A mechanistic study of XMAP215-mediated microtubule polymerization



 DESCRIPTION (provided by applicant): Microtubules (MTs) are essential for maintaining cell structure, forming the bipolar mitotic spindle, and facilitating intracellular transport/cellular movement. The -tubulin heterodimer is the building block of MTs. The GTP-hydrolysis dependent gain and loss of -tubulin at MT ends underlies MTs dynamics. While MTs are inherently dynamic in vitro, MT associated proteins, including the XMAP215 family, regulate MT dynamics in cells to facilitate the formation of complex dynamic MT-based structures such as the mitotic spindle. XMAP215 family members use an array of five tubulin-binding tumor overexpressed gene (TOG) domains to increase MT polymerization rates. Multiple models have been proposed for the XMAP215 family's TOG- dependent MT polymerization mechanism. A wrap around model suggests XMAP215 TOG domains interact with tubulin in a 1:1 or 1:2 XMAP215:tubulin complex. In contrast, a templating model proposes that XMAP215 forms a long linear structure that facilitates the addition of three or more tubulin heterodimers at MT plu ends. Structural analysis of pentameric TOG domain arrays suggests that TOG domains have differential architectures. Specifically, the N-terminal TOG domains are structurally similar, whereas the C-terminal TOG domains have unique, divergent architectural features that structurally predict an ability to engage laterally- associated -tubulin heterodimers and may underlie their respective abilities to promote MT polymerization in vitro. I hypothesize that XMAP215 TOG domains template MTs using structurally distinct features tuned to bind free, pseudo-polymerized, or lattice-incorporated tubulin, which allows them to collectively promote MT polymerization and drive proper mitotic spindle formation. I will test this hypothesis by (1) biochemically determining how TOG domains interact with different forms of tubulin, which will help elucidate XMAP215:tubulin binding stoichiometry and (2) determining how specific TOG domains contribute to XMAP215-dependent MT polymerization activity and mitotic spindle structure in cell culture. Together, these aims will allows us to refine the current models of how XMAP215 mechanistically interacts with tubulin to accelerate MT polymerization and maintain bipolar mitotic spindle structure.