Investigating the Regulation and Structural Features of the conserved Membrane-Binding F-BAR Protein Cdc15

Vanderbilt University, Nashville TN

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Abstract

? DESCRIPTION (provided by applicant): The final step in cell replication is the physical separation of daughter cells, or cytokinesis. Animal cells divide using an actin- and myosin-based contractile ring (CR). CR assembly, constriction, and disassembly are tightly regulated to protect the genomic complement. Cytokinesis failure or inappropriate location or timing of cytokinesis can lead to cell death or aneuploidy, a state that promotes tumorigenesis. The goal of this proposal is to enhance the molecular understanding of cytokinesis using the eukaryotic fission yeast Schizosaccharomyces pombe. S. pombe is an excellent model organism to study cytokinesis because this process is evolutionarily conserved from yeast to animal cells. Furthermore, S. pombe offers many technical advantages including a gene deletion collection and easy implementation of forward and reverse genetics, biochemistry, and microscopy. Facile genetic manipulation in S. pombe has allowed for large-scale screens to identify additional cytokinetic proteins, many of which are conserved in higher eukaryotes. The proposed research investigates the regulation and molecular mechanism of an essential cytokinetic protein, Cdc15. Cdc15 is the founding member of the Pombe Cdc15 Homology (PCH) / FCH-Bin/Amphiphysin/Rvs (F-BAR) family, which comprises proteins that link membranes via F-BAR domains to actin cytoskeleton remodeling through additional functional domains. Cdc15's N-terminal F-BAR domain recruits the formin Cdc12 to the division site and binds membranes, while its non-essential C-terminal SH3 domain interacts with numerous proteins involved in robust completion of cytokinesis. Cdc15 functions are cell cycle regulated by phosphorylation of >33 serines or threonines, most of which occur in the disordered central region. The phosphorylated sites match diverse kinase consensus sequences, suggesting that multiple signaling pathways converge on Cdc15. In Aim 1, kinases and phosphatases that regulate Cdc15 will be identified by screening gene deletion or temperature-sensitive mutants. A combination of mass spectrometry, mutagenesis, genetic analysis, biochemistry and microscopy will be used to characterize how the phosphorylation status of the specific residues (de)phosphorylated by a single kinase or phosphatase affects Cdc15 activity. The second aim of this proposal employs proteomics, structure-function analysis, microscopy, biochemistry, and genetic experiments to test the hypothesis that the disordered central region binds one or more proteins that participate in cytokinesis. The combined results of these aims will identify signalin pathways that temporally and spatially regulate animal cell cytokinesis and will better characterize the molecular machinery whose integrity is essential for successful cytokinesis. This knowledge will provide foundational insight for understanding the pathogenesis of disorders resulting from cytokinetic defects.