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.

专业吞噬细胞响应各种提示而发生细胞形状的变化，这种可塑性需要 以极端方式重塑细胞骨架的能力。历史上，许多关于细胞生物学的新见解 来自研究细胞内病原体，这些病原体通过调节亚细胞而进化生存 宿主途径的组织和功能。该提案的目标是更好地了解分枝杆菌如何 操纵宿主细胞骨架以及 WASH 复合物（一种 Arp2/3 成核促进因子）的作用 这个过程。我们发现了一种古老的分枝杆菌效应子 EsxM，它可以促进 通过与 WASHC4（WASH 复合体的成员）的假定相互作用来形成巨噬细胞细胞骨架。这些 变化通过迁移巨噬细胞增强了分枝杆菌疾病的传播。惊人的转变 EsxM 诱导的巨噬细胞形态和行为使我们能够探究肌动蛋白重排的程度 感染期间（目标 1）以及 WASH 复合物是否在调节细胞形状和 动力（目标 2）。尽管细胞骨架重排在迁移细胞中的重要性已被确​​定，但人们对此知之甚少。 了解分枝杆菌效应器在感染过程中如何操纵该轴。此外，WASH 人们主要研究了复合物在内吞运输中的作用，但对其潜在作用知之甚少 巨噬细胞迁移，或作为细胞内细菌的目标。因此，对宿主进行新的调查 需要细胞骨架和 WASH 复合体来了解它们在细胞迁移和免疫中的作用。这 该提议的总体假设是分枝杆菌效应子 EsxM 诱导肌动蛋白的变化 细胞骨架通过 WASH 复合体增强巨噬细胞的迁移能力。为了 为了检验这个假设，我将首先利用我们建立的斑马鱼模型，该模型提供遗传 易处理性和光学透明度。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 线观察 EsxM 在细胞迁移和先天环境中诱导的肌动蛋白细胞骨架的变化 免疫。我将结合高分辨率成像、药物治疗和遗传工具来功能性地 在这些过程中测试巨噬细胞迁移的模式。在目标 2 中，我将测试基因如何被破坏 使用斑马鱼基因敲除技术，WASH 复合物可改变体内巨噬细胞的形态和迁移。我也会 利用 WASH 复合物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。总的来说，这些研究都将利用斑马鱼系统来 实时提供对动态肌动蛋白细胞骨架的见解，并探讨 WASH 复合物在 巨噬细胞迁移。拟议工作的完成将代表我们在 了解肌动蛋白细胞骨架在感染情况下如何改变，以及 WASH 复合物如何改变 调节巨噬细胞生物学，而不仅仅是内吞运输。研究结果将凸显以下问题的重要性： 研究宿主与病原体的相互作用，以阐明宿主因子在感染和体内平衡过程中的功能。