectively, in tubulin-GTP solutions assembled under conditions supporting dynamic instability. At the steady state of assembly, Pi release is nonlinear with respect to time, proceeding at a rate determined by the following: (a) the intrinsic GTPase activity of the nonpolymerized tubulin-GTP, and (b) the microtubule number concentration, which decreases progressively. Direct observation of the time course of nucleated microtubule assembly indicates that Pi release is closely coupled to microtubule elongation, even during the initial stages of assembly when uncoupling of tubulin-GTP addition and GTP hydrolysis would be most evident. Studies of the inhibition and reversal of the growth phase by cytostatic drugs show no evidence of a burst of Pi release. We conclude that nucleotide hydrolysis can keep pace with tubulin-GTP addition rates of 200 molecules per second per microtubule and that extended caps of tubulin-GTP or tubulin-GDP-Pi are not generated in normal assembly, nor are they required to stabilize growing microtubules or to support the phenomenon of dynamic instability of microtubules at the steady state.     

Mechanism for oscillatory assembly of microtubules

Dampened oscillations of microtubule assembly can accompany polymerization at high tubulin subunit concentrations. This presumably results from a synchronization of dynamic instability behavior, which generates a large population of rapidly disassembling microtubules, that liberate tubulin-GDP oligomers. Subunits in oligomers cannot assemble until they dissociate, to allow GDP-GTP exchange. To determine whether rapidly disassembling microtubules generate oligomers directly, we measured the rate of dilution-induced disassembly of tubulin-GDP microtubules and the rate of dissociation of GDP from the so-formed tubulin-GDP subunits. The rate of GDP dissociation from liberated subunits was found to correspond to that of tubulin-GDP subunits (t1/2 = 5 s), rather than tubulin-GDP oligomers. This indicates that tubulin-GDP subunits are released from microtubules undergoing rapid disassembly. Oligomers apparently form in a side reaction from the high concentration of tubulin-GDP subunits liberated from the synchronously disassembling microtubule population. The rate of subunit dissociation is 0.11 s-1 with oligomers formed by concentrating tubulin-GDP subunits and 0.045 s-1 with oligomers formed by cold-induced microtubule disassembly. This difference provides evidence that the conformation of tubulin-GDP subunits released from rapidly disassembling microtubules differs from tubulin-GDP subunits that were not recently in the microtubule lattice.     

Effects of GDP on microtubules at steady state

We have re-examined the effect of varying GDP concentrations on the kinetics of GTP-induced assembly of microtubules from microtubule protein, and on the elongation of pre-existing microtubules subjected to a temperature jump relaxation from 21.5 to 37 degrees C. The assembly kinetics follow a simple model for assembly which involves a fast equilibrium of tubulin-GTP and tubulin-GDP coupled to the elongation process due to tubulin-GTP. The initial rate of the relaxation process is found to be dependent upon the GTP/GDP ratio, in confirmation of the results of Engelborghs and Van Houtte (Biophys. Chem. 14 (1981) 195). As an alternative to the interpretation previously advanced by them, involving modification of the reactivity of microtubule ENDs by GDP, we show that this result is consistent with the above model with one reasonable modification, namely, that the ratio of the affinities of tubulin for GTP and GDP should vary with temperature. The analysis shows that a decrease in this ratio of approx. 2-fold at 37 degrees C accounts for the observed effects. We conclude that more complex mechanisms involving consideration of modification of the reactivity of microtubule ENDs by GDP are not required to explain these results. This finding has important implications for current models of GDP-induced microtubule disassembly.     

Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates

Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995- 8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.     

The effect of podophyllotoxin on microtubule dynamics

We have investigated the effects of podophyllotoxin on the dynamic properties of microtubules assembled from pure tubulin dimer. Excess podophyllotoxin causes the complete disassembly of microtubules, through formation of a tubulin-GTP-podophyllotoxin ternary complex with a dissociation rate constant of 160 s-1 at 37 degrees C, similar to that found upon extensive isothermal dilution in this buffer system. Addition of substoichiometric concentrations of podophyllotoxin causes partial disassembly of microtubules through production of an equivalent amount of the ternary complex. Microtubule length measurements and incorporation of [3H]GTP-tubulin dimer show that podophyllotoxin can suppress the dynamic instability of tubulin dimer microtubules and that it acts substoichiometrically in so doing. We interpret the action of substoichiometric podophyllotoxin on microtubule ends in terms of effects on interconversion of growing and shrinking microtubules in a dynamic system in which tubulin-GTP-podophyllotoxin is kinetically analogous to tubulin-GTP in addition and to tubulin-GDP in dissociation. The ability to suppress dynamic instability may be one way in which drugs such as podophyllotoxin, acting at relatively low concentrations, are able to arrest cell growth and development in a selective way, without necessarily affecting the integrity of the major part of the cytoskeletal microtubule network.     

Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations

We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant.     

Regulation of the microtubule steady state in vitro by ATP.

ATP increases microtubule steady state assembly and disassembly rates in vitro in a concentration-dependent manner. Bovine brain microtubules, composed of 75% tubulin and 25% high molecular weight microtubule-associated proteins (MAPs), were purified by three cycles of assembly and disassembly in the absence of ATP. When assembled to steady state, these microtubules add dimers at one end and lose them at the other in a unidirectional assembly-disassembly process. In the presence of 1.0 mM ATP the unidirectional flow of tubulin from one end of the microtubules to the other increases as much as 20 fold, as revealed by loss of 3H-GTP from uniformly labeled microtubules under GTP chase conditions and by the rate of disassembly following addition of 50 microM podophyllotoxin. UTP, CTP and 5' adenylylimidodiphosphate (AMP-PNP) cannot substitute for ATP in producing this effect. Furthermore, the increase in steady state flow rate persists afer ATP is removed. Thus microtubules assembled in ATP and centrifuged through sucrose cushions to separate them from nucleotides continue to exhibit increased rates in the next assembly cycle in the absence of ATP. It is possible that an ATP-dependent microtubule protein kinase is responsible for the observed increase in tubulin flow rate. A kinase activity associated with brain MAPs has been reported to be cAMP-dependent (Sloboda et al., 1975). We have found an adenylate cyclase activity associated with these microtubules. Whether the adenylate cyclase is a contaminant or due to a specific microtubules-associated protein, and whether its activity is functionally linked to the increased rate of assembly and disassembly in the presence of ATP, remain to be determined.     

Microtubule assembly and disassembly at alkaline pH

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.     

On the nonlinear relationship between the initial rates of dilution-induced microtubule disassembly and the initial free subunit concentration

We have examined the dilution-induced in vitro disassembly kinetics of bovine brain microtubules, initially at steady state, using a wider range of dilutions (2-100-fold) than previously employed. In contrast to earlier results, as well as to the simple nucleation-condensation model for microtubule formation, the initial rate of dimer loss from microtubule ends was not a linear function of the initial concentration of unpolymerized tubulin. Over a 2-20-fold dilution range, plots of the initial rate of dimer loss versus the initial unpolymerized tubulin concentration were approximately linear. However, at greater dilutions, rates of microtubule depolymerization increased nonlinearly. For example, between a 10-fold dilution and a 100-fold dilution, the initial rate of dimer loss for microtubule-associated protein-containing microtubules increased by 300%, rather than a maximum of 11% expected on the basis of a linear rate plot. The nonlinear response was observed for dimer loss from opposite microtubule ends separately and with microtubules containing and lacking associated proteins. Qualitatively similar results were obtained using a wide range of experimental protocols, from which we can reasonably exclude methodological artifact as a basis for the data. We can also reasonably exclude the dissociation of the high molecular weight microtubule-associated proteins 1 and 2 from the microtubules as an explanation for the nonlinearity of the rate plots. The nonlinearity of the rate plots indicates that kinetic constants obtained under nonsteady state conditions of extreme microtubule dilution may not describe the steady state condition accurately.     

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.     

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.     

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.     

Mechanochemical model of microtubule structure and self-assembly kinetics

Microtubule self-assembly is largely governed by the chemical kinetics and thermodynamics of tubulin-tubulin interactions. An important aspect of microtubule assembly is that hydrolysis of the beta-tubulin-associated GTP promotes protofilament curling. Protofilament curling presumably drives the transition from tip structures associated with growth (sheetlike projections and blunt ends) to those associated with shortening (rams' horns and frayed ends), and transitions between these structures have been proposed to be important for growth-shortening transitions. However, previous models for microtubule dynamic instability have not considered such structures or mechanics explicitly. Here we present a three-dimensional model that explicitly incorporates mechanical stress and strain within the microtubule lattice. First, we found that the model recapitulates three-dimensional tip structures and rates of assembly and disassembly for microtubules grown under standard conditions, and we propose that taxol may stabilize microtubule growth by reducing flexural rigidity. Second, in contrast to recent suggestions, it was determined that sheetlike tips are more likely to undergo catastrophe than blunt tips. Third, partial uncapping of the tubulin-GTP cap provides a possible mechanism for microtubule pause events. Finally, simulations of the binding and structural effects of XMAP215 produced the experimentally observed growth and shortening rates, and tip structure.     

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.     

Microtubule dynamic instability: some possible physical mechanisms and their implications.

Video microscopic observation of a population of microtubules at steady state of assembly shows individual microtubules which interconvert between phases of growing and shrinking. The average duration of either phase is strongly affected by the tubulin concentration. Close to the steady-state (or 'critical') concentration, the mean excursion lengths may be of cellular dimensions, suggesting that dynamic instability can function as a control mechanism for the spatial organization of microtubule arrays. Numerical modelling, based on a limited number of assumptions, illustrates the transition behaviour, and the polar nature of this instability. The basic concept is that tubulin-GTP adds to a terminal position of the microtubule lattice and causes hydrolysis of the tubulin-GTP at a previously terminal lattice position [1, 2]. The predictions of this model can be evaluated experimentally. Further, examination of the consequences of introducing into the lattice a molecule such as a tubulin-drug complex, with altered capacity for helical propagation, provides a quantitative model for substoichiometric inhibition of microtubule dynamics and growth. This principle could have a more general relevance to mechanisms of regulation of microtubules within the cytoskeleton.     

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.