Regulation of Microtubule Dynamics - A Chemical Biology Approach

Microtubules (MTs) are components of the cytoskeleton involved in a number of cellular processes including intracellular transport, cell motility and formation of the mitotic spindle. Essential to the function of MTs is their ability to stochastically switch between phases of growth and shortening, called dynamic instability. This process is regulated by nucleotide occupancy and a conserved set of MT-associated proteins. Despite extensive study, little is known about the regulation of MT dynamics at the structural and molecular level. Using a chemical biology approach, the studies described in this thesis provide further insight into the regulation of MT dynamics.;Chapter 2 describes the use of hydrogen/deuterium exchange mass spectrometry (H/DX-MS) coupled with isothermal urea denaturation to understand the role of altered nucleotide occupancy on microtubule lattice instability. From this data, we were able to propose a new model that emphasizes the importance of intradimer flexibility in the assembly of MTs.;Chapter 3 involves using mass shift perturbation analysis and data-directed docking to discover the laulimalide binding site. From this data we were able to confirm the existence and location of a fourth distinct ligand binding site within alpha/beta-tubulin. The laulimalide binding site is unique in that it is found on the outside surface of the microtubule. Therefore, laulimalide may be a useful chemical probe for the study of MT-associated proteins.;Chapter 4 characterizes the cellular phenotype of laulimalide in HeLa cells. Laulimalide was found to exhibit unique effects in metaphase and interphase in comparison to a taxane MT stabilizer. These results provide evidence that laulimalide acts as its own class of MT stabilizing drug.;Chapter 5 involves the development of a method to isolate the MT proteome for the study of MT-interacting proteins. This method was successful in identifying a large number of MAPs and will be used for future quantitation studies.