Regulation of microtubule nucleation in Schizosaccharomyces pombe: recruitment of Mto1 to the site of the prospective eMTOC
Miller, Victoria Jane
Mto1 recruits γ-tubulin to the sites of cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe. The regulation of Mto1 localisation is central to re-modelling of the microtubule cytoskeleton during the cell cycle. This thesis describes how Mto1 is recruited to the cell equator during mitosis, thereby establishing the equatorial microtubule nucleation centre (eMTOC). F-actin is found to be required for Mto1 localisation to the cell equator and Mto1 is shown to co-localise with the cytokinetic actin ring (CAR). Yeast 2-hybrid screening and tandem-affinity purification were used to screen for additional proteins required for Mto1 localisation to the equator. Further candidate screening identified Myp2, a type II myosin present in the CAR, as being required for Mto1 localisation to the cell equator. Myp2 is shown to physically interact with Mto1 and to be required for formation of the post-anaphase microtubule array. The regulation of Mto1 localisation to the cell equator was also studied. Time-lapse microscopy reveals that Mto1 localisation to the equator does not require either the anaphase-promoting complex or the septation initiation network, both of which have been previously shown to be necessary for the recruitment of γ-tubulin to the eMTOC. Maintenance of the equatorial CAR has previously been attributed to the postanaphase array. The position of the CAR in the mto1-427 mutant strain, which fails to nucleate a PAA, is shown to be unaltered from wild-type strain during exponential growth, suggesting that the PAA does not centre the CAR during normal growth.