Imaging Ca2+, Membrane Potential and Exocytosis in Mast Cells

肥大细胞质膜中免疫球蛋白E受体的聚集导致细胞质Ca2+的增加和随后分泌颗粒内容物的胞外分泌。然而，在单个细胞中参与这些反应的生化和生理机制仍然知之甚少。在这个建议中，定量数字图像增强荧光显微镜将用于成像细胞质游离电离钙，膜电位和胞外分泌在单个肥大细胞。许多方法将被用来确定在肿瘤肥大细胞对抗原刺激的个体Ca2+反应中观察到的随机变化的生理基础。Ca2+内流和Ca2+从细胞内储存的释放对这些反应的相对贡献也将被评估。Ca2+的局部增加和瞬态梯度的可能性是响应于质膜的一个离散区域的刺激而产生的。这些研究将使用荧光指示剂fura-2和其他荧光探针来测量单个细胞的膜电位变化和胞外反应。这些实验对于理解参与分泌的离子机制将是重要的。

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.