Regulated Exocytosis of Lysosomes

描述(申请人提供)：现在看来，受调控的胞吐作用并不局限于特定的分泌细胞，而传统的溶酶体可以通过与质膜融合来响应细胞内游离钙的升高。研究这种普遍存在的溶酶体胞外途径的生理功能，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发挥着核心作用。我们以前的研究还发现，Synaptopagmin VII(SYT VII)参与了溶酶体胞吐和质膜再封闭的调节，SYT VII是synaptopagmin家族中广泛表达的可能的钙离子感受器。我们现在计划扩大我们对钙离子调节的溶酶体胞吐作用的生理作用的研究，并表征参与这一过程的独特的分子相互作用和动态推动力。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，检测具有影响溶酶体的遗传缺陷的原代皮肤成纤维细胞的再封闭能力。完成这一目标将使我们能够确定质膜再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)探讨非常规SNARE VAMP7和突触素亚型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是否在溶酶体胞吐作用所涉及的特定SNAR复合体的形成中发挥重要作用，并将阐明溶酶体SNARTIVAMP/VAMP-7的功能，该功能先前与与轴突生长相关的胞外事件有关。3)验证调节常规溶酶体钙依赖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，溶酶体相关颗粒在免疫反应中发挥关键作用。4)用全内反射荧光显微镜(TIR-FM)研究钙离子调节的溶酶体胞吐作用的时空动力学。这一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置图，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调节胞吐作用的基本生物学过程的理解，包括维持质膜完整性和免疫系统细胞中的脱颗粒。

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.