The liberation of Ca2+ from intracellular stores into the cytosol 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 imaging technology has revealed that Ca2+ liberation through inositol trisphosphate receptor/channels (IP3R) occurs discontinuously, as a hierarchy of Ca2+ signals involving single channels ('fundamental' events) and concerted openings of multiple channels ('elementary' events). These transient, localized free [Ca 2+] elevations arise through IP3R clustered at discrete functional release sites on the endoplasmic reticulum. Individual sites 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 that 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 pathological states. Our overall goals are to elucidate how cells generate the hierarchy of IP3-mediated 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. By utilizing advanced biophotonic tools - including confocal, multiphoton and total internal reflection microscopy, and photoreleased IP3 - we aim to: (i) Develop improved optical techniques so as to image Ca2+ flux through individual channels within the intact cell. (ii) Elucidate how the activity of IP3R at a release site is orchestrated to generate elementary Ca2v signals. (iii) Determine how cellular Ca2+ buffers modulate the coordination between release sites to generate global Ca2+ signals. (iv) Explore the principles by which spatio-temporal patterning of Ca 2+ transients encodes specific and selective cell signals. (v) Investigate the roles of the IPa/Ca2+ messenger pathway in neuronal signaling and in the pathogenesis of AIzheimer's disease.
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