PKC--CYTOPLASMIC REGULATOR OF EARLY DEVELOPMENT

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Understanding the biology of reproduction requires understanding of processes which occur at key developmental transitions such as meiotic resumption, fertilization, and gastrulation. One of the most widely-conserved aspects of these processes are dynamic remodelings of cellular architecture. Indeed, dramatic changes in cell architecture are intrinsic to virtually all developmental processes, both normal (eg., fertilization, organogenesis, and cell differentiation) and pathological (e.g. neoplasia, metastasis, and teratogenesis). The means by which such changes in cellular structure are orchestrated, however, are poorly understood, in spite of the benefits such understanding would yield to treatment of birth defects and cancer. The long term goal of the proposed research is to characterize the biochemical mechanisms by which protein kinase C (PKC) regulates changes of cell structure that accompany early amphibian development. PKC, an intracellular signalling enzyme, has been firmly implicated in tumorigenesis in humans and other organisms, thus, elucidation of its role in amphibian development is likely to have broad relevance. The amphibian system also has important advantages over other systems. First, an extensive literature documents the timing of the architectural changes and the intracellular signalling events which accompany early amphibian development, therefore, potential correlations between the two can be readily discerned. Second, the large size of amphibian oocytes and the large quantity obtainable from a single animal makes the system very practical for biochemical and ultrastructural studies. The proposed studies will exploit these advantages to define biochemical and molecular mechanisms involved in PKC-mediated events during the early development of Xenopus laevis. The experiments described in this proposal are designed to answer the following questions: 1) Does PKC mediate crucial changes in cytoplasmic architecture during the resumption of meiotic maturation and egg activation? 2) What are the endogenous substrates of PKC and when during unperturbed development are they phosphorylated? 3) What are the intracellular locations of PKC substrates? 4) When, during unperturbed development, do potential PKC activating events occur? 5) What are the spatial and temporal distributions of endogenous PKC activators and inhibitors? 6) Does phosphorylation of myosin or myosin light chains (MLC) by PKC mediate recruitment of myosin into the cortical network and induce cortical contraction? 7) Does phosphorylation of myosin or MLC by PKC regulate the formulation of the contractile ring? These questions encompass both biochemical and mechanistic cytological aspects of cell structure and should therefore provide considerable insight into the mechanisms by which a specific intracellular signalling molecule, PKC, mediates changes in cell structure.