Mechanism and in vivo function of LC3-associated phagocytosis: When neighbors help take out the trash

LC3相关吞噬细胞作用(LAP)是一种降解途径，它将包裹在吞噬体膜内的货物安全地移出。LAP参与清除细胞内细菌和清除细胞身体和碎片，以防止自身免疫反应。在LAP过程中，吞噬体膜上装饰着与自噬相关的蛋白质，包括Lc3。然而，LC3募集到吞噬小体的机制以及自噬相关蛋白在吞噬小体上的作用尚不清楚。因此，我们建议使用线虫体内细胞身体清除模型来确定LAP介导的降解机制。此外，基于我们在LAP和神经生物学方面的专业知识，我们将建立线虫作为模型来定义LAP在清除神经释放的DEBRIS中与疾病相关的功能。我们发表的数据表明，吞噬酶体内的身体膜需要Atg8/Lc3家族蛋白来及时分解。在目标1中，我们将定义膜包裹货物的存在如何传递以招募LC3到吞噬小体，以及LC3在分解身体膜方面所起的作用。这一目标依赖于我们建立的基于降解子的报告系统来专门标记吞噬小体内的身体膜，用于延时和相关光学和电子显微镜(CLEM)技术。目标1将确切地确定LAP是如何分解货膜的。我们发现吞噬溶酶体以一种依赖mTOR和ARL8的方式管状和囊化，以加速降解。在目标2中，我们将使用RNAi和Clem来测试吞噬酶体管形成是否在微管上的运动蛋白的帮助下发生。由于mTOR调节几条途径，我们将筛选进行吞噬酶体管连接的mTOR效应器。这一目的将揭示吞噬溶酶体管化的机制，为进一步了解吞噬溶酶体的降解提供了进一步的了解。外包降解对于高度分化的神经元可能是至关重要的，它需要清除受损的细胞器和蛋白质聚集体，以便在有机体的整个生命周期中保持功能。在目标3中，我们将测试邻近细胞是否通过LAP清除不需要的神经元碎片，并确定吞噬小体降解是否影响神经退行性运动神经元疾病，如ALS。这将确立LAP在神经元动态平衡和疾病状态中的意义。该项目将提供对体内吞噬体成熟的机械性见解，并测试LAP在神经退行性疾病中的作用。LAP介导的降解被从线虫到哺乳动物的各种细胞和组织使用。由于LAP缺乏与病理条件之间已经建立了联系，我们的体内项目可能为潜在的临床研究奠定基础，并最终对公众健康有利。

LC3-associated phagocytosis (LAP) is a degradative pathway that removes membrane-wrapped cargos within the safety of the phagolysosomal membrane. LAP is involved in removal of intracellular bacteria and clearance of cell corpses and debris to prevent autoimmune reactions. During LAP, the phagosomal membrane is decorated with proteins classically associated with autophagy, including LC3. However, the mechanism of LC3 recruitment to phagosomes and the role of autophagy-related proteins on phagosomes are not clear. Therefore, we propose to determine the mechanisms of LAP-mediated degradation using an in vivo model of cell corpse clearance in C. elegans. Furthermore, based on our expertise with LAP and neurobiology, we will establish C. elegans as a model to define the disease relevant functions of LAP in clearing neuronally released debris.Our published data shows that Atg8/LC3 family proteins are required for timely breakdown of the corpse membrane inside the phagolysosome. In Aim 1, we will define how the presence of membrane-wrapped cargo is communicated to recruit LC3 to phagosomes and what LC3 does to break down the corpse membrane. This aim depends on our established degron-based reporter system to specifically label the corpse membrane inside the phagosome for time-lapse and correlated light and electron microscopy (CLEM) techniques. Aim 1 will establish exactly how LAP breaks down cargo membranes.We discovered that phagolysosomes tubulate and vesiculate in an mTOR- and ARL8-dependent manner to accelerate degradation. In Aim 2, we will use RNAi and CLEM to test whether phagolysosome tubulation occurs with the help of motor proteins along microtubules. As mTOR regulates several pathways, we will screen for mTOR effectors that conduct phagolysosome tubulation. This aim will reveal the mechanism of phagolysosome tubulation, providing further insight into phagolysosomal degradation.Outsourcing degradation may be of pivotal importance for highly differentiated neurons, which need to clear damaged organelles and protein aggregates to stay functional throughout the whole life of the organism. In Aim 3, we will test whether neighboring cells clear unwanted neuronal debris by LAP and determine whether phagolysosomal degradation impacts neurodegenerative motoneuron diseases, such as ALS. This will establish the significance of LAP in the neuronal homeostasis and during disease conditions.This project will provide mechanistic insight into phagosome maturation in vivo and test the role of LAP in neurodegenerative diseases. LAP-mediated degradation is used by a variety of cells and tissues from nematodes to mammals. Due to the established links between LAP-deficiency and pathological conditions, our in vivo project may pave the ground for potential clinical studies, and eventually be beneficial for public health.