A cell-free system for exocytosis in the beta-cell

β细胞胞吐作用的无细胞系统

• 批准号: 6811319
• 负责人: SUSANNE G STRAUB-SHARP
• 金额: $ 15.8万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-15 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6811319
• 关键词: calcium flux; calcium indicator; cell free system; cell line; cyclic AMP; diacylglycerols; exocytosis; fluorescence microscopy; glucose transport; granule; guanosine triphosphate; insulin; laboratory rat; membrane fusion; membrane proteins; membrane structure; method development; norepinephrine; pancreatic islets; protein localization; protein protein interaction; protein structure function; somatostatin; tetanus toxin; time resolved data; tissue /cell culture

DESCRIPTION (provided by applicant): The most important aim of this grant application is to set up a cell-free system to study mechanisms of exocytosis at the single granule level in the pancreatic beta-cell. With this achieved, a powerful new method will be available to study docked granules and the fundamental mechanisms of insulin exocytosis. Initially it is proposed to develop and characterize this new method to study exocytosis on membrane sheets. beta-cells attached to coverslips will be "unroofed" in the absence of Ca2+ to expose a flat layer of plasma membrane with "docked" fluorescently labelled secretory granules. When stimulated by Ca2+, many of the granules undergo exocytosis that can be monitored by changes in fluorescent markers. Initially, the clonal beta-cell line, INS 832/13, a subclone of the INS-1 cells will be used. Subsequently, the study will be extended to primary rat beta-cells fluorescently labeled by an adenovirus approach. The system will be validated by measurement of the number of exocytotic events that occur when the membrane sheets are abruptly exposed to a stimulatory Ca2+ concentration, and when they are exposed to a stimulatory Ca2+ concentration after treatment with tetanus toxin (TTX) that blocks exocytosis. Similar studies will be performed in the presence of physiological inhibitors of secretion such as norepinephrine, and potentiators of exocytosis such as cyclic AMP and diacylglycerol. Validation will require that exocytosis is stimulated, inhibited and potentiated respectively by these conditions. Optimization of the system will be achieved by studying the effects of changes in sonication conditions, e.g. sonication intensity, time and buffer composition, and by changes in the buffer conditions under which exocytosis is stimulated. Subsequently, it will be possible to study 1. the mechanisms by which stimulation, inhibition and potentiation of exocytosis occur; 2. membrane-granule and protein/protein interactions important to the control of granule tethering, docking and exocytosis; and 3. physiological mechanisms such as time-dependent potentiation and biphasic insulin release. The method will also allow the study of some aspects of endocytosis.

描述（由申请人提供）：本赠款应用的最重要目的是建立一个无细胞的系统，以研究胰腺β细胞单个颗粒水平的胞吐作用机制。实现这一目标，将可以使用一种有力的新方法来研究停靠的颗粒和胰岛素胞吐作用的基本机制。最初，提议开发和表征这种研究膜片上胞吐作用的新方法。在没有CA2+的情况下，附着在盖玻片上的β细胞将被“未盖”，以揭露带有“停靠”荧光标记为分泌颗粒的质膜平坦的层。当通过Ca2+刺激时，许多颗粒会通过荧光标记物变化来监测的胞吞作用。最初，将使用克隆β细胞系832/13，即INS-1细胞的子插锁。随后，该研究将扩展到通过腺病毒方法标记的原代大鼠β细胞荧光。该系统将通过测量膜片突然暴露于刺激性Ca2+浓度时发生的胞吐事件的数量，以及用破伤风毒素（TTX）治疗后的刺激性Ca2+浓度时发生的。在存在分泌的生理抑制剂（例如去甲肾上腺素）以及胞吞作用的增强剂（如环状AMP和二酰基甘油）的情况下，将进行类似的研究。验证将要求通过这些条件分别刺激，抑制和增强胞吐作用。通过研究超声处理条件变化的影响，例如超声强度，时间和缓冲液组成，以及刺激胞吞作用的缓冲条件的变化。随后，可能会研究1。发生刺激，抑制和增强胞吐作用的机制； 2。膜颗粒和蛋白质/蛋白质相互作用，对控制颗粒的绑扎，对接和胞吐作用很重要；和3。生理机制，例如时间依赖性增强和双相胰岛素释放。该方法还将允许研究内吞作用的某些方面。