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 powerful 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 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 oliogomerizes on membranes, and how other contractile ring components are organized around this scaffold using sophisticated microscopy approaches. Cdc12 is the formin that nucleates the F-actin of the contractile ring but how it is regulated is unclear. We defined new functional domains and phosphoregulation within Cdc12 and here we will test whether these additional domains and phosphorylation events modulate the ability of Cdc12 FH1-FH2 to nucleate, possessively elongate, and bundle F-actin using spontaneous assembly and sedimentation assays, and multi-color single molecule TIRF imaging with dual labeling of actin and Cdc12 fragments. We will also test our hypothesis that polarity kinases inhibit the establishment of the Cdc15 scaffold at cell ends, forcing it to assemble in the cell middle. 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 and where 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 #
2R01GM101035-05
Application #
9103774
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Melillo, Amanda A
Project Start
2012-06-01
Project End
2020-04-30
Budget Start
2016-06-01
Budget End
2017-04-30
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37240
Willet, Alaina H; Bohnert, K Adam; Gould, Kathleen L (2018) Cdk1-dependent phosphoinhibition of a formin-F-BAR interaction opposes cytokinetic contractile ring formation. Mol Biol Cell 29:713-721
Neitzel, Leif R; Broadus, Matthew R; Zhang, Nailing et al. (2018) Characterization of a cdc14 null allele in Drosophila melanogaster. Biol Open 7:
Snider, Chloe E; Willet, Alaina H; Chen, Jun-Song et al. (2017) Phosphoinositide-mediated ring anchoring resists perpendicular forces to promote medial cytokinesis. J Cell Biol 216:3041-3050
McDonald, Nathan A; Lind, Abigail L; Smith, Sarah E et al. (2017) Nanoscale architecture of the Schizosaccharomyces pombe contractile ring. Elife 6:
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 26:237-243
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 14:534-546
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; 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
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
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

Showing the most recent 10 out of 15 publications