 |
Split Decisions: Regulation of Cell Division
Summary: Kathy 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 primarily interested in understanding the molecular mechanisms that regulate cell division. We use the fission yeast, Schizosaccharomyces pombe, as a model organism for our studies because 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 has been 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. These attributes allow a broad range of experimental techniques to be applied with ease, including biochemistry, genetics, proteomics, and live-cell imaging.
The entrance of eukaryotic cells into mitosis is driven by the activation of a cyclin-dependent kinase (Cdk) complex. When cells achieve a critical size required for cell division, the Cdc25 protein-tyrosine phosphatase activates the complex by dephosphorylating Cdk1 at Tyr15. Tyr15 phosphorylation is catalyzed by the Wee1 protein kinase. Active Cdk1 inhibits Wee1 and activates Cdc25 to allow a sharp rise in Cdk1 activity, phosphorylation of multiple Cdk targets, and progression into mitosis. Clp1 is a member of the evolutionarily conserved Cdc14 protein phosphatase family that reverses Cdk1 phosphorylation events. 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 currently 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 already discovered that Cdc2 phosphorylates and inhibits Clp1 during metaphase. When Cdk1 activity declines at the onset of anaphase, Clp1 autodephosphorylates and achieves maximum phosphatase activity. Once active, Clp1 dephosphorylates other Cdk1 targets, contributing to a coordinated exit from mitosis. In this manner, Clp1 activation is perfectly coordinated with the decline in Cdk1 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 Cdk1 activity inhibits cytokinesis, promoting Clp1 activity at the site of division overcomes Cdk1-mediated inhibition and drives cytokinesis forward. We are currently analyzing Clp1 phosphoregulation by yet other cell cycle kinases.
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 localizes to these distinct sites is unclear, however. We employed a proteomic approach to identify a large number of Clp1-interacting proteins. 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. Using genetics, fluorescence recovery after photobleaching, and live-cell imaging, we found that Clp1 influences the dynamic properties of cytokinetic proteins. Our findings explain why Clp1 is required to ensure the fidelity of cytokinesis and more broadly indicate how the mutual antagonism of Cdk1 and Clp1/Cdc14 phosphatase fine-tunes the timing of cytokinesis. In our list of Clp1 interacting proteins, we also identified the yeast equivalent of Borealin, Nbl1, a component of the chromosome passenger complex. Our studies on Nbl1 led to the unexpected discovery that the Aurora B kinase impacts Clp1 localization during cytokinesis. We continue to study how this occurs and what the roles of other Clp1 associated proteins are.
Cytokinesis is the final event of the cell cycle. It is regulated temporally and spatially such that a barrier forms precisely between replicated and segregated chromosomes. One major event in cytokinesis is the reorganization of the cell's actin cytoskeleton to form a contractile apparatus. One protein linked to assembly of this apparatus is the F-BAR protein Cdc15, and we have been studying its contribution to this process. F-BAR domains bind and curve membranes, and other domains in F-BAR proteins, typically an SH3 domain, connect them to the F-actin cytoskeleton. Upon entry into mitosis, Cdc15 dephosphorylation triggers a conformational change that results in its oligomerization and allows interaction with binding partners. Using mass spectrometry, we identified a large number of phosphorylation sites (>30); mutation of these sites to nonphosphorylatable residues resulted in precocious Cdc15 oligomerization and the formation of pre-ring assemblies of cytokinetic proteins. This in turn led to defects in subsequent steps of cytokinetic ring construction, constriction, and disassembly.
We are currently asking which protein kinases and phosphatases contribute to Cdc15 regulation and how these affect the ability of the F-BAR domain to oligomerize and bind membrane. When activated, Cdc15 recruits members of both the Arp2/3-dependent and the formin-dependent actin nucleation pathways to the medial region of the cell. This is not, however, sufficient for F-actin nucleation, and we are asking what other regulatory events govern assembly of the cytokinetic ring. We also found that the Cdc15 SH3 domain is important for recruiting a host of proteins that contribute to the structural integrity of the contractile ring. Our studies are beginning to unravel the complex web of protein-protein and protein-membrane interactions necessary for proper formation and constriction of the division apparatus.
Formation and constriction of the CR also require 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, wherein the final SIN kinase phosphorylates its scaffold to help recruit an intermediate kinase more efficiently. We have also obtained evidence that the scaffold is modified through ubiquitination, as a means of inhibiting cytokinesis prior to chromosome segregation. These results provide a better framework for understanding the coordination of cytokinesis with chromosome segregation.
Ubiquitination of substrate proteins induces an array of specific responses, depending on the extent and architecture of the modification. Specific ubiquitin configurations elicit unique cellular responses and affect essential processes, including protein degradation, DNA repair, chromatin remodeling, endocytosis, and cell cycle regulation. Due to the vital roles of ubiquitination, this process is highly regulated and requires a cascade of three enzymes, culminating in a substrate- and site-specific modification. Likewise, cleavage of ubiquitin moieties or chains by deubiquitinating enzymes (DUBs) is tightly regulated in space and time.
We took a multifaceted approach to investigate all S. pombe DUBs, combining the determination of endogenous localizations, evaluation of in vitro activity, and proteomic analysis of protein interactions. Our analysis revealed that DUBs are present in nearly every cellular compartment, and we identified a significant number of new DUB-interacting proteins and paradigms of DUB regulation. We are currently using quantitative mass spectrometry to identify DUB targets involved in cell division.
Grants from the National Institutes of Health provided partial support for these projects.
Last updated February 01, 2011
|
|
|
|
