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

lc3相关吞噬（LAP）是一种降解途径，在吞噬溶酶体膜的安全范围内去除膜包裹的货物。LAP参与细胞内细菌的清除和细胞尸体和碎片的清除，以防止自身免疫反应。在LAP过程中，吞噬体膜被典型的与自噬相关的蛋白质修饰，包括LC3。然而，LC3向吞噬体募集的机制以及自噬相关蛋白在吞噬体中的作用尚不清楚。因此，我们建议利用线虫体内细胞尸体清除模型来确定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，我们将建立秀丽隐杆线虫作为模型，以确定LAP在清除神经释放碎片方面的疾病相关功能。我们发表的数据表明Atg8/LC3家族蛋白是吞噬溶酶体内尸体膜及时分解所必需的。在目的1中，我们将定义被膜包裹的货物是如何被告知将LC3招募到吞噬体，以及LC3是如何分解尸体膜的。这一目标依赖于我们建立的基于降解的报告系统，该系统专门标记吞噬体内的尸体膜，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是如何分解货物膜的。我们发现吞噬溶酶体以mTOR-和arl8依赖的方式管状化和囊泡化以加速降解。在Aim 2中，我们将使用RNAi和CLEM来测试吞噬溶酶体是否在微管运动蛋白的帮助下发生管化。由于mTOR调节多种途径，我们将筛选进行吞噬溶酶体管化的mTOR效应物。这一目的将揭示吞噬酶体管化的机制，为吞噬酶体降解提供进一步的见解。外包降解可能对高度分化的神经元至关重要，这些神经元需要清除受损的细胞器和蛋白质聚集体才能在生物体的整个生命周期中保持功能。在Aim 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，并确定吞噬溶酶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在神经元稳态和疾病状态中的意义。该项目将提供体内吞噬体成熟的机制，并测试LAP在神经退行性疾病中的作用。从线虫到哺乳动物，各种细胞和组织都使用lap介导的降解。由于lap缺乏与病理状况之间的联系，我们的体内项目可能为潜在的临床研究奠定基础，并最终有益于公众健康。

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.