Regulation of microtubule dynamic instability by tubulin-GDP

The regulation of the spontaneous transitions between growth and shortening of microtubules is central to the biological function of dynamic instability. Here we examine the effects of controlled amounts of tubulin-GDP (Tu-GDP) on the dynamic properties of microtubules in vitro. The transphosphorylation equilibrium between GTP, GDP, UTP, and UDP in the presence of nucleoside-5'-diphosphate kinase (NDPK) was used to fix the ratio chi D = [Tu-GDP]/([Tu-GTP]) + [Tu-GDP]) in solution. Lower levels of Tu-GDP (chi D < 0.6) produce only a small increase in the apparent critical concentration, Cc'. However, at chi D > 0.6 a dramatic increase in Cc is observed. At steady state of assembly, low levels of Tu-GDP (chi D < 0.5) cause a significant reduction of the dynamic length redistribution of the microtubule population. The principal observable effect of Tu-GDP on the empirical parameters of microtubule dynamic instability is to decrease the duration of individual phases of microtubule growth and shortening, with relatively little effect on the intrinsic rates of growth and shortening. Observations in dark-field video microscopy reveal that the irregularities in the rates of growth (and shortening) are increased in the presence of Tu-GDP. At elevated levels of Tu-GDP, pauses occur frequently during the growth phase, microtubule dynamics cease to conform to a clear two-phase process, and the extents of growth and shortening excursions are strongly attenuated. The experimental results are well reproduced by computer simulation, using mechanisms defined in the lateral cap model for dynamic instability [Martin, S. R., Schilstra, M. J., & Bayley, P. M. (1993) Biophys. J. 65, 578-596], which includes the binding of Tu-GDP to the microtubule end in competition with Tu-GTP. In the presence of Tu-GDP, the growing-state lifetime is significantly attenuated, and the microtubule length versus time excursions simulated by the model show irregularities and complex multistate behavior, including pauses, as observed experimentally. These results suggest that Tu-GDP can modulate microtubule dynamics significantly under conditions where little bulk microtubule disassembly is induced. Tu-GDP therefore appears to exemplify the action of a relatively simple factor with the potential capability for regulation of microtubule dynamics in a cellular environment.