Single-molecule interrogation of microtubule dynamics mechanisms

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The microtubule (MT) cytoskeleton is essential to eukaryotic cells: microtubules are dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the biochemical properties of individual αβ-tubulin subunits and how they interact within the MT lattice. The quantitative mechanisms of MT dynamics have been difficult to understand because the MT end is a complex biochemical environment where individual tubulins adopt different conformations and can have different numbers of neighbor contacts, and because it has not been possible to measure individual interactions directly. With the goal of quantitatively examining the contributions of longitudinal and lateral interactions, nucleotide state, MT end configurations, and lattice-induced conformational changes to MT assembly and switching, we recently showed that interactions between yeast αβ-tubulin and the MT end can be observed at the single-molecule level and quantified with high temporal resolution using interferometric scattering (iSCAT) microscopy. Comparative studies of yeast and human tubulin are proposed to establish general mechanisms of microtubule dynamics. Aim 1 will use iSCAT to measure and quantify the interactions of human and yeast αβ-tubulin with the end of a stable microtubule seed, and how these interactions depend on nucleotide state. Aim 2 will use iSCAT and other techniques to measure how a mutation that perturbs the αβ-tubulin propensity for conformational change affects biochemical interactions with the microtubule end and microtubule growth and shrinking kinetics more generally. Aim 3 will use iSCAT and other techniques to measure how different doses of an αβ-tubulin mutant with its plus-end “blocked” affect microtubule elongation and catastrophe. The results will be used to construct a biochemical model for microtubule dynamics that will deepen the understanding of catastrophe.