UCSF

The microtubule (MT) cytoskeleton plays an essential role in mitosis, intracellular transport, cell shape, and cell migration. The assembly and disassembly of MTs, which can occur through the addition or loss of subunits at either the plus- or minus-ends of the polymer, is essential for MTs to carry out their biological functions. In Chapter 1, I describe my work on a recently described family of MT minus-end binding proteins called CAMSAP/Patronin/Nezha that act on MT ends to regulate their dynamics. Patronin, the single member of this family in Drosophila, was previously shown to stabilize MT minus-ends against depolymerization in vitro and in vivo. Here, I show that all three mammalian CAMSAP family members also bind specifically to MT minus-ends and protect them against kinesin-13-induced depolymerization. However, these proteins differ in their abilities to suppress tubulin addition at minus-ends and to dissociate from MTs. CAMSAP1 does not interfere with polymerization and tracks along growing minus-ends. CAMSAP2 and CAMSAP 3 decrease the rate of tubulin incorporation and remain bound, thereby creating stretches of decorated MT minus-ends. Using truncation analysis, I find that somewhat different minimal domains of CAMSAP and Patronin are involved in minus-end localization. However, I find that in both cases, a highly conserved C-terminal domain and a more variable central domain cooperate to suppress minus-end dynamics in vitro and that both regions are required to stabilize minus-ends in Drosophila S2 cells. These results show that members of the CAMSAP/Patronin family all localize to and protect minus-ends but have evolved distinct effects on MT dynamics. In Chapter 2, I describe work characterizing a set of inhibitors of a kinesin-12 family member called Kif15, which has a role in spindle organization during mitosis. Kif15 has been shown in previous work to cooperate with the tetrameric kinesin Eg5 to facilitate bipolar spindle formation; we were interested in the possible application of these molecules to spindle disruption in hopes that the combination of Eg5 and Kif15 inhibition might be clinically efficacious. I show that these small molecules have activity against Kif15 through an uncompetitive mechanism and that Kif15 inhibition with these molecules affects spindle organization and cell division in vivo.