The goal of this research is to understand the mechanisms of microtubule (MT) polymerases and depolymerases in regulating MT plus ends. During the mentored phase, 1 have gained extensive expertise in single molecule total internal reflection fluorescence (TIRF) microscopy to study mechanisms of MT polymerases, XMAP215/Dis1 proteins, and Cytoplasmic linker Associated proteins (CLASPs). These Studies indicate a fundamental role for TOG domains in regulating MT polymerization. My studies show tubulin dimers recruited by CLASP and XMAP215 TOG domains promote two unique types of MT regulatory activities by recruiting soluble tubulin either to polymerizing or depolymerizing MT ends. I have also attained extensive progress towards a medium resolution structure ofthe XMAP215 yeast ortholog, Stu2, bound to a tubulin dimer using cryo-electron microscopy (Cryo-EM) and single particle image analysis. As previously described in the original proposal, my research program for the independent phase will be carried out as an assistant professor at the University of California Davis, and will utilize a combination of TIRF microscopy and structural biology approaches. 1) I will explore deeper questions into the structures and mechanisms of TOG domains in XMAP215 and CLASP proteins. 2) I will study the structures of MT depolymerases (kinesins-13 and 8) in complex with tubulin dimers using cryo-EM andx-ray crystallography. I will also explore the role of conserved MT depotymerase features in the MT depolymerization mechanism using TIRF microscopy. 3) I will reconstitute the antagonism and/or interaction between MT polymerases and depolymerases using multi-color TIRF microscopy and cryo-EM. The latter studies aim to explore how this antagonism alters slow MT dynamics in interphase to fast MT dynamics at the onset of mitosis. I will also study the effect of phosphorylation by mitotic kinases on the activities of MT polymerases and depolymerases how it affects this antagonism. Hypotheses arising from biophysical and structural studies proposed in the independent phase will be validated in vivo using fission yeast as a model system for microtubule dynamics, in collaboration with Fred Chang's Laboratory (Columbia University).
Defects in regulation of the cell division.are directly linked to human genetic defects and cancers. Results from this study will broaden our understanding of this regulation and provide knowledge to develop new strategies to inhibit tumor growth. Strategies developed based on the basic knowledge attained from these studies can help identify more effective means for cancer therapy