Imaging Ca2+, Membrane Potential and Exocytosis in Mast Cells

免疫球蛋白E受体在肥大细胞质膜上的聚集导致胞浆内钙离子的增加，从而导致分泌颗粒内容物的胞吐。然而，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。在这项建议中，将使用定量数字图像增强荧光显微镜来成像单个肥大细胞的胞浆游离钙、膜电位和胞吐作用。许多方法将被用来确定观察到的肿瘤肥大细胞对抗原刺激的个体钙反应随机变化的生理学基础。我们还将评估细胞内钙离子的内流和释放对这些反应的相对贡献。我们将探讨响应于质膜离散区域的刺激而产生局部的钙离子增加和瞬时梯度的可能性。荧光指示剂Fura-2和其他荧光探针将用于这些研究，以测量单个细胞的膜电位和胞吐反应的变化。这些实验将对理解分泌过程中涉及的离子机制非常重要。

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.