Three-dimensional reconstruction of abscission

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Abscission is the final stage of cytokinesis, when the narrow intercellular bridge connecting the two post-mitotic daughter cells is severed. Recently, we discovered helices of ESCRT-III-dependent fila-ments forming within the intercellular bridges of HeLa cells that could drive cortical constriction during abscission. The first goal of this research proposal is to study the relative contributions of CEP55 and ALIX, two potential upstream targeting components of the ESCRT-III complex, and Spastin, to abscission in HeLa cells using electron tomography. The second goal is to extend our analysis of abscission to the early Caenorhabditis elegans embryo. We want to answer the basic question, is our suggested model of abscission conserved in other systems? Using the worm, we propose to generate reference 3D models of intermediate stages of abscission in wild-type embryos, characterize the role of C. elegans ESCRT-III components by RNA-mediated interference (RNAi), and perform electron tomographic analyses of staged mutant embryos. It is anticipated that this comparative approach will lead to further fundamental structural and mechanistic insights into the process of abscission.