ANALYSIS OF ESCRT FUNCTION IN ENDOLYSOSOMAL TRAFFICKING

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。在本提案中，我们将建立在 这一发现和测试的假设，ESCRT（特别是ESCRT-III蛋白）作为一个动态的 膜稳定系统，以保护穿过内溶酶体网络的脆弱膜， 超越。两个目标将利用和探索对两种实验上易处理和无菌的内溶酶体的反应。 能有效地激活ESCRT机制的干扰物在目标1中，我们将确定ESCRT如何 机械识别和抵消由L-亮氨酰-L-亮氨酸甲酯诱导的溶酶体膜应激 （LLOMe）。这将涉及表征负责接合ESCRT的膜应力， 分子途径，并确定“关键”ESCRT-III蛋白，描绘分子 修复所需的功能，并确定触发这种稳定反应的途径。在目标2中，我们将 研究ESCRT如何响应和修复二氧化硅诱导的上皮细胞和吞噬细胞膜损伤 细胞这将包括测试Fe 2+依赖性脂质过氧化在参与ESCRT中的作用， ESCRT组分在吞噬体膜上的相对作用和动力学，并检验 ESCRT限制吞噬细胞中的内溶酶体损伤，从而抑制与炎症相关的炎症。 通过这些途径运输的许多东西。从这项工作中获得的见解将适用于 了解ESCRT如何感知和响应广泛的膜应力。

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 proposal, we will build on this discovery and test the hypothesis that ESCRTs (and in particular ESCRT-III proteins) act as a dynamic membrane stabilizing system to protect vulnerable membranes across the endolysosomal network and beyond. Two aims will exploit and explore responses to two experimentally tractable and sterile endolysosomal disruptants that potently engage the ESCRT machinery. In Aim 1, we will determine how the ESCRT machinery recognizes and counteracts lysosomal membrane stress induced by L-leucyl-L-leucine methyl ester (LLOMe). This will involve characterizing the membrane stress responsible for engaging ESCRTs, defining the molecular pathway(s) involved and identifying “keystone” ESCRT-III proteins, delineating the molecular features required for repair, and identifying pathways that trigger this stabilizing response. In Aim 2, we will examine how ESCRTs respond to and repair silica induced membrane damage in epithelial and phagocytic cells. This will include testing a role for Fe2+-dependent lipid peroxidation in engaging ESCRTs, imaging the relative role and dynamics of ESCRT components on phagosomal membranes, and testing the hypothesis that ESCRTs limit endolysosomal damage in phagocytic cells and thereby dampen inflammation associated with the many things that transit through these pathways. The insights gained from this work will be applicable to understanding how ESCRTs sense and respond to a broad range of membrane stresses.