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， 构成这一网络的内噬体膜、吞噬体膜和溶酶体膜面临着来自 病原体和其他内化物质以及新陈代谢和化学应激。的后果 损伤根据特定的间隔和损伤的程度而不同，但广泛的溶酶体 膜通透性触发细胞死亡，而内小体和吞噬小体通过 颗粒物质和病原体导致炎症体激活和随后的细胞因子反应。一个 广泛应用的去除受损细胞器的策略涉及使用选择性自噬，参见 就像嗜血成性一样。然而，如果细胞器得到修复，则不需要移除。我们最近发现了一种 ESCRT(运输所需的内体分选复合体)机制在应对纳米技术方面的新作用 内溶酶体膜的鳞片破坏和促进其修复。 在这个项目中，我们建立在这一发现的基础上，并测试ESCRT(特别是 ESCRT-III蛋白)在维持内溶酶体的完整性和功能方面发挥着关键作用，它通过识别和 修复纳米级的膜损伤。ESCRT机器的这一作用有别于其广泛的 在腔内囊泡生物发生中的公认功能，并似乎适用于两种质膜 以及内部细胞器上。纳米级损伤涉及短暂的纳米级预孔或孔(S)，这些孔重新密封或， 超过关键门槛，扩展到允许不受限制的内容交换。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。此行政补充申请中所要求的设备将 允许我们升级现有的CSU-W1旋转圆盘共聚焦显微镜系统，以提供均匀的激光 当荧光标记的分子响应时，照明以及快速和精确的光操纵 保护细胞免受内溶酶体膜应激的影响。这种增强的照明控制将使 完成这一项目所需的量化分析，并提供关于ESCRT和合作如何 分子可感知和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.