ANALYSIS OF ESCRT FUNCTION IN ENDOLYSOSOMAL TRAFFICKING

摘要 内溶酶体网络是细胞外物质进入细胞的门户。因此， 构成这一网络的内体、吞噬体和溶酶体的膜面临着来自 病原体和其他内化物质以及代谢和化学应激。后果 损伤根据具体的隔室和损伤程度而变化，但广泛的溶酶体 膜透化引发细胞死亡，而细胞膜透化引起的内体和吞噬体的有限破坏， 颗粒物质和病原体导致炎性小体活化和随后的细胞因子应答。一 广泛使用的去除受损细胞器的策略涉及使用选择性自噬， 是嗜血癖然而，如果细胞器被修复，则去除是不必要的。我们最近发现了一个 ESCRT（运输所需的内体分选复合物）机制在应对纳米 内溶酶体膜的规模破坏和促进其修复。 在这个项目中，我们正在建立在这一发现和测试的假设，ESCRT（特别是 ESCRT-III蛋白）在维持内溶酶体的完整性和功能中起关键作用， 修复纳米级膜损伤。ESCRT机制的这一作用不同于其广泛的 在管腔内囊泡生物发生中的公认功能，并且似乎适用于质膜和 和内部细胞器。纳米级损伤涉及寿命短的纳米尺寸的预孔或重新密封的孔， 在临界阈值以上，扩展以允许无限制的内容交换。我们使用了一系列的化学物质， 物理和生物应激源来定义信号以及分子和物理机制 潜在的ESCRT介导的修复。本行政补充申请中要求的设备将 使我们能够升级现有的CSU-W1旋转圆盘共聚焦显微镜系统， 照明沿着荧光标记分子响应时的快速和精确的光操纵 保护细胞免受内溶酶体膜应激。这种增强的照明控制将使 完成该项目所需的定量分析，并深入了解ESCRT和合作 分子感知并响应广泛的生理和病理生理膜应激。

Abstract The endolysosomal network is the portal by which extracellular material enters the cell. As such, the membranes of the endosomes, phagosomes, and lysosomes that comprise this network face challenges from pathogens and other internalized materials as well as from metabolic and chemical stresses. Consequences of damage vary according to the specific compartment and degree of damage, but extensive lysosomal membrane permeabilization triggers cell death while limited disruption of endosomes and phagosomes by particulate material and pathogens leads to inflammasome activation and ensuing cytokine responses. A widely deployed strategy for removing damaged organelles involves the use of selective autophagy, referred to as lysophagy. Removal is, however, unnecessary if organelles are instead repaired. We recently discovered a new role for the ESCRT (endosomal sorting complex required for transport) machinery in responding to nano- scale disruptions in endolysosomal membranes and promoting their repair. In this project, we are building on this discovery and testing the hypothesis that ESCRTs (and in particular ESCRT-III proteins) play a key role in maintaining endolysosomal integrity and function by recognizing and repairing nanoscale membrane damage. This role for the ESCRT machinery is distinct from its widely recognized function in intralumenal vesicle biogenesis and appears applicable at both the plasma membrane and on internal organelles. Nanoscale damage involves short-lived nm-size pre-pore or pore(s) that reseal or, above a critical threshold, expand to allow unrestrained content exchange. We are using a range of chemical, physical, and biological stressors to define the signals as well as molecular and physical mechanisms underlying ESCRT-mediated repair. The equipment requested in this administrative supplement application will allow us to upgrade our existing CSU-W1 spinning disc confocal microscope system to provide uniform laser illumination along with rapid and precise photomanipulation of fluorescently tagged molecules as they respond to and protect cells from endolysosomal membrane stress. This enhanced control of illumination will enable the quantitative analyses needed to complete this project and provide insights into how ESCRTs and cooperating molecules sense and respond to a broad range of physiologic and pathophysiologic membrane stress.