The long-term goal is to understand the molecular mechanisms of calcium (Ca2+) wave formation and of the regulation of cellular functions by these waves. Critical cellular events such as granule exocytosis, cytoskeletal rearrangement and gene transcription are regulated by Ca2+ waves. The Ca2+ fertilization wave of Xenopus laevis is widely used as an experimental model system to study how these waves are generated and how they control cellular activities. Several hypotheses exist as to how the intracellular messengers inositol (1,4,5)-trisphosphate (IP3) and Ca2+ are involved in the initiation and propagation of Ca2+ waves. The proposed studies will test these hypotheses of the roles of IP3 and Ca2+ in the formation of the Xenopus Ca2+ fertilization wave. The principal investigator has developed a novel technology that combines capillary electrophoresis with a biological IP3 detector to measure IP3 concentrations in single cells. This extraordinarily sensitive method quantitates IP3 concentrations in small, selected regions of cytoplasm. The current detection limits for the system are l00 nM of IP3 in 10 nl of cytoplasm (1% of the volume of a Xenopus oocyte or egg). The application of this strategy to single cells will achieve physiological measurements not previously possible. For the first time, the concentration of IP3 will be measured at the subcellular level. To determine how IP3 and Ca2+ initiate the Ca2+ fertilization wave, the concentrations of these messengers will he measured simultaneously in small regions of individual cells. Since IP3 degradation terminates the Ca2+ signal, the rates and pathways for IP3 degradation will be measured in these cells. The research will then address the mechanism of wave propagation by mapping the concentrations of IP3 and Ca2+ as the fertilization wave traverses the cell. The results of this research will establish fundamentally important knowledge of the fertilization and development of biological organisms.
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