Recent developments in the application of confocal microscopy to the study of intracellular Ca2+ imaging have demonstrated the existence of complex patterns of intracellular Ca2+ release generated in Xenopus oocytes by the activation of hormone receptor pathways. In particular, the patterns of Ca2+ foci and spiral waves of Ca2+ observed, behaved in a fashion consistent with the notion that intracellular Ca2+ release is an excitable process. Studying these patterns will generate information about the underlying cellular machinery which contributes to Ca2+ signalling. The relevance of the proposed studies to the field of Ca2+ signalling lies in the fact that with great spatial and temporal resolution one can study discrete entities (i.e. Ca2+ foci and/or spiral Ca2+ waves) which fill a gap in the understanding of the steps that follow hormone receptor activation to Ca2+ sensitive effectors downstream in the cascade of cellular signal events. Precise knowledge of when and where intracellular cellular Ca2+ is mobilized is relevant to all enzymatic processes which use Ca2+ as their messenger (e.g. Ca2+ - dependent kinases, phosphatases, proteases). This proposal presents evidence that the production of inositol trisphosphate (IP3), the activation of the IP3 receptor (IP3R) and Ca2+ itself, are critical to the formation of Ca2+ release patterns. A multidisciplinary approach which uses techniques of molecular biology, confocal imaging, and electrophysiology is proposed. Simultaneous confocal Ca2+ imaging and immunofluorescence will be used to determine the distribution of Ca2+ activity patterns in relation to the localization of the IP3R and to the Ca2+ -ATPases of the endoplasmic reticulum (ER). Ca2+ pattern formation will be studied by manipulating the expression levels of the IP3R, over- expressing Ca2+ -ATPases to manipulate the size of the Ca2+ stores, and by manipulating the production of IP3 by expression of different hormonal receptor pathways. These components of the system are more likely to have an impact in the formation of Ca2+ patterns. The Xenopus oocyte translation system has been chosen because of the ease with which foreign cDNAs are introduced and expressed, and because confocal Ca2+ imaging in these preparation has yielded excellent temporal and spatial resolution in the imaging of Ca2+ patterns.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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University of Virginia
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