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; 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.

专业的吞噬细胞响应各种线索就会发生细胞形状，这种可塑性需要 以极端方式重塑细胞骨架的能力。从历史上看，许多对细胞生物学的新见解有 从研究通过调节亚细胞来生存的细胞内病原体的出现 主机通路的组织和功能。该提议的目的是更好地了解分枝杆菌 操纵宿主细胞骨架和促进ARP2/3成核促进因子的WASH复合物的作用 这个过程。我们发现了一个古老的分枝杆菌效应子ESXM，该效应促进了变化 巨噬细胞细胞骨架通过与WASHC4的推定相互作用，WASTC4是WASH复合物的成员。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引起的巨噬细胞形态和行为使我们探究了肌动蛋白重排的程度 在感染期间（AIM 1）以及洗涤复合物在调节细胞形状和 运动性（目标2）。尽管在迁移细胞中建立了细胞骨架重排的重要性，但几乎没有 了解分枝杆菌效应子如何在感染过程中操纵该轴。另外，洗涤 复杂的研究主要是因为其在内吞运输中的作用，但对其潜在作用知之甚少 在巨噬细胞迁移或作为细胞内细菌的靶标。因此，对主机的新调查 需要细胞骨架和清洗络合物来了解它们在细胞迁移和免疫力中的作用。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复合物导致巨噬细胞的迁移能力增强。为了 为了检验这一假设，我将首先利用我们已建立的斑马鱼模型，该模型提供遗传 障碍性和光学透明度。在AIM 1中，我将利用已建立的巨噬细胞F-肌动蛋白记者斑马鱼 在细胞迁移和先天的背景下，观察ESXM诱导的肌动蛋白细胞骨架的变化线 免疫。我将结合高分辨率成像，药理学治疗和遗传工具 在这些过程中，巨噬细胞迁移的测试模式。在AIM 2中，我将测试遗传破坏 洗涤复合物使用斑马鱼敲除在体内改变巨噬细胞的形态和迁移。我也会 利用鼠标线破坏了洗涤复合物以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 实时提供有关动态肌动蛋白细胞骨架的见解，并询问Wash复合物中的作用 巨噬细胞迁移。拟议工作的完成将代表我们的重大进步 了解在感染的背景下如何改变肌动蛋白细胞骨架以及如何洗涤复合物 仅除了内吞贩运之外，可以调节巨噬细胞生物学。这些发现将突出 研究宿主 - 病原体相互作用，以阐明感染和稳态期间宿主因子的功能。