The coordination between cell growth and division is a highly regulated process that is intimately linked to the cell cycle. Efforts to identify an independent mechanism that measures cell size have been unsuccessful. Instead, we propose that size control is an intrinsic function of the basic cell cycle machinery.
My work shows that in the fission yeast Schizosaccharomyces pombe Cdc25 accumulates in a size dependent manner. This accumulation of Cdc25 occurs over a large range of cell sizes. Additionally, experiments with short pulses of cycloheximide have shown that Cdc25 is an inherently unstable protein that quickly returns to a size dependent equilibrium in the cell suggesting that Cdc25 concentration is dependent on size and not time. Thus, Cdc25 can act as a sizer for the cell. However, cells are still viable when Cdc25 is constitutively expressed suggesting that there is another sizer in the case that Cdc25 expression is compromised.
Cdc13 is a likely candidate due to the similar characteristics to Cdc25 and the ability to activate Cdc2. Cdc13 accumulates during the cell cycle in a manner similar to Cdc25. I show that in the absence of Cdc2 tyrosine phosphorylation, the cell size is sensitive to Cdc13 activity showing that Cdc13 accumulation can determine when cells enter mitosis. These results suggest a two sizer model where Cdc25 is the main sizer with Cdc13 acting as a backup sizer in the event of Cdc25 expression is compromised.
Additionally, in the absence of Cdc2 phosphorylation by the kinases Wee1 and Mik1, mitotic entry is regulated by the activity of Cdc2. In the absence of Cdc2 phosphorylation, this activity is regulated by binding of cyclins to Cdc2. Under these circumstances, the activity of Cdc13 can regulate mitotic entry provide further evidence that Cdc13 could be a sizer of the cell in the case where Cdc25 expression is compromised.
The results I present in this dissertation provide the groundwork for understanding how cells regulate size and how this size regulation affects cell cycle control in S. pombe . The results show how the intrinsic cell cycle machinery can act as a sizer for the G2/M transition in S. pombe . Interestingly, this mitotic commitment pathway is well conserved suggesting a general solution for size control in eukaryotes at the G2/M transition. Understanding the mechanism of how protein concentration is regulated in a size dependent manner will give much needed insight into how cells control size. Elucidating the mechanism for size control will capitalize on decades of research and deepen our understanding of basic cell biology.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1792 |
| Date | 17 June 2015 |
| Creators | Keifenheim, Daniel L. |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved. |
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