| Stu2 is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Overall, I provide evidence that Stu2 regulates the dynamics of microtubule plus-ends and their critical interactions with cortical sites in the cytoplasm. Conditional stu2 mutant cells arrest prior to anaphase. In addition, cells lacking Stu2 display a checkpoint-independent block in spindle elongation suggesting a defect in the polar microtubule arrays. Depletion of Stu2 also leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. Consistent with a role for Stu2 in cytoplasmic microtubule function, I found that loss of Stu2 causes a spindle orientation defect.; In a two-hybrid screen for proteins that interact with Stu2, our lab identified the microtubule-binding proteins, Bik1 and Bim1 (Chen et al., 1998). In addition to their localization to the microtubule-organizing center, I found that Stu2 and Bik1 also localize to the tips of both the nuclear and cytoplasmic microtubules. I used co-immunoprecipitation to confirm the two-hybrid interactions and identify one between Bik1 and Bim1. Also using the two-hybrid system, I mapped the domains of Stu2, Bik1, and Bim1 that are sufficient for an interaction with each other. At a non-permissive temperature, stu2-10 bik1Δ cells are inviable due to defects in bipolar spindle assembly suggesting that a functional Stu2-Bik1 complex may be necessary for this process. I have also found that loss of STU2 function alters the localization of Kar9, a protein required for spindle orientation, in the same manner as a deletion of BIM1 (Miller et al., 2000). These results suggest a model in which Stu2, Bik1, and Bim1 associate with one another to regulate microtubule-dependent processes in the cell. |
