Mechanisms of non-apoptotic programmed cell death and corpse clearance

非凋亡性程序性细胞死亡和尸体清除的机制 项目总结/摘要 许多细胞在发育和体内平衡过程中被编程死亡，但并非所有细胞都通过死亡而死亡。 凋亡此外，癌细胞已被证明对促增殖疗法具有抗性。因此有 这对于理解细胞死亡的非凋亡机制以及死亡细胞如何发出清除信号是重要的。 此外，尸体清除和吞噬溶酶体降解对于避免炎症和自体免疫是重要的。 免疫疾病，但我们对所有这些过程的理解存在重大差距。 我们建立了C.线虫极体是非凋亡细胞，其经历未知形式的 程序性坏死（Fazeli等人，Cell Rep 2018）1.使用这个遗传模型，我们建议确定 导致Aim 1中坏死细胞死亡和膜完整性丧失的机制，最初集中在非 基于一名大学生生成的初步数据， 学生.这些有针对性的筛选将确定极体死亡是否与 已建立的程序性坏死模式或定义了一种未发现的细胞死亡类型，这可能会让我们了解 在健康和疾病期间人类的未表征细胞死亡模式。 我们发现，极体外化磷脂酰丝氨酸（PS），并通过胚胎细胞清除， LC 3相关吞噬作用1.然而，在坏死细胞中调节PS暴露的信号通路是 不知道。在一名本科生的初步数据中，我们已经确定了至少四种多余的脂质， PS暴露所需的扰码，并通过使它们全部突变来减少PS暴露。这 这种方法是目标2中的几种方法之一，它将使我们能够定义调节吞噬的信号通路 以及Atg 8/LC 3家族蛋白向吞噬体的募集。这些信号通路 可能在其他类型的细胞死亡中是保守的，包括凋亡，为细胞凋亡提供了广泛的见解。 清理尸体的机制 我们还发现了Atg 8/LC 3募集到吞噬溶酶体的第一个作用：促进分解 尸体的细胞膜。然而，Atg 8/LC 3如何转导信号以及哪些因素 进行膜分解仍然是一个谜。在目标3中，我们梳理了 Atg 8/LC 3蛋白及其相互作用，以确定尸体内膜破裂的机制。 吞噬溶酶体膜破裂是核酸和蛋白质降解的限制因素 在吞噬溶酶体货物，这是特别重要的MHC II类表达免疫细胞，其中 溶酶体分解产物用于抗原呈递。LC 3相关吞噬作用的缺陷 与哺乳动物的自身免疫性疾病有关，决定了C。线虫细胞促进尸体 膜破裂可以为免疫过程中人类的保守机制提供新的见解 信号传导、炎症和自身免疫性疾病以及病原体清除。

Mechanisms of non-apoptotic programmed cell death and corpse clearance Project Summary/Abstract Many cells are programmed to die during development and homeostasis, but not all cells die via apoptosis. Additionally, cancer cells have proven resistant to apoptosis-promoting therapies. Therefore, it is important to understand non-apoptotic mechanisms of cell death, as well as how dying cells signal for clearance. Furthermore, corpse clearance and phagolysosomal degradation are important to avoid inflammation and auto- immune disease, but there are major gaps in our understanding of all these processes. We established that C. elegans polar bodies are non-apoptotic cells that undergo an unknown form of programmed necrosis(Fazeli et al., Cell Rep 2018)1. Using this genetic model, we propose to identify the mechanisms that lead to necrotic cell death and loss of membrane integrity in Aim 1, focusing initially on non- apoptotic caspases and scramblase regulators based on preliminary data generated by an undergraduate student. These targeted screens will identify whether polar body death shares common mechanisms with established modes of programmed necrosis or defines an undiscovered type of cell death, which may give insight into uncharacterized cell death modalities in humans during health and disease. We found that polar bodies externalize phosphatidylserine (PS) and are cleared by embryonic cells using LC3-associated phagocytosis1. However, the signaling pathways that regulate PS exposure in necrotic cells are not known. In preliminary data from an undergraduate student, we have identified at least four redundant lipid scramblases that are required for PS exposure and have reduced PS exposure by mutating them all. This approach is one of several in Aim 2 that will allow us to define the signaling pathways that regulate engulfment of the corpse as well as the recruitment of Atg8/LC3 family proteins to the phagosome. These signaling pathways may be conserved in other types of cell death, including apoptosis, providing wide-ranging insights into the mechanisms of corpse clearance. We also discovered the first role for Atg8/LC3 recruitment to the phagolysosome: promoting breakdown of the corpse membrane within the lumen1. However, how Atg8/LC3 transduces the signal and which factors carry out membrane breakdown remain a mystery. In Aim 3, we tease apart the localization and roles of Atg8/LC3 proteins and their interactors to define the mechanisms of corpse membrane breakdown within the phagolysosome. Membrane breakdown is the limiting factor for the degradation of nucleic acids and proteins within phagolysosomal cargos, which is especially important for MHC class II-expressing immune cells, where lysosomal breakdown products are used for antigen presentation. As defects in LC3-associated phagocytosis have been tied to autoimmune disease in mammals, determining how C. elegans cells promote corpse membrane breakdown can provide new insights into conserved mechanisms in humans during immune signaling, inflammation, and autoimmune disease in addition to pathogen clearance.