INTRACELLULAR DYNAMICS OF CALCIUM SIGNALS AND EXOCYTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 钙信号和胞吐作用的调节是所有动物细胞生理学的中心问题。我们通过在电可兴奋和不可兴奋的哺乳动物细胞系：PC12嗜铬细胞瘤细胞、TSA上皮细胞和胰管上皮细胞中的生物物理实验寻求对这种信号的定量理解。指导这项工作的两个长期假设：(A)钙离子清除和胞吐作用的调节在不同的细胞中采取不同的形式，并根据每个细胞的生理作用而调整；(B)几个细胞内细胞器对细胞内钙动力学做出重大贡献。本授权期的目的是：(1)验证分泌颗粒积累和释放钙离子在生理反应中对细胞内钙信号有重要贡献的假说。(2)测定受体诱导的三磷酸肌醇(IP3)升高的幅度，并验证通过IP3的钙信号是通过IP3 5-磷酸酶快速代谢IP3，然后迅速重新摄取钙进入内质网钙库而终止的假说。以及(3)检验细胞骨架轨迹和快速细胞骨架重塑参与了分泌颗粒从储备池到分泌能力池的动员这一假设。这项工作需要一系列生物物理技术，包括：离子电流的膜片钳；胞吐作用的安培和电容测量；基因靶向探针、指示剂和细胞蛋白质的转基因；指示剂的比率光度法和荧光共振能量转移(FRET)；视频荧光成像；全内反射显微镜(TIRF)；共聚焦显微镜；以及定量动力学模型。利用虚拟细胞进行建模将涉及细胞内钙离子和IP3的产生和分解的分室动力学。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Calcium signaling and the regulation of exocytosis are central issues in the physiology of all animal cells. We seek quantitative understanding of such signaling through biophysical experiments in electrically excitable and non-excitable mammalian cell lines: PC12 pheochromocytoma cells, tsA epithelial cells, and pancreatic duct epithelial cells. Two long-term hypotheses guide this work: (a) that Ca2+ clearance and the regulation of exocytosis take different forms in different cells and are tuned to the physiological role of each cell; and (b) that several intracellular organelles make significant contributions to cellular Ca2+ dynamics. The aims in this grant period are: (1) To test the hypothesis that accumulation and release of Ca2+ by secretory granules can make significant contributions to cellular Ca2+ signaling during physiological responses. (2) To measure the amplitude of receptor-evoked inositol 1,4,5, trisphosphate (IP3) elevations and to test the hypothesis that Ca2+ signaling via IP3 is terminated by rapid metabolism of IP3 by IP3 5- phosphatase followed by rapid reuptake of Ca2+ into the endoplasmic reticulum Ca2+ stores. And (3) To test the hypothesis that cytoskeletal tracks and fast cytoskeletal remodeling participate in the mobilization of secretory granules from reserve pools into secretion-competent pools. The work requires a range of biophysical techniques including: patch clamp of ion currents; amperometric and capacitance measurements of exocytosis; transfection of genetically targeted probes, indicators, and cellular proteins; ratiometric photometry and fluorescence resonance energy transfer (FRET) of indicators; video fluorescence imaging; total internal reflection microscopy (TIRF); confocal microscopy; and quantitative kinetic modeling. Modeling by Virtual Cell will concern the compartmental dynamics of Ca2+ and production and breakdown of IP3.