Subnanoscale mechanics of microtubule dynamic instability

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Eukaryotic microtubules (MTs) are cellular filaments that form the mitotic spindle, define the shape of axons and dendrites, and provide tracks for intracellular transport. MTs undergo stochastic switching between phases of growth and shrinkage driven by the hydrolysis of GTP nucleotides by tubulin dimers, building blocks that constitute the MT lattice. This semistable behavior of MTs, also known as dynamic instability, is crucial for MT function. The detailed mechanism by which GTP binding and hydrolysis control the MT assembly and disassembly is still poorly understood, with two aspects standing out as essentially unresolved: (a) the missing link between GTP binding by tubulin and its conformation, and (b) the mechanism of destabilization of the MT lattice by GTP hydrolysis.This project therefore aims at characterizing, by fully atomistic simulations, the mechanics and energetics of (a) a single tubulin dimer depending on its nucleotide state and (b) a complete MT complex in response to GTP hydrolysis. Using preliminary data obtained during the preparatory phase, we will first employ protein structural analysis to assess what conformational changes in tubulin are induced by GTP binding. Atomistic free energy calculations will then be used to characterize the energetics of the tubulin dimer along this conformational pathway. The obtained free energy landscapes will enable us to judge which of the currently available experiment-based hypotheses of MT assembly is most plausible in terms of mechanics. Second, we propose to develop and apply new, atomistic models of complete MTs using the most recent high-resolution cryo-electron microscopy (cryo-EM) data to study the structural transitions that the MT lattice undergoes upon GTP hydrolysis. By using free energy calculations, we intend to obtain a quantitative understanding of how and how much single dimers in the lattice contribute to the energetics of this transition. The obtained energy barriers will enable us to interpret at the atomic level experimentally observable differences between the extreme states of the MT lattice: pre-hydrolysis and fully hydrolyzed.Considering the rapidly growing number of structural studies of tubulin and MTs only over the past 2 years, high-resolution structural data are expected to be available toward the end of the proposed project such that the full potential of our results can be assessed. We have also established a collaboration with the experimental lab of Eva Nogales (University of California, Berkeley, USA) who is currently working on revealing intermediate (post-hydrolysis) states of the MT lattice and their modulation by cellular factors using high-end cryo-EM. The collaboration would provide a solid basis for validating our results and allow us to complement the cryo-EM analysis of MTs with new atomistic details on the dynamics and energetics of hydrolysis-driven transitions in the MT lattice.