Long-range, through-lattice coupling improves predictions of microtubule catastrophe

Abstract

Microtubules are cylindrical polymers of αβ-tubulin that play critical roles in fundamental processes such as chromosome segregation and vesicular transport. Microtubules display dynamic instability, switching stochastically between growth and rapid shrinking as a consequence of GTPase activity in the lattice. The molecular mechanisms behind microtubule catastrophe, the switch from growth to rapid shrinking, remain poorly defined. Indeed, two-state stochastic models that seek to describe microtubule dynamics purely in terms of the biochemical properties of GTP- and GDP-bound αβ-tubulin predict the concentration dependence of microtubule catastrophe incorrectly. Recent studies provide evidence for three distinct conformations of αβ-tubulin in the lattice that likely correspond to GTP, GDP.Pi, and GDP. The incommensurate lattices observed for these different conformations raise the possibility that in a mixed nucleotide state lattice, neighboring tubulin dimers might modulate each other’s conformations and hence each other’s biochemistry. We explored whether incorporating a GDP.Pi state or the likely effects of conformational accommodation can improve predictions of catastrophe. Adding a GDP.Pi intermediate did not improve the model. In contrast, adding neighbor-dependent modulation of tubulin biochemistry improved predictions of catastrophe. Because this conformational accommodation should propagate beyond nearest-neighbor contacts, our modeling suggests that long-range, through-lattice effects are important determinants of microtubule catastrophe.

Microtubule instability driven by longitudinal and lateral strain propagation

“…In contrast, 208 simultaneous compaction/expansion of the two PFs has no statistically significant effect on the 209 7/17stability of the double-PF system with a relative change of ∆∆G assoc = −1.0 ± 1.5 k B T (equilibrium 210 constant fold-change by ∼0.37), implying that the lateral bond is stabilized once the conformational 211 mismatch is resolved. Our results, therefore, directly support the previous ideas that a structural 212 conflict at the lateral interface would weaken it or locally increase the rate of GTPase activity [36,37]. 213 However, not only do we propose that both ideas would be equivalent, but we also estimate the 214 magnitude of lateral bond destabilization and predict that it would be a transient and reversible 215 effect.…”

“…Supplementary Material). Remarkably, this energy is very close to both the energy harvested by 136 MTs upon GTP hydrolysis [19,32] and the maximal excess energy that can be stored in a MT previously speculated that such a structural conflict would either weaken the lateral interactions 150 between incompatible dimers or increase the rate of GTPase activity [36,37]. In the latter case, the 151 hydrolysis-triggered compaction of an expanded dimer located next to a compacted dimer would be 152 more favorable.…”

Microtubules form by progressively faster tubulin accretion, not by nucleation-elongation

“…The longitudinal affinity we obtained is in the range of that obtained from biochemical models for microtubule growing and shrinking (e.g. (Gardner et al, 2011;Kim and Rice, 2019;Piedra et al, 2016;VanBuren et al, 2002) ). The corner and lateral affinities are both about 10-fold stronger than in those models, a difference that might reflect the different buffer conditions (high glycerol concentration) in 'nucleation' assays relative to 'dynamics' assays.…”

Insights into allosteric control of microtubule dynamics from a buried β‐tubulin mutation that causes faster growth and slower shrinkage

“…Microtubule dynamics were measured by time‐lapse DIC microscopy, as described previously . Temperature was maintained at 30°C using a Weather Station with enclosure fit to the Olympus IX81 microscope body.…”

“…The flow chambers were prepared as described previously . Samples of WT or β:T238A αβ‐tubulin along with 43 nM EB1‐GFP in imaging buffer (BRB80 + 0.1 mg/ml BSA + 0.2 mM MgCl 2 +1 mM GTP + 50 mM KCl + 0.1% Methylcellulose + antifade reagents [glucose, glucose oxidase, catalase]) were flowed into the chamber.…”