Regulated Exocytosis of Lysosomes

描述(申请人提供)：当受伤时，真核细胞在几秒钟内重新密封他们的质膜。这一过程对于避免胞浆因子的损失以及恢复细胞内和细胞外环境之间的关键屏障至关重要。钙离子通过伤口内流触发溶酶体胞吐，这是质膜修复所必需的事件。胞吐作用被认为是通过添加内膜补片或通过减轻膜张力，促进自发的双层修复来介导质膜的重新密封。然而，最近的研究表明，钙离子的内流也会触发由造孔蛋白引起的损伤的修复。当插入质膜时，造孔蛋白会产生稳定的病变，不能通过贴片或降低膜张力来重新密封。对这一过程的研究表明，受损细胞中的钙内流显著刺激内吞作用，其动力学与细胞重新密封的动力学过程一致。其他结果表明，跨膜孔和机械损伤是通过内吞作用从质膜上移除的，这一过程需要溶酶体酶-酸性鞘磷脂酶的胞吐作用。这些新的发现代表了我们对质膜修复理解的重大概念上的进步，因为它们表明溶酶体胞吐的作用是释放作用于细胞表面的关键水解酶，而不是增加补丁或缓解膜张力。我们假设钙内流触发溶酶体酸性鞘磷脂酶的胞吐作用，该酶在细胞表面裂解鞘磷脂，产生神经酰胺，并诱导内体的形成，将病变携带到细胞中进行降解。为了验证这一假说，我们将追求两个特定的目标：1)确定质膜损伤过程中溶酶体酸性鞘磷脂酶的胞吐是否导致神经酰胺的生成和内小体的形成，以及这种损伤修复途径是否存在于肌肉纤维中；2)确定在质膜损伤过程中产生的内小体在细胞内的命运。除了阐明细胞如何在病原体产生的膜破坏剂的攻击下存活外，该项目还将为人类严重疾病的病理机制提供新的见解，包括溶酶体储存疾病和肌肉营养不良。 公共卫生相关性：质膜修复对于细胞损伤后的生存至关重要。这项提议的目标是研究哺乳动物细胞通过内吞作用去除其膜上伤口的机制。这些结果将阐明细胞是如何在病原体产生的毒素的攻击下存活下来的，并将增加我们对缺陷导致严重人类疾病的知识，包括溶酶体储存疾病和肌肉营养不良。

DESCRIPTION (provided by applicant): When wounded, eukaryotic cells reseal their plasma membrane in a few seconds. This process is essential to avoid loss of cytosolic factors, and for restoring the critical barrier between the intracellular and extracellular environments. Calcium influx through wounds triggers lysosomal exocytosis, an event required for plasma membrane repair. Exocytosis was thought to mediate plasma membrane resealing by addition of an endomembrane patch, or by relieving membrane tension, facilitating spontaneous bilayer restoration. However, it recently became clear that calcium influx also triggers the repair of lesions caused by pore-forming proteins. When inserted in the plasma membrane, pore-forming proteins generate stable lesions that cannot be resealed by a patch, or by reducing membrane tension. An investigation of this process revealed that calcium influx in injured cells markedly stimulates endocytosis, with a kinetics that coincides with cell resealing. Additional results suggested that trans-membrane pores and mechanical lesions are removed from the plasma membrane by endocytosis, and that this process requires exocytosis of a lysosomal enzyme, acid sphingomyelinase. These new findings represent a major conceptual advance in our understanding of plasma membrane repair, since they indicate that the role of lysosomal exocytosis is to release a critical hydrolase that acts on the cell surface, and not to add a patch or relieve membrane tension. We hypothesize that calcium entry triggers exocytosis of lysosomal acid sphingomyelinase, which cleaves sphingomyelin at the cell surface, generating ceramide and inducing formation of endosomes that carry the lesions into the cells for degradation. To test this hypothesis, we will pursue two specific aims: 1) Determine if exocytosis of lysosomal acid sphingomyelinase during plasma membrane wounding leads to ceramide generation and endosome formation, and whether this injury repair pathway is present in muscle fibers; 2) Determine the intracellular fate of the endosomes generated during plasma membrane wounding with pore-forming toxins. In addition to clarifying how cells survive attack by membrane damaging agents produced by pathogens, this project will provide new insight on mechanisms underlying the pathology of serious human diseases, including lysosomal storage diseases and muscular dystrophy. PUBLIC HEALTH RELEVANCE: Plasma membrane repair is essential for cellular survival after injury. The goal of this proposal is to investigate the mechanism by which mammalian cells remove wounds on their membrane by endocytosis. The results will clarify how cells survive attack by toxins produced by pathogens, and will increase our knowledge of defects leading to serious human diseases including lysosomal storage diseases and muscular dystrophy.