Asymmetric cell division, in which the two daughter cells adopt different fates, plays a key role in embryonic development and in tissue regeneration in adult life. The goal of this project is to obtain detailed molecular information about intrinsically asymmetric cell divisions using the nematode C. elegans as a model system. The research addresses two general questions: How are intracellular asymmetries established? How are mitotic spindles aligned along the axis of asymmetry? The research focuses on the six PAR proteins, which are required for a series of reproducible asymmetric divisions in the lineage leading to the C. elegans germline. Mutation of these proteins disrupts both polarized distributions of cellular components and proper alignment of mitotic spindles in the early embryo. Four of the PAR proteins and an interacting protein, PKC-3, are localized to the cell periphery at the poles of asymmetrically dividing cells, defining discrete cortical domains in the anterior (PAR-3, PAR-6 and PKC-3) and in the posterior (PAR1- and PAR-2). The proposed experiments address three broad and overlapping questions: How do the PAR proteins become localized? How do the PAR proteins influence the asymmetric distribution of other molecules? How do the PAR proteins contribute to spindle orientation? Specific aims include 1) Determination of the functional relationships between PAR-3, PAR-6, PKC-3 and two other proteins that influence their activity, PAR-5 and Cdc-42. In this aim a combination of in vitro binding studies and in vivo tests of mutations in binding domains will lead to insights into molecular mechanisms for PAR protein action. 2) Identification of additional components of the polarity system. 3) Identification of substrates of the PAR-1, PAR-4 and PKC-3 serine/threonine kinases. Because the PAR proteins are only a small part of the polarity system, understanding their mechanism of action requires the identification of proteins that influence their activities or which they influence. In these two aims, a combination of reverse genetics, protein interaction screens, biochemical fractionation and kinase assays will result in the discovery of some of these proteins. The recent discovery of broad conservation of PAR-1, PAR-3 and PAR-6 in polarity systems in flies and mammals indicates that our analysis of the PAR proteins will provide basic information that will be applicable to studies of human growth and development.
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