Regulated Exocytosis of Lysosomes

溶酶体的调节胞吐作用

• 批准号: 6545189
• 负责人: Norma Windsor Andrews
• 金额: $ 34.93万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6545189
• 关键词: SDS polyacrylamide gel electrophoresis; calcium ion; cell mediated lymphocytolysis test; cell membrane; collagen; cytotoxic T lymphocyte; exocytosis; fibroblasts; fluorescence microscopy; genetically modified animals; intermolecular interaction; laboratory mouse; lysosomes; mast cell; membrane proteins; synaptotagmin; tissue /cell culture; western blottings; wound healing

DESCRIPTION (provided by applicant): It is now aparent that regulated exocytosis is not restricted to specialized secretory cells, and that conventional lysosomes can respond to elevations in intracellular free Ca2+ by fusing with the plasma membrane. Investigating the physiological function of this ubiquitous lysosomal exocytic pathway, we found that it plays a central role in the mechanism by which mammalian cells reseal their plasma membrane after injury. Our previous studies also implicated synaptotagmin VII (Syt VII), a widely expressed member of the synaptotagmin family of putative Ca2+ sensors, in the regulation of lysosomal exocytosis and plasma membrane resealing. We now plan to expand our studies of the physiological role of Ca2+-regulated lysosomal exocytosis, and to characterize unique molecular interactions and dynamic propel-ties involved in this process. Our specific aims are: 1) To examine the resealing capacity of primary skin fibroblasts with genetic defects affecting lysosomes, using a collagen matrix contraction model of plasma membrane wounding and repair. Completion of this aim will allow us to determine if defects in plasma membrane resealing are a component of the serious symptoms associated with a series of lysosome-related genetic syndromes. 2) To investigate the role of the unconventional SNARE VAMP7 and of the synaptotagmin isoform Syt VII in the regulation of lysosomal exocytosis and plasma membrane repair. These studies will determine if Syt VII plays an essential role in the formation of specific SNARE complexes involved in lysosomal exocytosis, and will clarify the function of the lysosomal SNARE TIVAMP/VAMP-7, previously implicated in exocytic events associated with neurite outgrowth. 3) To verify if molecular interactions that regulate Ca2+-dependent exocytosis of conventional lysosomes also control secretion of the lysosome-related secretory granules of cytotoxic T lymphocytes (CTLs) and mast cells. These studies will provide important new information on the mechanisms regulating the exocytosis of lysosome-related granules that play critical roles in the immune response. 4) To investigate the spatial-temporal dynamics of Ca2+-regulated lysosomal exocytosis by total internal reflection fluorescence microscopy (TIR-FM). This powerful imaging technique will allow us to obtain quantitative parameters on the motion and distribution of lysosomes near the plasma membrane, map fusion sites on individual cells, and determine the diffusion coefficient of lysosomal membrane proteins after fusion. Taken together, the information generated by this project will significantly improve our understanding of fundamental biological processes involving regulated exocytosis of lysosomes, including maintenance of plasma membrane integrity and degranulation in cells of the immune system.

描述（由申请人提供）：现在显然，受调节的胞吐作用并不限于专门的分泌细胞，并且常规溶酶体可以通过与质膜融合来对细胞内游离Ca2+的升高做出反应。通过研究这种普遍存在的溶酶体胞吐途径的生理功能，我们发现它在哺乳动物细胞损伤后重新密封其质膜的机制中发挥着核心作用。我们之前的研究还表明突触结合蛋白 VII (Syt VII) 是假定的 Ca2+ 传感器突触结合蛋白家族中广泛表达的成员，参与溶酶体胞吐作用和质膜重新密封的调节。我们现在计划扩大对 Ca2+ 调节的溶酶体胞吐作用的生理作用的研究，并表征该过程中涉及的独特分子相互作用和动态推进。我们的具体目标是：1）使用质膜损伤和修复的胶原基质收缩模型来检查具有影响溶酶体的遗传缺陷的原代皮肤成纤维细胞的重新密封能力。完成这一目标将使我们能够确定质膜重新密封缺陷是否是与一系列溶酶体相关遗传综合征相关的严重症状的一个组成部分。 2) 研究非常规 SNARE VAMP7 和突触结合蛋白亚型 Syt VII 在调节溶酶体胞吐作用和质膜修复中的作用。这些研究将确定 Syt VII 在参与溶酶体胞吐作用的特定 SNARE 复合物的形成中是否发挥重要作用，并将阐明溶酶体 SNARE TIVAMP/VAMP-7 的功能，该功能先前与神经突生长相关的胞吐事件有关。 3) 验证调节传统溶酶体 Ca2+ 依赖性胞吐作用的分子相互作用是否也控制细胞毒性 T 淋巴细胞 (CTL) 和肥大细胞的溶酶体相关分泌颗粒的分泌。这些研究将为调节溶酶体相关颗粒胞吐作用的机制提供重要的新信息，这些颗粒在免疫反应中发挥关键作用。 4）通过全内反射荧光显微镜（TIR-FM）研究Ca2+调节的溶酶体胞吐作用的时空动态。这种强大的成像技术将使我们能够获得质膜附近溶酶体运动和分布的定量参数，绘制单个细胞上的融合位点，并确定融合后溶酶体膜蛋白的扩散系数。总而言之，该项目产生的信息将显着提高我们对涉及溶酶体胞吐作用调节的基本生物过程的理解，包括维持质膜完整性和免疫系统细胞脱颗粒。