Regulated Exocytosis of Lysosomes

描述（由申请人提供）：当真核细胞受伤时，在几秒钟内重新封闭其质膜。这个过程对于避免细胞质因子的损失，以及恢复细胞内和细胞外环境之间的关键屏障是必不可少的。钙通过伤口流入引发溶酶体胞吐，这是质膜修复所必需的。胞外分泌被认为是通过增加膜内贴片或缓解膜张力介导质膜重封，促进自发的双层修复。然而，最近的研究表明，钙的流入也会引发由气孔形成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，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，其动力学与细胞重封一致。其他结果表明，跨膜孔隙和机械损伤是通过内吞作用从质膜上清除的，这一过程需要溶酶体酶（酸性鞘磷脂酶）的胞吐作用。这些新发现代表了我们对质膜修复理解的重大概念进展，因为它们表明溶酶体胞外分泌的作用是释放一种作用于细胞表面的关键水解酶，而不是增加贴片或缓解膜张力。我们假设钙的进入触发溶酶体酸性鞘磷脂酶的胞外分泌，该酶在细胞表面切割鞘磷脂，产生神经酰胺并诱导形成内体，这些内体将病变带到细胞中进行降解。为了验证这一假设，我们将追求两个具体目标：1)确定在质膜损伤过程中溶酶体酸鞘磷脂酶的胞吐是否导致神经酰胺的产生和内体的形成，以及这种损伤修复途径是否存在于肌肉纤维中；2)决定形成孔的毒素在质膜损伤过程中产生的核内体在细胞内的命运。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，该项目还将为包括溶酶体贮积病和肌肉萎缩症在内的严重人类疾病的病理机制提供新的见解。

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.