The entry of Ca2+ ions into the cytosol from the extracellular fluid and from endoplasmic reticulum (ER) stores is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as electrical excitability, secretion, proliferation and cell death. Improved optical technology now enables visualization of a hierarchy of Ca2+ signaling events, ranging from openings of single-channel Ca2+-permeable channels ('fundamental'events), concerted openings of clustered channels ('elementary'events) and propagating Ca2+ waves. The localized free [Ca2+] elevations arising through individual and clustered channels serve autonomous signaling functions, and their activity may further be coordinated through Ca2+ diffusion and Ca2+-induced Ca2+ release to propagate global cellular Ca2+ waves: Fundamental and elementary events thus form hierarchical building blocks underlying the complex spatiotemporal Ca2+ signals hat permit graded and selective regulation of cell functions. Elucidation of their generation, interaction and functional consequences is, therefore, pivotal to understand the physiological functioning of the ubiquitous Ca2+ messenger pathway and its involvement in disease. Our overall goals are to elucidate how cells generate the hierarchy of Ca2+ signals, how these are utilized for specific and localized regulation of effector responses, and how disruptions in the signaling pathway may be involved in disease pathogenesis. By utilizing advanced biophotonic tools - including confocal, multi-focal and total internal reflection microscopy, in conjunction with photolysis of caged second messengers and neurotransmitters we aim to: (i) Develop improved optical techniques so as to image Ca2+ flux through individual channels in the plasma membrane and ER of intact cells, (ii) Utilize simultaneous imaging of hundreds of single-channels to explore differences in their gating, and study self- and inter-channel modulation by Ca2+ microdomains. (iii) Elucidate how the activity of individual IP3R at a release site is orchestrated to generate elementary Ca2+ puffs, (iv) Investigate the hierarchy of spatio-temporal patterning of the IPs/Ca2+ messenger pathway in neuronal signaling, and in the pathogenesis of Parkinson's disease. Calcium serves a 'life or death'function in virtually all cells of the body, regulating processes as diverse as the heartbeat and synaptic transmission between brain cells and is implicated in numerous diseases including Alzheimer's and Parkinson's. Our goal is to elucidate the hierarchical mechanisms by which Ca2+ signals are generated at levels from single molecules to the whole cell, with the dual aims of better understanding their normal functioning and how disruptions in Ca2+ signaling, may lead to disease.
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