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- W2014906536 abstract "Understanding how microtubules (MTs) assemble and disassemble is vital to understanding fundamental cellular processes, such as mitosis and polarization, and their regulation via proteins and therapeutic drugs. Our current understanding of assembly kinetics is based on the classic one-dimensional (1D) assembly model of Oosawa, which assumes that the multiprotofilament MT can be modeled as if it were a single protofilament. In the classic 1D model the subunit off-rate is independent of free subunit concentration, an assumption that has yet to be confirmed experimentally. Using TIRF-microscopy and laser tweezers assays to measure microtubule self-assembly from GMPCPP-tubulin in vitro with nanoscale accuracy, we find that the off-rate is not constant but rather increases with increasing free subunit concentration. Consistent with this observation, we find that a simple two-dimensional (2D) model predicts the increasing off-rate with subunit concentration due to a shift in tip structure from relatively blunt at low concentrations to relatively tapered at high concentrations, which we confirmed experimentally by TIRF-microscopy. Because both the on-rate and off-rate increase with free tubulin concentration, the 2D model requires an association rate constant that is an order-of-magnitude larger than the 1D model. We tested this prediction by measuring the variability in assembly rate, and found that the on- and off-rates are consistent with the 2D model predictions and are an order-of-magnitude higher than predicted by the 1D model. In summary, we find that the kinetic rates of microtubule self-assembly have been severely underestimated in the literature, by at least an order-of-magnitude. Because net growth results from a small difference between large on-rates and off-rates, the net rate can be significantly altered by weak microtubule associated protein and therapeutic drug interactions with the microtubule." @default.
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- W2014906536 date "2011-02-01" @default.
- W2014906536 modified "2023-10-16" @default.
- W2014906536 title "Kinetics of Microtubule Assembly" @default.
- W2014906536 doi "https://doi.org/10.1016/j.bpj.2010.12.3098" @default.
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