Mechanistic Analysis of Microtubule Dynamics and Regulation
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Microtubules are critical components in a cells cytoskeletal network, known to form the mitotic spindle which allows for chromosome segregation and cell division and organizing the cytoplasm of non-dividing cells. The quick reorganization of the cytoskeleton relies heavily on the underlying behavior of microtubules, known as dynamic instability. Dynamic instability, the rapid switch between growing and shrinking states of the microtubule, depends on the functional GTPase behavior of the microtubule polymerizing subunits, αβ-tubulin. Recent studies have noted the presence of multiple conformational states of αβ-tubulin in the microtubule lattice, in addition to major conformational changes that occur in αβ-tubulin within the microtubule as compared to free αβ-tubulin in solution. In Chapter 2, I will discuss a study in which I explored the role of the conformational cycle and its impact on microtubule dynamic instability. By studying a mutation in β-tubulin, T238A, I have shown that nucleotide hydrolysis and conformational changes in the lattice are tightly linked and provide allostery throughout the microtubule. Uncoupling the two cycles disrupts the allostery which greatly impacts the rapid transitions normally seen in dynamic instability that allow for fast and decisive structural rearrangements. In Chapter 3, I will discuss a study that aimed to dissect the molecular mechanisms of the yeast microtubule polymerase, Stu2p. In this project, I developed an all-yeast in vitro reconstitution system using total internal reflection fluorescence microscopy which enabled me to study a variety of Stu2 mutants, in the presence of wild-type and mutant yeast tubulin samples. Here, I discovered how the tubulin conformational state can impact Stu2 function and determined a new property of Stu2 in its ability recognize and bind either the microtubule lattice or free tubulin. From these findings, I have proposed a new alternating engagement mechanism to explain how Stu2 functions processively at the microtubule plus end to increase the growth rates of microtubules. Finally, in Chapter 4 I will summarize my work on both of these projects and discuss both future directions and preliminary results looking to solve the structure of human β:T238A microtubules using cryo-EM.