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.     

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.     

Dynamic instability of microtubules: Monte Carlo simulation and application to different types of microtubule lattice.

Dynamic instability is the term used to describe the transition of an individual microtubule, apparently at random, between extended periods of slow growth and brief periods of rapid shortening. The typical sawtooth growth and shortening transition behavior has been successfully simulated numerically for the 13-protofilament microtubule A-lattice by a lateral cap model (Bayley, P. M., M. J. Schilstra, and S. R. Martin. 1990. J. Cell Sci. 95:33-48). This kinetic model is now extended systematically to other related lattice geometries, namely the 13-protofilament B-lattice and the 14-protofilament A-lattice, which contain structural "seams". The treatment requires the assignment of the free energies of specific protein-protein interactions in terms of the basic microtubule lattice. It is seen that dynamic instability is not restricted to the helically symmetric 13-protofilament A-lattice but is potentially a feature of all A- and B-lattices, irrespective of protofilament number. The advantages of this general energetic approach are that it allows a consistent treatment to be made for both ends of any microtubule lattice. Important features are the predominance of longitudinal interactions between tubulin molecules within the same protofilament and the implication of a relatively favorable interaction of tubulin-GDP with the growing microtubule end. For the three lattices specifically considered, the treatment predicts the dependence of the transition behavior upon tubulin concentration as a cooperative process, in good agreement with recent experimental observations. The model rationalizes the dynamic properties in terms of a metastable microtubule lattice of tubulin-GDP, stabilized by the kinetic process of tubulin-GTP addition. It provides a quantitative basis for the consideration of in vitro microtubule behaviour under both steady-state and non-steady-state conditions, for comparison with experimental data on the dilution-induced disassembly of microtubules. Similarly, the effects of small tubulin-binding molecules such as GDP and nonhydrolyzable GTP analogues are readily treated. An extension of the model allows a detailed quantitative examination of possible modes of substoichiometric action of a number of antimitotic drugs relevant to cancer chemotherapy.     

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.     

Guanosine-5′-triphosphate hydrolysis and tubulin polymerization

Summary GTP hydrolysis associated with polymerization is a distinctive feature of microtubule assembly. This reaction may be fundamentally linked to the dynamic properties of microtubules in vivo. Kinetic analysis of the connection between microtubule assembly and associated GTP hydrolysis indicates that these two events are kinetically uncoupled, GTP hydrolysis occurring after tubulin incorporation in the microtubule. As a consequence, the combination of the diffusionnal incorporation of GTP in microtubules at steady-state and of subsequent GTP hydrolysis results in the formation of a steady-state GTP cap at microtubule ends. The interplay between GTP and GDP at microtubule ends is examined. Inhibition by GDP of steady-state GTP hydrolysis at microtubule ends and of microtubule elongation is understood within a tight reversible binding of GDP at microtubule ends generating inactive elongation sites. Nucleotides are freely exchangeable at microtubule ends. This result indicates that the nature of the nucleotide present at microtubule ends must be considered in a model for microtubule assembly.These data are pooled in order to define the general features of a model describing microtubule assembly and treadmilling in terms somewhat different from previously proposed models.     

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.     

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.     

Microtubule dynamics.

Although compelling evidence has been obtained for heterogeneity in the structure of subunits in microtubules, it has not been possible to prove that this results from the presence of tubulin-GDP and tubulin-GTP in polymers. There are reasons to exclude the existence of even a monolayer of tubulin-GTP subunits at microtubule ends. Dynamic behavior appears to be best accounted for by a mechanism in which tubulin-GDP in microtubules exists in two conformations. The mechanism of microtubule-associated protein binding to microtubules and the role of phosphorylation on this reaction are discussed.     

Temperature-jump studies of microtubule dynamic instability

Evidence for a slowly dissociating tubulin-GTP cap at microtubule ends was derived from observation of a delay for attaining a maximum disassembly rate, after the temperature of steady state microtubules was rapidly decreased from 36 to 34 degrees C. The possibility that the microtubules were capped by a single tubulin-GTP subunit on each subhelix was ruled out, by comparison of the disassembly kinetics following a temperature decrease and dilution. The existence of a subpopulation of microtubules that underwent irreversible or near irreversible disassembly was demonstrated by a 30-s lag for attainment of a maximum assembly rate, after steady state microtubules were shifted from 34 to 36 degrees C. A dynamic instability model predicts that a maximum assembly rate will be delayed until disappearance of a subpopulation of microtubules that disassemble before being recapped. Analysis indicates that the 30-s lag resulted because approximately 2% of the mass in the steady state microtubule population was uncapped and disassembling and not readily recapped. The half-time for recapping of disassembling microtubules, by addition of tubulin-GTP subunits to ends, was equal to or greater than 20 s. Since tubulin-GDP dissociated from microtubules at a rate of about 4500 s-1, slow recapping resulted in dramatic shortening of disassembling microtubules.     

Stabilization of microtubules by tubulin-GDP-Pi subunits

Microtubule dynamic instability has been accounted for by assuming that tubulin subunits at microtubule ends differ from the tubulin-GDP subunits that constitute the bulk of the microtubule. It has been suggested that this heterogeneity results because ends contain tubulin subunits that have not yet hydrolyzed an associated GTP molecule. Alternatively, in a recent model it was proposed that ends contain tubulin-GDP-Pi subunits from which Pi has not yet dissociated. The models differ in their predicted response to added ligands: because GDP in subunits in microtubules does not exchange with nucleotide in solution, the heterogeneity from a tubulin-GTP cap will not be eliminated by added GTP; however, the dissociability of Pi in tubulin-GDP-Pi subunits will allow a heterogeneity resulting from a tubulin-GDP-Pi cap to be eliminated by added excess Pi. Elimination of the heterogeneity is expected to be manifested by an elimination of dynamic instability behavior. Using video microscopy to study the kinetic behavior of individual microtubules under reaction conditions where dynamic instability is the dominant mechanism for microtubule length changes, we have determined the effects of 0.167 M Pi on the rate of subunit addition in the elongation phase, the rate of subunit dissociation in the rapid shortening phase, and the rates of the phase transitions from elongation to rapid shortening and from rapid shortening to growing. Since 0.167 M Pi did not decrease the subunit dissociation rate in the rapid shortening phase or the rate of the phase transition from growing to rapid shortening, our results provide no support for the hypothesis that tubulin-GDP-Pi subunits are responsible for dynamic instability behavior of microtubules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of DAPI in microtubule reactions at steady-state

The new fluorophor for tubulin, DAPI, is shown to bind to a site different from the exchangeable nucleotide binding site (E site) and to inhibit GTP hydrolysis by the tubulin-colchicine complex within an uncompetitive scheme. Moreover the dissociation rate constant of tubulin for microtubule ends at 32 degrees C was found largely decreased in the presence of saturating amounts of the probe while the association rate constant was little affected. These data on the kinetic parameters of tubulin interactions in the presence of DAPI, together with the inhibition of GTP hydrolysis by microtubules at the steady state are understood as the main cause for microtubule stabilization at steady-state by DAPI.     

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.     

Force generation by cytoskeletal filament end-tracking proteins

Force generation in several types of cell motility is driven by rapidly elongating cytoskeletal filaments that are persistently tethered at their polymerizing ends to propelled objects. These properties are not easily explained by force-generation models that require free (i.e., untethered) filament ends to fluctuate away from the surface for addition of new monomers. In contrast, filament end-tracking proteins that processively advance on filament ends can facilitate rapid elongation and substantial force generation by persistently tethered filaments. Such processive end-tracking proteins, termed here filament end-tracking motors, maintain possession of filament ends and, like other biomolecular motors, advance by means of 5'-nucleoside triphosphate (NTP) hydrolysis-driven affinity-modulated interactions. On-filament NTP hydrolysis/phosphate release yields substantially more energy than that required for driving steady-state assembly/disassembly of free filament ends (i.e., filament treadmilling), as revealed by an energy inventory on the treadmilling cycle. The kinetic and thermodynamic properties of two simple end-tracking mechanisms (an end-tracking stepping motor and a direct-transfer end-tracking motor) are analyzed to illustrate the advantages of an end-tracking motor over free filament-end elongation, and over passive end-trackers that operate without the benefit of NTP hydrolysis, in terms of generating force, facilitating rapid monomer addition, and maintaining tight possession of the filament ends. We describe an additional cofactor-assisted end-tracking motor to account for suggested roles of cofactors in the affinity-modulated interactions, such as profilin in actin-filament end-tracking motors and EB1 in microtubule end-tracking motors.     

Mechanism of GTP hydrolysis at microtubule ends

The kinetics of tubulin subunits incorporation into microtubules and the kinetics of inorganic phosphate release have been measured in parallel. Correlation of the two measurements indicates that the tubulin GTPase activity is due to GTP hydrolysis and exchange at the end of the microtubules. In some cases where the free GTP available in the medium is in-sufficient the rate of GTP hydrolysis is limited by the rate of tubulin-GTP association at the end of the microtubules. The affinity constant of GTP for the microtubule end appears to be 100 times lower than the affinity constant of the tubulin-GTP complex.     

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.     

End-stabilized microtubules observed in vitro: stability, subunit, interchange, and breakage.

We report a reliable method to prepare, in vitro, microtubules that are stabilized at both ends by axonemal structures, and report studies of their properties. Such "end-stabilized" microtubules neither grow nor shorten over times of several hours when tubulin subunits are present in the surrounding solution. When subunits are removed, the microtubules eventually break. Breakage occurs within a sinuous and flexible region, a few microns in length, that begins at a single point on the microtubule and grows. When breakage does occur, the resulting two free ends shorten very rapidly until the flexible part has depolymerized and the region of straight microtubule is reached. The remainder of the microtubule then shortens at rates comparable to those ordinarily observed in dynamic instability. Formation of the flexible region can be reversed if subunits are added to the buffer prior to breakage. End-stabilized microtubules are a useful tool for studying interactions of molecules with the microtubular wall. They may be a good model for interpreting stabilizing events that happen in the cell. A preliminary study of the effects of microtubule poisons on the wall is presented.     

GDP-Tubulin Incorporation into Growing Microtubules Modulates Polymer Stability

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

Towards an understanding of microtubule function and cell organization: an overview.

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.     

The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends

During cell division the replicated chromosomes are segregated precisely towards the spindle poles. Although many cellular processes involving motility require ATP-fuelled force generation by motor proteins, most models of the chromosome movement invoke the release of energy stored at strained (owing to GTP hydrolysis) plus ends of microtubules. This energy is converted into chromosome movement through passive couplers, whereas the role of molecular motors is limited to the regulation of microtubule dynamics. Here we report, that the microtubule-depolymerizing activity of MCAK (mitotic centromere-associated kinesin), the founding member of the kinesin-13 family, is accompanied by the generation of significant tension-remarkably, at both microtubule ends. An MCAK-decorated bead strongly attaches to the microtubule side, but readily slides along it in either direction under weak external loads and tightly captures and disassembles both microtubule ends. We show that the depolymerization force increases with the number of interacting MCAK molecules and is ～1?pN per motor. These results provide a simple model for the generation of driving force and the regulation of chromosome segregation by the activity of MCAK at both kinetochores and spindle poles through a 'side-sliding, end-catching' mechanism.     

EBs recognize a nucleotide-dependent structural cap at growing microtubule ends

Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions?of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.