INTRACELLULAR DYNAMICS OF CALCIUM SIGNALS AND EXOCYTOSIS

钙信号和胞吐作用的细胞内动力学

• 批准号: 7956413
• 负责人: BERTIL HILLE
• 金额: $ 1.63万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956413
• 关键词: Animals; Calcium Signaling; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; Confocal Microscopy; Electric Capacitance; Endoplasmic Reticulum; Epithelial Cells; Exocytosis; Fluorescence Resonance Energy Transfer; Funding; Grant; Inositol; Institution; Ions; Kinetics; Mammalian Cell; Measurement; Measures; Metabolism; Microscopy; Modeling; Organelles; Pancreatic duct; Pheochromocytoma; Photometry; Physiological; Physiology; Production; Proteins; Regulation of Exocytosis; Research; Research Personnel; Resources; Role; Secretory Vesicles; Signal Transduction; Source; Techniques; Testing; Transfection; United States National Institutes of Health; Work; fluorescence imaging; inositol-1,4,5-trisphosphate 5-phosphatase; patch clamp; ratiometric; receptor; research study; response; reuptake; virtual

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 钙信号传导和胞吐作用的调节是所有动物细胞生理学的核心问题。我们通过在电兴奋性和非兴奋性哺乳动物细胞系（PC12 嗜铬细胞瘤细胞、tsA 上皮细胞和胰管上皮细胞）中进行生物物理实验，寻求对此类信号传导的定量理解。两个长期假设指导这项工作：（a）Ca2+清除和胞吐作用的调节在不同细胞中采取不同的形式，并根据每个细胞的生理作用进行调整； (b) 几种细胞内细胞器对细胞 Ca2+ 动力学做出了重大贡献。本资助期的目标是： (1) 检验分泌颗粒积累和释放 Ca2+ 可以对生理反应期间细胞 Ca2+ 信号传导做出重大贡献的假设。 (2) 测量受体诱发的肌醇 1,4,5, 三磷酸 (IP3) 升高的幅度，并检验以下假设：IP3 5-磷酸酶快速代谢 IP3，随后将 Ca2+ 快速重新摄取到内质网 Ca2+ 库中，从而终止通过 IP3 的 Ca2+ 信号传导。 (3)检验细胞骨架轨迹和快速细胞骨架重塑参与分泌颗粒从储备池动员到分泌能力池的假设。这项工作需要一系列生物物理技术，包括：离子电流膜片钳；胞吐作用的电流和电容测量；基因靶向探针、指示剂和细胞蛋白的转染；指示剂的比率光度测定和荧光共振能量转移（FRET）；视频荧光成像；全内反射显微镜（TIRF）；共焦显微镜；和定量动力学建模。 Virtual Cell 建模将关注 Ca2+ 的区室动力学以及 IP3 的产生和分解。