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Split Decisions: Regulation of Cell Division
Summary: Kathleen Gould is interested in understanding how cells divide. Her laboratory utilizes a genetically tractable yeast, Schizosaccharomyces pombe, as a model organism to study the molecular mechanisms of cytokinesis.
My laboratory is interested in understanding the molecular mechanisms that regulate cell division. We use primarily the fission yeast, Schizosaccharomyces pombe, as a model organism for our studies, since the machinery operating core biological processes such as the cell cycle has been conserved throughout evolution. S. pombe also offers several experimental advantages over higher eukaryotic cells. Its genome is sequenced, a complete gene deletion set is available, the localization of the proteome has been determined, and many conditionally lethal mutations have been isolated in cell cycle regulators. Also, a full range of techniques can be applied with ease, including biochemical, genetic, and live-cell imaging.
The entrance of eukaryotic cells into mitosis is driven by the activation of a cyclin-dependent kinase (Cdk) complex that in S. pombe is Cdc2 partnered with the mitotic B-type cyclin, Cdc13. As S. pombe cells achieve a critical size required for cell division, the Cdc25 protein-tyrosine phosphatase activates the complex by catalyzing Cdc2 dephosphorylation at Tyr15. This phosphorylation event is catalyzed by the Wee1 protein kinase. Cdc2 acts to both inhibit Wee1 and activate Cdc25 to allow a sharp rise in its own activity that induces mitosis. Clp1 is a member of the evolutionarily conserved Cdc14 protein phosphatase family that reverses Cdc2 phosphorylation. In a project funded by the National Institutes of Health, we found that Clp1 turns off the Cdc2 autoamplification loop by binding and dephosphorylating Cdc25. We are investigating the precise mechanism whereby Clp1 inhibits Cdc25 activity since this will be relevant to the control of Cdc25 protein activities in the G1 phase of mammalian cells.
Because it is so important to regulate Cdk activities, we infer that regulation of its opposing phosphatase will be equally relevant to proper cell cycle control. Thus, we have investigated how Clp1 activity is regulated. We found that Cdc2 phosphorylates and inhibits Clp1 during metaphase. When Cdc2 activity declines at the onset of anaphase, Clp1 is able to autodephosphorylate and achieve maximum phosphatase activity. Once active, Clp1 dephosphorylates other Cdc2 targets, contributing to a coordinated exit from mitosis. In this manner, Clp1 activation is perfectly coordinated with the decline in Cdc2 activity. Clp1 is also a target of the septation initiation network (SIN) that induces the onset of cytokinesis. The SIN kinase, Sid2, phosphorylates Clp1 directly, promoting its cytoplasmic retention during anaphase. Because Cdc2 activity inhibits cytokinesis, promoting Clp1 activity at the site of division overcomes Cdc2-mediated inhibition and drives cytokinesis forward.
Clp1 localizes to many subcellular compartments, such as kinetochores, the mitotic spindle, and the division site, where it antagonizes Cdk1 activity. The mechanism by which Clp1 functions at these distinct sites is unclear, however. We employed a proteomic approach to identify a large number of Clp1-interacting proteins and have been characterizing them. One identified protein is Mid1, an anillin-related protein required for correct cytokinetic actin ring (CR) positioning. We found that Mid1 recruits Clp1 to the CR. By constructing and evaluating a mid1 mutant selectively unable to recruit Clp1, we identified the functional consequences of Clp1 activity at the CR, including complete dephosphorylation of the essential CR component Cdc15, which stabilizes the dynamic properties of both Cdc15 and myosin II. Our findings explain why Clp1 is required to ensure the fidelity of cytokinesis and more broadly indicate how the mutual antagonism of Cdc2 and Clp1/Cdc14 phosphatase fine-tunes the timing of cytokinesis.
Cytokinesis is the final event of the cell cycle. It is regulated temporally and spatially such that a barrier is formed between replicated and segregated chromosomes. One major event in cytokinesis is the reorganization of the cell's actin cytoskeleton to form the CR. One protein linked to ring establishment is Cdc15, and we have been studying its contribution to this process. We found that Cdc15 binds and recruits members of both the Arp2/3-dependent and the formin-dependent actin nucleation pathways to the medial region of the cell. Cdc15 also binds the membrane via its F-BAR domain. These functions are phosphorylation dependent, and we have mapped more than 20 phosphorylation events on Cdc15 that appear to regulate its localization and binding interactions. Mutants in key phosphorylation sites begin to organize the ring prematurely, indicating the importance of strict temporal control of Cdc15 phosphorylation. Additionally, the binding activities of Cdc15 that occur through its F-BAR domain are not sufficient for proper cytokinesis. We found that the Cdc15 SH3 domain is also important in recruiting proteins to the contractile ring to add to its structural integrity. Using two-hybrid and proteomic strategies, we identified a set of proteins that together prevent fragmentation of the CR during its constriction. Our results have begun to unravel the complex web of protein-protein and protein-membrane interactions necessary for proper CR formation and constriction, which in turn are necessary for cytokinesis.
Formation and constriction of the CR also requires a GTPase-signaling pathway, the SIN. The SIN and molecules that regulate its activity are assembled in a signaling center at the spindle pole body (SPB) on the scaffold proteins, Sid4 (septation initiation defective) and Cdc11. How information flows through the SIN is incompletely understood. We have found evidence for a positive amplification loop, whereby the final SIN kinase phosphorylates its scaffold to help recruit an intermediate kinase more efficiently. Additionally we have obtained evidence that the scaffold is modified through ubiquitination, as a means of inhibiting cytokinesis before chromosomes are completely segregated. These results provide a better framework for understanding the coordination of cytokinesis with chromosome segregation.
We had discovered some years ago that many aspects of the cell cycle are orchestrated from the SPB. However, what constitutes the SPB or how its surface organizes signaling modules, such as the SIN, is not understood. Through proteomics, we identified a novel essential SPB protein, Ppc89, that appears to be the core constituent of the SPB. Overproduction of Ppc89 induces outgrowth of the SPB and consequent accumulation of all of its components; its depletion results in loss of the SPB. We are studying how Ppc89 interacts with other SPB components, through proteomics and fluorescent resonance energy transfer (FRET). This analysis will lead to a model of signaling compartmentalization.
Another mechanism important for mitotic exit and cytokinesis is regulated proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that mediates polyubiquitination and degradation of key cell cycle regulators to drive cells through and out of mitosis. We purified the mitotic APC/C and, in collaboration with Thomas Walz (Harvard Medical School), used cryoelectron microscopy and single-particle reconstruction techniques to determine its three-dimensional structure. We then used antibodies to epitope-tagged components to map the localization of 12 of the 13 core components and the APC/C activator, Slp1, to provide the first structural overview of APC/C organization. We are continuing these strategies to improve the resolution of the structure and also using cross-linking, mass spectrometry, and electron microscopy approaches to define where within the structure activators, substrates, and inhibitors bind.
Last updated March 13, 2009
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