CAREER:Regulation of Membrane-Cytoskeletal Dynamics during Cytokinesis

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

This is a five-year CAREER award. Intellectual merit: Cytokinesis is achieved by the formation of a cleavage furrow in anaphase that bisects the mitotic spindle between the separated chromosomes, cytoplasm and organelles. The goal of this research activity is to gain insight into the mechanisms required to establish cleavage furrows, identify factors required to regulate furrow formation and to determine how DIP-1, a novel dynamin/DYN-1 interacting protein, coordinates this process. The underlying hypothesis is that the cleavage furrow is established by clustering of lipid raft proteins by an unknown signal(s) from the spindle. This signal triggers DIP-1 to regulate the targeting and assembly of dynamin/DYN-1, a known raft component, at the equatorial membrane of the cell during the metaphase-anaphase transition. Dynamin/DYN-1 subsequently directs actin filament formation, which assemble into the acto-myosin ring and in turn, promotes cleavage furrow invagination and completion. This hypothesis is based on the observations that: 1) The target of the signal for cytokinesis is the equatorial cortex; 2) DYN-1 localizes to equatorial membranes and newly formed cleavage furrows; 3) Dynamin is a known lipid raft component; 4) Dynamin can influence actin assembly; and 5) DIP-1, like DYN-1, regulates the establishment of cleavage furrow formation as well as completion. Based on these observations, the experimental focus of this project is on the role of DIP-1 in establishing the cleavage furrow and regulating DYN-1 function during cytokinesis in animal cells, using the nematode, C. elegans, as a model. Preliminary studies in dip-1 (RNAi) embryos show that (i) the spindle midzone microtubules assemble properly, but buckle and disappear during late anaphase (60%); (ii) the formation of cytoplasmic, spherical tubulin::GFP aggregates are observed (40%); (iii) the actin cortex is disorganized and membrane ruffling during cytokinesis is almost absent; and (iv) DIP-1 contains a BRCT motif commonly used to mediate interactions with other BRCT motif-containing proteins, such as BRCA2 and ECT2, known cytokinesis proteins. The strategy of this study is to: 1) Determine the cellular function of DIP-1; 2) Characterize the membrane-cytoskeletal consequences of DIP-1 depletion during cytokinesis; 3) Determine if DIP-1 directly binds to DYN-1; and 3) Determine the cell cycle localization of DIP-1::GFP and identify protein motifs and additional factors that regulate DIP-1::GFP localization during mitosis. These studies should provide new insight into the regulation and function of the membrane-cytoskeletal events that occur during cytokinesis with an understanding of the specific role of DIP-1 in this process. Broader Impact: This CAREER project also involves a significant, integrated educational component, designed to benefit both the students involved directly with the work and for the public, especially for local Native American communities. The high school, undergraduate and graduate students involved in or introduced to this research will gain hands-on experience using in vivo microscopy techniques in conjunction with genetics, biochemistry and molecular biology. Since live movies of cell division in C. elegans embryos are quite exciting and easy to comprehend, Dr. Skop's work is especially welcoming to all educational backgrounds. Since "systems biology" will be taught and performed in the laboratory, the introduction to these techniques will be skills that are highly sought after in the fields of genomics, proteomics and biology. Information about Dr. Skop's work and her "systems biology" course (Genetics 875) will be disseminated to the academic and high school communities through online resources related to her laboratory and courses.