Microtubule-associated proteins in fission yeast

The highly conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs) play a central role in cytoplasmic microtubule organisation and mitotic spindle formation. The fission yeast S. pombe has two family members, Alp14 and Dis1. Both localise to interphase microtubules, spindle pole bodies (the yeast equivalent of the centrosome), and kinetochores. Here we present the characterisation of Alp14 and Dis1 during interphase. We find that Alp14 localisation resembles that of Mal3, a canonical plus end tracking protein. Deletion results in a decrease in the number and length of interphase microtubule bundles at low temperatures. Alp14 is temperature sensitive. At the restrictive temperature we find that an interphasic intranuclear microtubule bundle forms, nucleated from the region of the spindle pole bodies and kinetochores. This intranuclear bundle has a structure and displays dynamics similar to that of a normal interphase bundle and is able to move the nucleus. Dis1 localises to interphase microtubules but does not show plus end tracking behaviour. Deletion has no apparent effect on the organisation of interphase microtubules, but Dis1 is cold sensitive and at the restrictive temperature the cells become blocked in mitosis with aster-like spindles. Deletion of both alp14 and dis1 is lethal. We investigate the functional redundancy between Alp14 and Dis1 during interphase. Over-expression of Dis1 in alp14 deletion cells can partially rescue the mutant microtubule phenotype. Conversely, attenuated expression of Dis1 in an alp14 deletion background results in almost complete loss of interphase microtubules. We conclude that the presence of at least one of the Dis1/XMAP215 homologues is essential for the maintenance of interphase microtubule arrays. Similar to Alp14, Tip1 is a microtubule plus-end tracking protein, homologous to human CLIP170. Together with the EB1 homologue, Mal3, Tip1 spatially regulates microtubule dynamics, ensuring that the cylindrical cell shape of S. pombe is maintained. In the second part of this thesis the characterisation of the protein SPCC736.15 (Toi4), identified in a screen for Tip1-interacting proteins is presented. During interphase, Toi4p-GFP localises to the central regions of the cell cortex. Shortly before mitosis, Toi4p-GFP begins to accumulate at the cell ends. Concurrent with the onset of mitosis, there is exclusion of Toi4p-GFP from the region of the cell cortex where the actomyosin ring forms and the cell subsequently divides. The S. cerevisiae homologue of Toi4p is Pil1p, which is proposed to be the major component of an endocytic organelle termed the eisosome. We tested for such a role for Toi4 in S. pombe, however we detect no link between Toi4 and endocytosis, suggesting that the homologues, although they have a similar localisation pattern, may perform different functions

The Fission Yeast XMAP215 Homolog Dis1p Is Involved in Microtubule Bundle Organization

Microtubules are essential for a variety of fundamental cellular processes such as organelle positioning and control of cell shape. Schizosaccharomyces pombe is an ideal organism for studying the function and organization of microtubules into bundles in interphase cells. Using light microscopy and electron tomography we analyzed the bundle organization of interphase microtubules in S. pombe. We show that cells lacking ase1p and klp2p still contain microtubule bundles. In addition, we show that ase1p is the major determinant of inter-microtubule spacing in interphase bundles since ase1 deleted cells have an inter-microtubule spacing that differs from that observed in wild-type cells. We then identified dis1p, a XMAP215 homologue, as factor that promotes the stabilization of microtubule bundles. In wild-type cells dis1p partially co-localized with ase1p at regions of microtubule overlap. In cells deleted for ase1 and klp2, dis1p accumulated at the overlap regions of interphase microtubule bundles. In cells lacking all three proteins, both microtubule bundling and inter-microtubule spacing were further reduced, suggesting that Dis1p contributes to interphase microtubule bundling