Imaging Ca2+, Membrane Potential and Exocytosis in Mast Cells

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Aggregation of receptors for immunoglobulin E in the plasma membrane of mast cells leads to an increase in cytoplasmic Ca2+ and the subsequent exocytosis of secretory granule contents. However, the biochemical and physiological mechanisms involved in these responses in individual cells are still poorly understood. In this proposal quantitative digital image-enhanced fluorescence microscopy will be used to image cytoplasmic free ionized calcium, membrane potential and exocytosis in individual mast cells. A number of approaches will be used to determine the physiological basis for the stochastic variations observed in the individual Ca2+ responses of tumor mast cells to antigen stimulation. The relative contributions of Ca2+ influx and release of Ca2+ from intracellular stores to these responses will also be assessed. The possibility that localized increases and transient gradients of Ca2+ are generated in response to stimulation of a discrete region of the plasma membrane will be explored. The fluorescent indicator fura-2 and other fluorescent probes will be used in these studies to measure changes in membrane potential and exocytotic responses of individual cells. These experiments will be important in understanding the ionic mechanisms involved in secretion.