SPATIAL AND TEMPORAL ASPECTS OF INSP3 SIGNALING

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Inositol 1,4,5-trisphosphate (InsP3) is utilized by virtually all cells as an intracellular messenger within a signaling pathway controlling many diverse functions including neurotransmitter, hormone and growth factor responses, secretion and muscle contraction. Disruptions of this pathway have been implicated in disorders including manic depressive illness, tumorigenesis and teratogenesis, and the relevance of this system to clinical studies will certainly grow as we come to understand it better. It is well established that InsP3 functions principally by liberating Ca2+ ions sequestered within intracellular stores. Furthermore, advances in techniques for monitoring cytosolic Ca2+ have revealed great complexities in the patterns of its liberation; Ca2+ may be released as local 'puffs', or as repetitive circular and spiral waves. These spatial and temporal aspects of InsP3 signaling are undoubtedly important for determining whether Ca2+ signals remain localized to sub-cellular regions or act globally, for the 'digital' encoding of information as frequency of repetitive waves, and for propagation of signals within and between coupled cells as Ca2+ waves. In the presence of InsP3 the cell cytoplasm acts as an excitable medium, formed from multiple discrete and autonomous sites that release 'quanta' of Ca2 in a regenerative manner in response to dual positive and negative feedback by Ca2+. Ca2+ waves are thus akin to a chemical action potential, and their characteristics will be determined by three factors; the functional properties of release sites, the ability of Ca2+ ions to act as diffusible messengers within and between sites, and the spatial organization of release sites. Our overall goals are to study each of these aspects, with the aim of elucidating how they contribute to the final Ca2+ dynamics evoked by InsP3 signaling. We will use Xenopus oocytes as a convenient and well characterized model cell system, utilizing non- metabolizable InsP3 analogues and photolysis of caged InsP3 to evoke Ca2+ liberation that will then be monitored with high spatial and temporal resolution by video-rate confocal microscopy. Functional studies of release sites will include the roles of Ca2+ feedback in evoking graded and regenerative release, the stochastic triggering of regenerative responses, processes underlying activation and inactivation of release, and variability between release sites. The mobilities and range of action of InsP3 and Ca2+ will be determined in intact cells, and we will study the effects of endogenous and exogenous buffers on Ca2+ diffusion and consequent changes in dose-dependence and dynamics of puffs and waves. High resolution imaging of spontaneous Ca2+ puffs and 'hot spots' evoked by photoreleased InsP3 will allow mapping of the three-dimensional distribution and morphology of Ca2+ release sites, and their presence will be correlated with that of InsP3 receptors and e.r. structure. Finally, in collaboration with theoretical groups, these quantitative data will be used to model the effects of Ca2+ mobility and the spatial organization of discrete release sites on the cellular dynamics of InsP3-mediated Ca2+ signaling.