We propose to use the powerful genetics and the impressive cytological properties of the early C. elegans embryo to investigate cytokinesis and its relationship to mitotic spindle orientation in a developing animal. The early C. elegans embryo offers two key advantages for these studies: (i) the ability to rapidly identify genes required for cytokinesis and for mitotic spindle orientation in early embryonic cells, and (ii) the ability to visualize with high resolution the subcellular localization of functionally important proteins in the large (approximately 22 x 55 micron) 1-cell stage zygote. We have three long term goals: (1) To use genetic and molecular methods to define cytokinesis as a series of discrete molecular interactions that execute cytokinesis in the early embryo. (2) To determine the mechanistic relationship between the termination of cytokinesis and the mechanisms that orient mitotic spindles during asymmetric divisions in early embryonic cells. (3) To identify motor proteins important for cytokinesis and the generation of asymmetric cell divisions. These studies will provide significant insight into the molecular basis for human pathologies: cytoskeleton/plasma membrane interactions have proven relevant to our understanding of cancer and of other significant diseases, including muscular dystrophy, deafness, and sterility. In preliminary studies, we have identified a gene called cyk-1 that is required for a late step in cytokinesis. This is the first gene identified in C. elegans that is specifically required for cytokinesis in the early embryo. Intriguingly, the CYK-1 protein localizes to the leading edge of the cleavage furrow late in cytokinesis, where we hypothesize it bridges the actin and tubulin cytoskeleton. While CYK-1 provides a starting point for identifying functionally protein/protein interactions that occur during cytokinesis, we first propose to identify as comprehensively as possible the genes required for cytokinesis and mitotic spindle orientation. To this end, we have begun a large-scale screen for temperature-sensitive, embryonic-lethal mutants, and we are using a functional genomics approach that involves the use of a recently discovered technology called RNA interference. We will molecularly clone genes that are most specifically required for cytokinesis and mitotic spindle orientation. By using genetic and molecular epistasis experiments, and by examining how the different proteins we identify interact, we will define the molecular interactions and pathways that control these fundamental cellular and developmental processes.
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