Regulated Exocytosis of Lysosomes

描述（由申请人提供）：现在很明显，受调节的胞外分泌并不局限于专门的分泌细胞，传统的溶酶体可以通过与质膜融合来响应细胞内游离Ca2+的升高。通过研究这种普遍存在的溶酶体胞外通路的生理功能，我们发现它在哺乳动物细胞损伤后重新封闭质膜的机制中起着核心作用。我们之前的研究也表明突触塔蛋白VII （Syt VII）是广泛表达的突触塔蛋白家族成员，被认为是Ca2+传感器，在溶酶体胞吐和质膜重封的调节中。我们现在计划扩大我们对Ca2+调节溶酶体胞吐的生理作用的研究，并表征这一过程中独特的分子相互作用和动态推进关系。我们的具体目标是：1)利用质膜损伤和修复的胶原基质收缩模型，研究影响溶酶体遗传缺陷的原代皮肤成纤维细胞的再密封能力。完成这一目标将使我们能够确定质膜重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)探讨非常规SNARE VAMP7和synaptotagmin亚型Syt VII在溶酶体胞外分泌和质膜修复中的调控作用。这些研究将确定Syt VII是否在参与溶酶体胞吐的特定SNARE复合物的形成中起重要作用，并将阐明溶酶体SNARE TIVAMP/VAMP-7的功能，该功能先前涉及与神经突生长相关的胞吐事件。3)验证调节常规溶酶体Ca2+依赖性胞吐的分子相互作用是否也控制细胞毒性T淋巴细胞（ctl）和肥大细胞溶酶体相关分泌颗粒的分泌。这些研究将为在免疫应答中起关键作用的溶酶体相关颗粒的胞吐调节机制提供重要的新信息。4)利用全内反射荧光显微镜（TIR-FM）研究Ca2+调控溶酶体胞吐的时空动态。这种强大的成像技术将使我们能够获得溶酶体在质膜附近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。综上所述，该项目产生的信息将显著提高我们对涉及溶酶体胞吐调节的基本生物学过程的理解，包括维持免疫系统细胞的质膜完整性和脱颗粒。

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.