Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study.

Abstract

This paper reports an experimental study, of the interference of GTP hydrolysis in the mechanism of microtubule assembly, following the model and theory previously published [Hill, T. L. & Carlier, M.-F. (1983) Proc. Natl. Acad. Sci. USA 80,[7234][7235][7236][7237][7238]. Results from dilution experiments show that microtubules depolymerize faster below the critical concentration than expected with a reversible polymerization model. The experimental plot of flux versus tubulin concentration exhibits a slope discontinuity at the critical copcentration, in agreement with the theory. Theoretical points calculated by the Monte Carlo method can be fitted qualitatively to the data. A consequence of this peculiar dynamic behavior of microtubules is that the ratio of tubulin dissociation and association rate constants measured, respectively, below and above the critical concentration does not yield the true value of the critical concentration. It is emphasized that the presence of GTP at microtubule ends is necessary to maintain the stability of the polymer.It is striking that two essential polymer components of the cytoskeleton of eukaryotic cells, actin filaments and microtubules (MTs), share many structural and physicochemical properties. In particular, the NTP bound to the protomer (ATP to actin, GTP to tubulin) undergoes hydrolysis during polymerization (1-3). NTP hydrolysis is also associated with maintenance of the polymers. Indeed, in the presence of low concentrations of GTP, MTs eventually depolymerize (4) and GDP is unable to promote polymerization (2). Therefore, actin filaments and MTs must be considered as steadystate polymers. However, GTP hydrolysis associated with MT assembly does not appear necessary for polymerization because hydrolysis is a monomolecular kinetic process uncoupled from polymerization (5), a result also more recently reported on actin (6, 7). Correlatively, MTs can be obtained and stabilized in the presence of nonhydrolyzable analogs of GTP (8) and thus be maintained in an equilibrium state.Although GTP hydrolysis is involved in MT assembly and steady state, until now Oosawa's model, which applies to equilibrium polymers-i.e., those undergoing linear reversible polymerization-satisfactorily accounted for the kinetic and thermodynamic data for MT assembly (4, 9).In an effort to explore the consequences of nucleotide hydrolysis in the polymerization process, Wegner first developed a model (10) pointing to the possibility of "head-to-tail polymerization" driven by ATP hydrolysis in actin polymerization. The same phenomenon was observed experimentally on MTs and called "treadmilling" by Margolis and Wilson (11).Possible implications of treadmilling in the regulation of MTs in vivo have been suggested (12)(13)(14)(15). On the basis of Wegner's model, the thermokinetic theory of MT and actin polymerization has been developed (16). In this model, GTP hydrolysis is tightly coupled to the polymerization process. As a consequence, only GDP is bound to the polymer and the critical concen...

Regulation of tubulin levels and microtubule assembly in Saccharomyces cerevisiae: consequences of altered tubulin gene copy number.

“…This property, taken together with the placement of nucleating centers, can explain not only quantitative but also qualitative elements of microtubule organization. The notion that total tubulin levels are important to this mechanism is supported by in vitro experiments (5), by model building (20), and by demonstrations that cells appear to have mechanisms for regulating tubulin levels in response to changes in microtubule assembly (2,7). Other factors are likely to be involved as well.…”

Internal Dynamics of Dynactin CAP-Gly Is Regulated by Microtubules and Plus End Tracking Protein EB1

“…10 suggest that packing alone is not sufficient to explain higher mobility of CAP-Gly assembled on microtubules because increased nano-to microsecond dynamics is found throughout the entire sequence rather than being limited to surface residues. We also note that the binding affinities of CAP-Gly to EB1 and microtubules are drastically different (high nanomolar versus mid-micromolar (12,19), and unpublished data), 3 and it will be instructive in the future to examine the possible connection between the thermodynamic binding parameters and the internal dynamics of CAP-Gly bound to EB1, assembled on MTs, and of CAP-Gly/EB1 complex associated on MTs. Such studies were reported in other systems (34 -36), where relationships 3 S. Ahmed and J. C. Williams, unpublished data.…”

“…heterodimers and are highly dynamic structures, undergoing continuous cycles of polymerization/depolymerization driven by the hydrolysis of GTP (2,3). Intracellular transport along the microtubules is accomplished by microtubule-associated motor proteins from dynein and kinesin families (4).…”

Asymmetric Behavior of Severed Microtubule Ends After Ultraviolet–Microbeam Irradiation of Individual Microtubules In Vitro

“…Rescue is thought to occur when a rapidly shortening end becomes recapped with GTP-tubulin, a process which is infrequent in comparison to the rate of GDP-tubulin dissociation. Although the mechanism and location of GTP hydrolysis within a microtubule is controversial and unresolved (2,4,5,7,8,12,34,36,40,45), there is substantial support for the GTP cap hypothesis: (a) the bulk of polymer is GDP-tubulin (17,29,30,36,46,47); (b) elongation and rapid shortening are distinctly different phases (6,21,34,45); (c) GDP-tubulin subunits do not support elongation in buffers which permit dynamic instability (3); (d) rapid shortening occurs within seconds at both ends when GTP-tubulin association is prevented by dilution (Voter, W. A, and H. P. Erickson, manuscript in preparation; Walker, R. A., and E. D. Salmon, unpublished observations); (e) addition of a GTPase system to microtubules at steady-state results in polymer disassembly (3); (f) for both ends, dissociation during the rapid shortening phase typicaUy occurs at a constant rate as expected for a homogeneous core of GDP-tubulin subunits (21,45); (g) for both ends, there is a substantial dissociation rate during the elongation phase without any apparent phase transition (45); and (h) the critical concentration for elongation is similar at the two ends, suggesting that there is reversible dissociation (of GTPtubulin subunits) at both ends (45).…”