In fission yeast, microtubule (MT) nucleation is regulated in space and time. In interphase, MTs are nucleated in the cytoplasm to regulate cell polarity, whereas in mitosis, nucleation takes place inside the nucleus to form a mitotic spindle. We hypothesize that several non-exclusive mechanisms may be responsible for this differential regulation of MT nucleation. Two fission yeast proteins, Mto1 and Pcp1, are involved in MT nucleation in interphase and mitosis, respectively. These proteins share a sequence motif, called CM1 that is responsible for interaction with the γ-tubulin complex (γ-TuC). In the first part of my project, I tested whether sequence differences between Mto1 and Pcp1 CM1 regions contribute to the differential regulation of MT nucleation in interphase vs. mitosis. I showed that the two CM1 regions are interchangeable and play no role in differential regulation of Mto1 and Pcp1. By generating Pcp1-9A1 mutant, where conserved residues within the Pcp1 CM1 region was replaced with alanines, I showed that Pcp1 CM1 region is required for its function. Moreover, using CM1 regions from two human proteins that are implicated in schizophrenia and microcephaly development, MMGL and CDK5RAP2, I showed that human CM1 domains could rescue yeast protein function, demonstrating that the CM1 region is conserved across evolution. In the second part of my project, I focused on regulation of cytoplasmic MT nucleation. In fission yeast, cytoplasmic MT nucleation occurs from several distinct sites in the cell and is promoted by the Mto1/2 complex. The Mto1/2 complex is composed of multiple copies of Mto1 and Mto2 and interacts with the γ-TuC. Disruption of the interaction of Mto1/2 with the γ-TuC, or of the Mto1-Mto2 interaction, results in a complete loss of interphase cytoplasmic nucleation. As cells enter mitosis, Mto2 is hyperphosphorylated, and the Mto1-Mto2 interaction is disrupted, leading to abolishment of cytoplasmic nucleation. This led to a hypothesis that Mto2 phosphorylation regulated the Mto1/2 complex mitotic disassembly. I showed that Mto2 phosphorylation is used to control levels of cytoplasmic nucleation in both interphase and mitosis. During interphase, I found that Mto2 is phosphorylated in order to reduce levels of MT nucleation. When Mto2 phosphorylation is prevented by mutation of phosphorylatable residues to alanines, Mto1/2 mutant complexes show a more robust interaction with the γ-TuC, and more MTs are nucleated in the cytoplasm. During mitosis, hyperphosphorylation of Mto2 plays a role in the disassembly of Mto1/2 complexes. In particular, while the interaction of wild-type Mto2 with Mto1 is disrupted during mitosis, Mto2-alanine mutants, in which phosphorylation was nearly abolished, are still able to interact with Mto1 in mitosis. Interestingly, Mto1/2 complexes containing Mto2-alanine mutants are still disassembled in mitosis by disruption of Mto2 self-interaction. I used SILAC phosphoproteomics to show that Mto2-alanine is still phosphorylated in mitosis, suggesting the Mto2 self-interaction might also be controlled by phosphorylation. While doing so, I developed a novel SILAC quantification method that is particularly useful for quantification of multiply phosphorylated proteins and peptides. Using data obtained by SILAC, I generated additional Mto2 alanine mutants with more phosphorylation sites mutated. Preliminary analysis showed that these mutants are similar to the alanine mutants analysed previously; however, more analysis is required to generate more definitive conclusions. In summary, in this study I have uncovered the functional conservation of the CM1 region from yeast to human. I also showed that Mto2 phosphorylation regulates cytoplasmic MT nucleation in both interphase and mitosis, by regulating the Mto2-Mto1 interaction and the Mto2-Mto2 self-interaction and therefore remodelling the Mto1/2 complex.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:695070 |
| Date | January 2014 |
| Creators | Borek, Weronika Ewa |
| Contributors | Sawin, Ken ; Ohkura, Hiro |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/16873 |
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