An Isolated Clasp TOG Domain Suppresses Microtubule Catastrophe and Promotes Rescue

Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the CLASP family that reduce catastrophe and/or increase rescue also contain arrayed TOGs. How CLASP TOGs contribute to activity is poorly understood. Using S. cerevisiae Stu1 as a model CLASP, I report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. To begin with, I introduce microtubules, their dynamics and regulatory proteins in Chapter 1. In Chapter 2, I discuss how the two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. I also explore the structure of TOG2 and how it reveals a CLASP-specific residue that likely dictates distinctive tubulin-binding properties. Next, in Chapter 3, I study how, contrary to the expectation that TOGs must work in arrays, the isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function. Finally, in Chapter 4, I will summarize my work and provide insight into future directions.