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. This goal of this project is to obtain detailed molecular information about intrinsically asymmetric cell divisions using the nematode C. elegans ass a model system. The research addresses two general questions: 1) How are intracellular asymmetries established? 2) How are mitotic spindles aligned along the axis of asymmetry? The research focuses on the six par genes, which are required for a series of reproducible asymmetric divisions in the lineage leading ot the C. elegans germ line. Mutations in these genes disrupt both polarized distributions of cellular components and proper alignment of mitotic spindles in the early embryo. The proposed experiments address three broad and overlapping questions: 1) How do the PAR proteins contribute to intracellular asymmetry? 2) How are the PAR proteins localized? 3) With what other cellular components do the PAR proteins interact to control asymmetric cell divisions? Specific aims include: 1) Molecular cloning of par-4, par-5 and par-6 and the determination of the distribution of their protein products in the early embryo. For cloning, we will use a variety of techniques including transposon tagging, germline transformation rescue, and production of antisense RNA phenocopies. Protein distributions will be determined by immunofluorescence microscopy using antibodies raised against fusion proteins made in bacteria. 2) Determination of the functional relationships among the par genes. We will compare PAR protein distributions by double label immunofluorescence microscopy in wild type and par mutant embryos. 3) Investigations of the mechanisms of PAR protein localization and action. We will assay the distribution par mRNAs, ectopically expressed PAR proteins, and tagged PAR protein fragments to test hypotheses about localization. 4) Identification of proteins that interact with the PAR proteins. This will be achieved by a combination of genetic screens for enhancers and suppressors of par mutations and """"""""interaction cloning"""""""" using filter binding assays and the yeast two hybrid system. The recent discovery of yeast and human homologues of par-1 suggests conservation of mechanisms to establish intracellular asymmetries. Because of this we expect our analysis to provide basic information that will be applicable to studies of human growth and development.
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