Cytokinesis, the physical division of one cell into two daughter cells, is the final stage of the cell reproductive cycle and the least well understood. Correctly timing the process of cytokinesis so that it occurs only after chromosome replication and segregation is necessary to prevent catastrophic genomic instability and accordingly, cytokinesis is strictly regulated in concert with other events of the cell cycle. We have made significant progress in identifying and characterizing proteins essential for cytokinesis using a leading model organism for cytokinesis studies, the fission yeast Schizosaccharomyces pombe. We now propose to gain a better understanding of how these myriad proteins work together and under cell cycle control to mediate successful cell division. We will focus on two essential, conserved proteins that are necessary for assembling an actomyosin-based contractile ring that is used to pinch cells in two - 1) Cdc15 and 2) Cdc12. Cdc15 is a scaffold of the contractile apparatus and it links the actomyosin contractile ring to the plasma membrane through its F-BAR domain. We found that bulk dephosphorylation of Cdc15 at mitotic entry induces a conformational switch in the protein that allows it to oligomerize, bind the membrane and act as a stable membrane-anchored scaffold for cytokinetic ring assembly. We will now determine how Cdc15 interacts with membranes, how it oligomerizes to form a scaffold structures, and how it might influence the membrane at the division site guided by a newly obtained X-ray crystal structure of its F-BAR domain. We will study how Cdc15 is coordinated with a second F-BAR protein to achieve the proper coupling between the membrane and actin polymerization during cytokinesis. Cdc12 is the formin that nucleates the F-actin of the contractile ring. How formins are deployed for cytokinesis is incompletely understood also, and we will test the hypothesis that transient mitotic phosphorylation of the cytokinetic formin Cdc12 relieves its autoinhibition so that it functions precisely at the correct cell cycle stage. We will complement these focused mechanistic studies with proteomic and large-scale genetic screens designed to establish a functional interaction network of cytokinesis components. Although some of the details will vary between organisms, these studies will have a major impact for understanding how cytokinesis is orchestrated in multiple species including humans.

Public Health Relevance

Correctly timing the process of cytokinesis so that it occurs only after chromosome replication and segregation is necessary to prevent catastrophic genomic instability. We have uncovered a primary mechanism that governs when the cytokinetic ring forms and a further understanding of this key mechanism will influence the search for cancer therapeutics aimed at manipulating the coordination of cell cycle events.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM101035-01
Application #
8271883
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Hamlet, Michelle R
Project Start
2012-06-01
Project End
2016-03-31
Budget Start
2012-06-01
Budget End
2013-03-31
Support Year
1
Fiscal Year
2012
Total Cost
$285,161
Indirect Cost
$96,411
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
McDonald, Nathan A; Gould, Kathleen L (2016) Characterization of Cytokinetic F-BARs and Other Membrane-Binding Proteins. Methods Mol Biol 1369:181-9
McDonald, Nathan A; Takizawa, Yoshimasa; Feoktistova, Anna et al. (2016) The Tubulation Activity of a Fission Yeast F-BAR Protein Is Dispensable for Its Function in Cytokinesis. Cell Rep 14:534-46
Makarova, Maria; Gu, Ying; Chen, Jun-Song et al. (2016) Temporal Regulation of Lipin Activity Diverged to Account for Differences in Mitotic Programs. Curr Biol 26:237-43
Chen, Jun-Song; Beckley, Janel R; Ren, Liping et al. (2016) Discovery of genes involved in mitosis, cell division, cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains. Yeast 33:507-17
Ren, Liping; Willet, Alaina H; Roberts-Galbraith, Rachel H et al. (2015) The Cdc15 and Imp2 SH3 domains cooperatively scaffold a network of proteins that redundantly ensure efficient cell division in fission yeast. Mol Biol Cell 26:256-69
Willet, Alaina H; McDonald, Nathan A; Gould, Kathleen L (2015) Regulation of contractile ring formation and septation in Schizosaccharomyces pombe. Curr Opin Microbiol 28:46-52
McDonald, Nathan A; Vander Kooi, Craig W; Ohi, Melanie D et al. (2015) Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis. Dev Cell 35:725-36
Willet, Alaina H; McDonald, Nathan A; Bohnert, K Adam et al. (2015) The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12. J Cell Biol 208:391-9
Ullal, Pranav; McDonald, Nathan A; Chen, Jun-Song et al. (2015) The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles. J Cell Biol 211:653-68
Chen, Jun-Song; Beckley, Janel R; McDonald, Nathan A et al. (2014) Identification of new players in cell division, DNA damage response, and morphogenesis through construction of Schizosaccharomyces pombe deletion strains. G3 (Bethesda) 5:361-70

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