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。在这项提案中，我们将在 这一发现并检验了ESCRT(尤其是ESCRT-III蛋白)作为一种动力作用的假设 膜稳定系统，以保护跨内溶酶体网和 更远一点。两个目标将开发和探索对两种实验上可驯化和无菌的内溶酶体的反应 有效地启动ESCRT机器的干扰剂。在目标1中，我们将确定ESCRT如何 机械识别和对抗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触ESCRT的膜应力，定义 分子途径(S)参与并鉴定“Keystone”ESCRT-III蛋白，描绘其分子 修复所需的特征，并确定触发这种稳定反应的途径。在目标2中，我们将 检测ESCRT对二氧化硅致上皮细胞和吞噬细胞膜损伤的反应和修复 细胞。这将包括测试依赖于Fe2+的脂质过氧化在参与ESCRT中的作用，成像 ESCRT组分在吞噬体膜上的相对作用和动力学，并检验以下假设 ESCRT限制吞噬细胞的内溶体损伤，从而抑制与以下相关的炎症 通过这些通道的许多东西。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压力。

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.