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Regulation of Cell Division
Summary: Kathleen Gould is interested in understanding how cells divide. Her laboratory utilizes the genetically tractable yeast, Schizosaccharomyces pombe, as a model organism to study the molecular mechanisms of mitotic exit.
One fundamental decision a cell makes is whether to proliferate. If a cell commits to the division pathway, a highly coordinated series of events is set into motion. Each cell division cycle consists of DNA replication (S phase); the separation of the duplicated chromosomes and cellular constituents in mitosis (M phase); two gap phases: G1, which occurs prior to S phase, and G2, which occurs prior to M phase; and the final event, cytokinesis. Strict regulation of the initiation and completion of S and M phases ensures that genetic information and other critical cellular components are duplicated and divided evenly between daughter cells with each cell cycle.
My laboratory is interested in understanding the molecular mechanisms that regulate cell division. We use 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 and S. pombe 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 have also investigated how Clp1 activity itself is regulated. We found that Cdc2 phosphorylates and inhibits Clp1 during metaphase. At the onset of anaphase, decreased Cdc2 activity allows Clp1 to autodephosphorylate and achieve maximum phosphatase activity. Once active, Clp1 dephosphorylates other Cdc2 targets, contributing to a coordinated exit from mitosis. 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.
Clp1 localizes to many subcellular compartments, such as kinetochores, the mitotic spindle, and the division site, where it is involved in several facets of cell cycle control. 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 is necessary for Clp1 localization to the CR. We identified the functional consequences of Clp1 activity at the CR, including complete dephosphorylation of the essential CR component Cdc15 and regulation of the dynamic properties of both Cdc15 and myosin II to provide stability to this structure. Our findings explain why Clp1 is required to ensure the fidelity of S. pombe cytokinesis.
Another mechanism important for mitotic exit 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.
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 of the first events in cytokinesis is the reorganization of the cell's actin cytoskeleton to form the CR. One protein linked to ring establishment is Cdc15. Cdc15 interacts directly through its F-BAR domain with members of both the Arp2/3-dependent and the formin-dependent actin nucleation pathways and is required for their medial recruitment during mitosis. It also binds the membrane via its F-BAR domain. However, these activities of Cdc15 are not sufficient for proper cytokinesis. We have found that the Cdc15 SH3 domain is also important. Using two-hybrid and proteomic strategies, we have identified at least two proteins that bind Cdc15 SH3 and influence CR organization. Our results suggest that a complex web of protein-protein and protein-membrane interactions contributes to proper CR formation and constriction, which in turn are necessary for cytokinesis.
In unrelated studies, we identified and characterized Cdc5 as an essential pre-mRNA-splicing factor and component of the nineteen complex (NTC). The NTC joins with small nuclear ribonucleoprotein subunits (snRNPs) just prior to the first catalytic step of pre-mRNA splicing, the excision of noncoding introns from a pre-mRNA. However, how snRNPs and the NTC are organized within a larger unit to execute the catalytic steps of pre-mRNA splicing is not known. Using a tandem affinity purification (TAP) epitope on Cdc5, we purified a stable 37S form of the S. pombe spliceosome that contains most known U2 and U5 snRNP proteins; the U2, U5, and U6 snRNAs; numerous uncharacterized splicing factors that we identified by mass spectrometric analysis; and lariat intermediate RNAs. In a collaboration with Thomas Walz funded by the National Institutes of Health, we used cryoelectron microscopy and single-particle reconstruction techniques to determine the three-dimensional structure of this complex. This structure provides a first view of the overall organization of a spliceosome at an intermediate step in the splicing reaction.
Last updated: December 6, 2007
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