The role of fission yeast microtubule associated protein, Alp14, in microtubule stabilisation and spindle assembly checkpoint maintenance
Accurate and timely chromosome segregation of replicated DNA during cell division is crucial to cell integrity. This process requires mitotic spindles to physically interact with sister kinetochores located at the centromeres. Proteins that function to facilitate this crucial interaction include regulators of microtubule dynamics such as microtubule associated proteins. This thesis primarily describes the study of fission yeast microtubule associated protein, Alp 14 by domain analysis. We report the surprising finding that despite high structural conservation, the two TOG domains in Alp 14 are required for distinct mechanisms. From our consistent data, the first TOG domain (TOG1 - resides at the N-terminus) is shown to be required for the spindle assembly checkpoint. Its deletion results in failure to maintain the spindle checkpoint in spindle damaged conditions, while its overproduction causes hyper-activation of the checkpoint without spindle damage. The second TOG domain (TOG2 - resides near the centre of the proteins), on the other hand, is required for microtubule stabilisation. Further analysis of the role of TOG 1 in the spindle assembly checkpoint suggests that Alp 14 may maintain the checkpoint via the outer kinetochore Nuf2/Ndc80 complex. Alp 14 is found to bind to Nuf2, while deletion of TOG 1 causes Nuf2 and Ndc80 to be delocalised from kinetochore in spindle damaged conditions. In this thesis, we have also studied the interaction of Alp 14 with kinesins Klp5 and Klp6. Our results show that Klp5 and Klp6 are essential for accurate chromosome segregation, promote microtubule depolymerisation and suggest that the kinesins are required for tension-generation at the kinetochore in metaphase. Surprisingly, although the function of Klp5/Klp6 in microtubule regulation is opposed to that of Alpl4/Disl, the proteins collaborate to ensure accurate chromosome segregation.