Roles for the WASH complex in macrophage migration and function

WASH 复合物在巨噬细胞迁移和功能中的作用

• 批准号: 10708762
• 负责人: Mollie Sweeney
• 金额: $ 4万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708762
• 关键词: Actins; Animals; Bacteria; Behavior; Biology; Bulla; CRISPR/Cas technology; Cell Shape; Cell physiology; Cells; Cellular biology; Clinical; Complex; Cues; Cytoskeleton; Data; Disease; F-Actin; Filopodia; Genetic; Genus Mycobacterium; Goals; Granuloma; Homeostasis; Human; Immune System Diseases; Immunity; Infection; Infection Control; Integration Host Factors; Invaded; Investigation; Knock-out; Knowledge; Length; Macrophage; Macrophage Cell Biology; Mediating; Membrane; Microscopy; Modeling; Morphology; Mus; Mutant Strains Mice; Mycobacterium Infections; Mycobacterium marinum; Mycobacterium tuberculosis; Natural Immunity; Optics; Outcome; Pathogenesis; Pathway interactions; Pattern; Phagocytes; Pharmacological Treatment; Phenotype; Play; Process; Reporter; Resolution; Role; Set protein; Shapes; Site; Sorting; System; Testing; Therapeutic; Time; Translating; Tuberculosis; Work; Zebrafish; cell motility; developmental disease; genetic manipulation; high resolution imaging; human disease; human pathogen; in vivo; insight; member; migration; mouse model; mycobacterial; novel; pathogen; protein complex; response; tool; trafficking; wound

Professional phagocytes undergo shifts in cell shape in response to a variety of cues, and this plasticity requires the ability to remodel the cytoskeleton in extreme ways. Historically, many new insights into cell biology have emerged from studying intracellular pathogens that have evolved to survive by modulating the subcellular organization and function of host pathways. The goal of this proposal is to better understand how mycobacteria manipulate the host cytoskeleton and the role of the WASH complex, an Arp2/3 nucleation promoting factor, in this process. We have discovered an ancient mycobacterial effector, EsxM, that promotes changes to the macrophage cytoskeleton through a putative interaction with WASHC4, a member of the WASH complex. These changes enhance the dissemination of mycobacterial disease via migrating macrophages. The striking shift in macrophage morphology and behavior induced by EsxM have led us to probe the extent of actin rearrangements during infection (Aim 1) and whether the WASH complex may have unknown roles in regulating cell shape and motility (Aim 2). Although the importance of cytoskeletal rearrangement in migrating cells is established, little is known about how mycobacterial effectors may manipulate this axis during infection. Additionally, the WASH complex has been studied primarily for its role in endocytic trafficking, but less is known about its potential role in macrophage migration, or as a target of intracellular bacteria. Thus, new investigations into the host cytoskeleton and the WASH complex are needed to understand their roles in cell migration and immunity. The overall hypothesis of this proposal is that mycobacterial effector EsxM induces changes in the actin cytoskeleton that lead to enhanced migratory capacity of macrophages via the WASH complex. In order to test this hypothesis, I will first take advantage of our established zebrafish model which offers genetic tractability and optical transparency. In Aim 1, I will utilize an established macrophage F-actin reporter zebrafish line to observe changes in the actin cytoskeleton induced by EsxM in the context of cell migration and innate immunity. I will combine high resolution imaging, pharmacological treatment, and genetic tools to functionally test modes of macrophage migration during these processes. In Aim 2 I will test how genetic disruption of the WASH complex changes macrophage morphology and migration in vivo using zebrafish knockouts. I will also take advantage of a mouse line in which the WASH complex is disrupted to study its role in mycobacterial infection in a mammalian model. Overall, these studies will both take advantage of the zebrafish system to provide insights into the dynamic actin cytoskeleton in real time, and interrogate a role for the WASH complex in macrophage migration. The completion of the proposed work will represent a significant advancement in our understanding of how the actin cytoskeleton is altered in the context of infection, and how the WASH complex regulates macrophage biology beyond endocytic trafficking alone. The findings will highlight the importance of studying host-pathogen interactions to elucidate the function of host-factors during infection and homeostasis.

专业吞噬细胞会因各种提示而改变细胞形状，而这种可塑性需要 以极端的方式重塑细胞骨架的能力。从历史上看，对细胞生物学的许多新见解 来自对细胞内病原体的研究，这些病原体已经进化成通过调节亚细胞来生存 寄主途径的组织和功能。这项提议的目标是更好地了解分枝杆菌如何 操纵宿主细胞骨架和WASH复合体的作用，WASH复合体是Arp2/3成核促进因子，在 这一过程。我们已经发现了一种古老的分枝杆菌效应器EsxM，它促进了 巨噬细胞的细胞骨架可能通过与WASHC4的相互作用而被激活，WASHC4是WASH复合体的一个成员。这些 变化通过迁移的巨噬细胞加强分枝杆菌疾病的传播。中国经济的显著转变 EsxM诱导的巨噬细胞的形态和行为使我们探索了肌动蛋白重排的程度 在感染过程中(目标1)以及洗涤复合体是否可能在调节细胞形状和 运动性(目标2)。尽管细胞骨架重排在细胞迁移中的重要性已经确定，但几乎没有 了解分枝杆菌效应器如何在感染期间操纵这个轴。此外，洗涤 人们主要研究复合体在胞内转运中的作用，但对其潜在作用知之甚少。 在巨噬细胞移动中，或作为细胞内细菌的目标。因此，对东道主的新调查 细胞骨架和WASH复合体是了解它们在细胞迁移和免疫中的作用所必需的。这个 这一提议的总体假设是分枝杆菌效应物EsxM诱导肌动蛋白的变化 通过WASH复合体导致巨噬细胞迁移能力增强的细胞骨架。按顺序 为了验证这一假设，我将首先利用我们建立的斑马鱼模型，该模型提供了基因 易操纵性和光学透明性。在目标1中，我将利用已建立的巨噬细胞F-肌动蛋白报告斑马鱼 观察EsxM在细胞迁移和先天性背景下诱导的肌动蛋白细胞骨架的变化 豁免权。我将结合高分辨率成像、药物治疗和遗传工具在功能上 在这些过程中巨噬细胞迁移的测试模式。在《目标2》中，我将测试基因如何破坏 利用斑马鱼基因敲除改变巨噬细胞形态和体内迁移。我也会 利用WASH复合体被破坏的小鼠品系来研究其在分枝杆菌中的作用 哺乳动物模型中的感染。总体而言，这些研究都将利用斑马鱼系统来 提供对动态肌动蛋白细胞骨架的实时洞察，并询问洗涤复合体在 巨噬细胞迁移。拟议工作的完成将标志着我们的 了解肌动蛋白细胞骨架在感染的背景下如何改变，以及WASH复合体是如何改变的 调节巨噬细胞生物学，而不仅仅是内吞运输。这些发现将突显出 研究宿主-病原体的相互作用，以阐明宿主因素在感染和动态平衡过程中的作用。