Presentation description

Stu2 is an essential microtubule-associated protein (MAP) conserved from yeast to humans that is involved in regulating microtubule dynamics. Microtubules are filaments that extend from the opposite poles of a dividing cell and attach to the kinetochore of a chromosome to form a mitotic spindle. Stu2 is also found to have several other roles, including nucleation of microtubules at the microtubules organizing center and regulating kinetochore-microtubules attachment. Hence so, Stu2 is an essential player in the partitioning of chromosomes. It is challenging to study Stu2 functions apart since mutations in STU2 have pleiotropic effects. Our approach to address this issue is to utilize the inducible protein-protein tethering system FRB/FKBP. We tag Stu2 with FRB and tag a target protein with FKBP upon the addition of the small molecule rapamycin. FRB and FKBP tightly bind to each other, tethering Stu2 to the target protein. The concept behind this approach is that we can study various Stu2 functions by increasing its local concentration at different regions of the mitotic spindle, leading to an overactivation of its function at that location. We are creating a panel of FKBP-tagged target proteins spanning the whole spindle and kinetochore. Our data so far show that tethering Spc110 at the spindle pole body (Yeast's Microtubule Organizing Center) and Bim1 (MAP that binds to the plus end of a microtubule) is lethal. Using fluorescence microscopy, we found that in the case of tethering Stu2 to Bim1, cells are unable to form a proper spindle suggesting that they are either defective in microtubule formation or maintaining their stability. In the case of tethering Stu2 to Spc110, the cell cycle is halted in metaphase, suggesting chromosome misorientation defects. Stu2 is also thought to act in microtubule nucleation at the microtubule organizing center. These cells may nucleate too many microtubules, thus leading to these defects. We plan to utilize a mix of genetic and biochemical tools to dissect these mutants to understand what Stu2 function is disrupted.