Regulated Exocytosis of Lysosomes

描述（由申请人提供）：当受伤时，真核细胞在几秒钟内重新密封其质膜。这一过程对于避免胞质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。通过伤口的钙内流触发溶酶体胞吐，这是质膜修复所需的事件。胞吐作用被认为是通过增加内膜片或通过缓解膜张力来介导质膜重新密封，从而促进自发的双层恢复。然而，最近变得清楚的是，钙内流也触发由成孔蛋白引起的损伤的修复。当插入质膜时，成孔蛋白产生稳定的损伤，这些损伤不能通过补片或降低膜张力来重新密封。对这一过程的研究表明，受损细胞中的钙流入显着刺激内吞作用，其动力学与细胞重新密封一致。其他结果表明，跨膜孔和机械损伤从质膜的内吞作用，这一过程需要胞吐的溶酶体酶，酸性鞘磷脂酶。这些新发现代表了我们对质膜修复理解的一个重大概念性进展，因为它们表明溶酶体胞吐作用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。我们假设钙进入触发溶酶体酸性鞘磷脂酶的胞吐作用，该酶在细胞表面切割鞘磷脂，产生神经酰胺并诱导内体的形成，内体将病变携带到细胞中进行降解。为了检验这一假设，我们将追求两个具体目标：1）确定在质膜损伤期间溶酶体酸性鞘磷脂酶的胞吐作用是否导致神经酰胺产生和内体形成，以及这种损伤修复途径是否存在于肌纤维中; 2）确定在质膜损伤期间用成孔毒素产生的内体的细胞内命运。除了阐明细胞如何在病原体产生的膜损伤剂的攻击下存活外，该项目还将为严重人类疾病（包括溶酶体贮积病和肌营养不良症）的病理学机制提供新的见解。

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.