Recombinant αβ-Tubulin and a Simple Computational Model Shed Light on the Molecular Mechanisms of Microtubule Dynamics
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Microtubules (MTs) are essential to all eukaryotic organisms. They help segregate chromosomes and organize the cytoplasm. MTs are hollow barrels of the protein αβ-tubulin that exhibit a non-equilibrium behavior called dynamic instability: the stochastic switching of single polymers from a state of gradual growth to one of rapid disassembly. Dynamic instability underlies the MT cytoskeleton's rapid reorganizability and enables its diversity of functions. MTs can be reconstituted from purified αβ-tubulin and have been studied in vitro for over 40 years. Over this time, huge strides have been made in the development of an understanding of dynamic instability. Nevertheless, the mechanistic basis of important phenomena like GTP-dependent assembly and GTP hydrolysis-induced conformational change and catastrophe (the switch from growing to shrinking) remain controversial or unexplained. In Chapter 2, I discuss a study in which we used a computational model to investigate the consequences of a new way of thinking about the effect of nucleotide-state on αβ-tubulin and MT assembly. Our results suggest that GDP exposure on the MT plus-end can frustrate elongation and lead to catastrophe. We therefore predicted that GDP to GTP exchange on the MT plus-end might reduce the frequency of catastrophe. We tested our prediction by analyzing the effects of a mutant αβ-tubulin and a GTP analog designed to increase the rate of terminal nucleotide exchange on MT dynamics in vitro. Our experimental results support the results from our model. Thus, we believe that GDP exposure on the MT plus-end increases the likelihood of catastrophe, and can be countered by GDP to GTP exchange. In Chapter 3, I discuss a comparison of yeast and porcine MT dynamics in vitro. My measurements reveal striking differences between yeast and mammalian MT dynamics, and provide new constraints for models of MT dynamics. I conclude my thesis in Chapter 4 with my view of what my work means, what remains to be done and what paths my work has opened for further exploration.