Regulated Exocytosis of Lysosomes

描述（由申请人提供）：现在很明显，受调节的胞吐作用并不局限于特化的分泌细胞，并且常规溶酶体可以通过与质膜融合来响应细胞内游离Ca 2+的升高。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质膜的机制中起着核心作用。我们以前的研究还涉及synaptotagmin VII（Syt VII），一个广泛表达的成员synaptotagmin家族的推定的Ca 2+传感器，在调节溶酶体胞吐和质膜resealing。我们现在计划扩大我们的研究的生理作用的Ca 2+调节的溶酶体胞吐作用，并表征独特的分子相互作用和动态propel-ties参与这一过程。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，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再密封能力。这一目标的完成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组成部分。2)研究非常规SNARE VAMP 7和突触结合蛋白亚型Syt VII在调节溶酶体胞吐和质膜修复中的作用。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的Ca 2+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）和肥大细胞的溶酶体相关分泌颗粒的分泌。这些研究将提供重要的新信息的机制，调节溶酶体相关的颗粒，在免疫反应中发挥关键作用的胞吐作用。4)利用全内反射荧光显微镜（TIR-FM）研究钙离子调控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定量参数的运动和分布的溶酶体附近的质膜，地图上的融合位点的单个细胞，并确定融合后的溶酶体膜蛋白的扩散系数。总之，该项目产生的信息将显着提高我们对基本生物学过程的理解，包括溶酶体的调节胞吐作用，包括维持质膜完整性和免疫系统细胞的脱粒。

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.