Tubulin oligomers and microtubule oscillations

Abstract

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 ( = a, j 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 regimes 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.The assembly of microtubules or other biopolymers is commonly thought to involve two interacting species, the polymer and the monomer. This suffices to explain the pseudo-first-order assembly kinetics that is frequently observed (reviewed in [l]). In the case of microtubules a third assembly state can be found, the oligomer. Its role in microtubule assembly has remained elusive. Initial electron microscope evidence suggested a role as nucleating centers (reviewed in [2]). Later X-ray studies called this view into question [3], leaving open the possibility that oligomers contributed to microtubule elongation [4]. The observation by cryo-electron microscopy of oligomers as primary breakdown products [5] suggested yet another function, namely that of a temporary storage form of nonpolymeric tubulin generated by disassembling microtubules. Whatever the role, it was clear that it must somehow be related to the assembly competence of tubulin which is activated by GTP but inactivated by GDP produced during microtubule assembly. Only the tubulin subunits are capable of quickly exchanging bound GDP by GTP [6], thus rendering them active, whereas GDP bound to microtubules is non-exchangeable.When studying microtubule assembly using synchroton X-ray scattering, we made two observations bearing on the role of oligomers. One was that the assembly of oligomers and microtubules had different physico-chemical properties but both aggregates drew on the pool of tubulin subunits [7, 81. The second was that in certain conditions the assembly kinetics deviated from the simple exponential approach to equilibrium. This could take the form of a short-lived assembly overshoot and decay, or of assembly overshoot, decay In a previous report [I 01 we have studied the dependence of microtubule oscillations in solution on various nucleotides and MAPs and we have proposed a reaction model which includes the disassembly of microtubules into oligomers that act as transient storage aggregates of nonpolymerizable tubulin. In the present study, we describe the structural transitions as seen by X-ray scattering and re...

Modeling oscillatory microtubule polymerization

“…Then microtubules spontaneously nucleate and polymerize to long rigid polymers made up of α − β tubulin dimers. An increasing number of long microtubules in a solvent causes an increasing turbidity and the amount of polymerized tubulin-t may be monitored by measuring this turbidity [6] or by X-ray scattering [8]. The nucleation of microtubules is a rather complex process and it is still a matter of debate whether the nucleation rate depends in experiments only on the initial concentration of tubulin-t, c t , or during the polymerization on the temporally varying c t [3,30].…”

“…In the latter case oscillations occur only as a transient, because GTP is either consumed or some reactions steps may be inhibited due to the accumulation of GDP [8]. If both possibilities are combined, the length of a transient regime depends on the initial concentrations of GTP and GDP and on the capacity to regenerate GTP.…”

“…Microtubule assembly and disassembly proceeds in several steps [1][2][3]5,7,8]. Aggregation of guanosine triphosphate (GTP) liganded tubulin dimers, the so-called tubulin-t, to microtubules is started by heating up tubulin solutions to a temperature of about 30 − 37 o C in the presence of GTP.…”

“…However, the reaction steps, such as nucleation, growth and decay of microtubules or the rate limiting factors of oligomer decay or tubulin regeneration, which have been identified to be crucial for oscillations [5][6][7][8]11], are taken into account. Moreover we address the question whether microtubule oscillations occur transiently or in a persistent manner beyond a Hopf bifurcation.…”

“…[2,3] is attracting considerable attention, both experimentally and theoretically [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18]. Two phenomena in this area, the dynamical instability of microtubules [4] and the oscillatory polymerization [5][6][7][8][9][10][11][12] challenge theoretical modeling already for a while [13][14][15][16][17][18].…”