Toward understanding cyclin requirements for mitotic entry in the mammalian somatic cell cycle [electronic resource]

The mammalian somatic cell cycle relies upon a complex network of core machinery, some of which have redundant or overlapping functions. That cyclins are essential components in the mitotic circuit has been well established, but many molecular and spatiotemporal requirements remain unknown. In this thesis, I present three lines of study in which we examined cyclin requirements for mitotic onset using mammalian tissue culture cells. These studies utilize diverse approaches to better understand requirements for cyclin synthesis, localization, and post-translational changes in regulating mitotic entry in the mammalian somatic cell cycle. In the first study, we show that G2-phase cyclin synthesis is not required for mitotic entry. Protein inhibition and cyclin perturbation studies spanning the last five decades have supported a requirement for G2-phase protein synthesis of cyclins and other pro-mitotic proteins for mitotic entry; however, previous protein inhibition approaches have relied on inhibiting protein synthesis using cycloheximide (CHX), which could constitute enough of a stress to activate a p38-mediated antephase checkpoint. The antephase checkpoint prevents G2-phase cells from entering mitosis in the face of a range of stress agents, thereby constituting a second possible interpretation for why CHX-treated cells arrest in G2 phase. Here we show that CHX-treated G2-phase cells are able to enter mitosis when administered a p38 inhibitor, demonstrating that CHX secondarily activates a p38-mediated antephase checkpoint, which is responsible for preventing CHX-treated cells from entering mitosis. Sufficient cyclin synthesis for mitotic onset is apparently completed earlier, by the end of S phase. Thus, the ~4--6 hours of G2 phase preceding mitotic entry is not due to cyclin and other pro-mitotic protein synthesis requirements as has been the classical view of the cell cycle field, but rather appears to be necessary for preparations necessary to satisfy checkpoint requirements and other preparations for mitosis. The subsequent studies presented pertain to regulatory aspects controlling cyclin A2's dual roles in S phase and mitosis. Cyclin A2, the somatic form of cyclin A, is implicated in two essential aspects of the cell cycle, DNA synthesis and mitosis, but it is not known how cyclin A2 coordinates these two distinct functions. A mainly nuclear protein, cyclin A2 also accumulates in the cytoplasm in late S phase and during G2 phase, and we show that this cytoplasmic pool is required for mitotic entry. In addition, we examined cyclin A2 phosphorylation in the context of cell cycle regulation, and preliminary data suggests that phosphorylation is a key regulatory mechanism capable of controlling cyclin A2 localization and therefore its function. Collectively, these studies contribute to our understanding of cyclin requirements for mitotic entry, describing how the mitotic onset is in part governed by preparatory activities including localization-dependent cyclin functions and those that satisfy late-stage checkpoint requirements.

Keywords: Chemical and Systems Biology.
Advisor: Ferrell James Ellsworth primary advisor. Thesis advisor
Elias Joshua
Meyer Tobias advisor. Thesis advisor