: Microtubule Self-Assembly Kinetics
5 September 2013 14:00 in CM101
Microtubule assembly and disassembly is vital for many fundamental cellular processes. Our current understanding of microtubule assembly kinetics is based on a one-dimensional assembly model, which assumes that each protofilament of a microtubule behaves independently. In this model, the subunit disassociation rate from a microtubule tip is independent of free subunit concentration. Using Total-Internal-Reflection-Fluorescence (TIRF) microscopy and a laser tweezers assay to measure in vitro microtubule assembly with nanometer resolution accuracy, we now find that the subunit dissociation rate from a microtubule tip increases at higher free subunit concentrations. This is because there is a shift in microtubule tip structure from relatively blunt at low free concentrations to relatively tapered at high free concentrations, which we confirmed experimentally by TIRF microscopy. Because both the association and the dissociation rates increase at higher free subunit concentrations, we find that the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature. In addition, we performed Brownian dynamics simulations to determine the theoretically predicted rate constants, free energies, and entropic penalties of subunit addition and loss. Together, our studies lead to a major revision of kinetic estimates of microtubule assembly, and provide a new perspective on how microtubule-associate proteins and anticancer drugs might control assembly.
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