Septins are a family of GTP-binding proteins implicated in mammalian cell division. Since these proteins form heterologous complexes and filaments in interphase cells, it has been assumed that depletion of any or all septins in a given cell type will give rise to the same phenotype. I demonstrate that while all septins expressed in HeLa cells localize to the cleavage furrow and midbody during cytokinesis, and co-immunoprecipitate throughout cell division, they do not all have identical roles during this process. Specific depletion of SEPT2 or SEPT11 caused defects in the early stages of cytokinesis, ultimately resulting in binucleation. Similar results were observed upon simultaneous depletion of all septins. In sharp contrast, SEPT9 was dispensable for the early stages of cell division, but was critical for the final separation of daughter cells. I demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody. Immunofluorescence microscopy suggests that SEPT9 may act to compartmentalize the exocyst at the site of abscission, analogous to the role performed by septins in Saccharomyces cerevisiae. I provide evidence that the N-terminal region of SEPT9, which is absent from the shorter SEPT9 isoforms, plays an important role in abscission. I describe a long-anticipated link between a mammalian septin and the cell cycle machinery by showing that the N-terminal region of SEPT9 is phosphorylated at threonine 24 upon mitotic entry by cyclin-dependent kinase 1. This creates a binding site for the WW domain of the peptidyl-prolyl isomerase Pin1. I provide evidence that Pin1 induces a conformational change in the N-terminal region of SEPT9 that is important for the completion of cytokinesis. I propose that mitotic regulation of SEPT9 by Cdk1 and Pin1 regulates an interaction between SEPT9 and an unidentified protein that is critical for abscission.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/42489 |
| Date | 15 November 2013 |
| Creators | Estey, Mathew |
| Contributors | Trimble, William S. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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