Roles for the WASH complex in macrophage migration and function

专职吞噬细胞会根据各种线索改变细胞形状，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 从研究细胞内病原体中产生，这些病原体通过调节亚细胞 宿主途径的组织和功能。这项提案的目的是为了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（Arp 2/3成核促进因子）的作用 这个过程我们已经发现了一种古老的分枝杆菌效应子EsxM，它促进了 通过与WASH复合物的一个成员WASHC 4的假定相互作用，在巨噬细胞细胞骨架中起作用。这些 这些变化通过迁移巨噬细胞增强了分枝杆菌疾病的传播。这一惊人的转变 EsxM诱导的巨噬细胞形态和行为使我们能够探测肌动蛋白重排的程度 在感染过程中（目的1），以及WASH复合物是否可能在调节细胞形状和 运动性（目的2）。虽然细胞骨架重排在迁移细胞中的重要性已经确立， 已知分枝杆菌效应子在感染期间如何操纵该轴。此外，WASH 人们主要研究复合物在内吞运输中的作用，但对其潜在作用知之甚少 在巨噬细胞迁移中，或作为细胞内细菌的靶标。因此，对宿主的新调查 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。的 该建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，导致通过WASH复合体增强巨噬细胞的迁移能力。为了 为了验证这一假设，我将首先利用我们建立的斑马鱼模型， 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 细胞迁移和先天性巨噬细胞的背景下，观察由EsxM诱导的肌动蛋白细胞骨架的变化。 免疫力我将结合联合收割机高分辨率成像，药物治疗，和基因工具， 测试这些过程中巨噬细胞迁移的模式。在目标2中，我将测试基因破坏是如何产生的。 WASH复合物使用斑马鱼敲除改变体内巨噬细胞形态和迁移。我也会 利用WASH复合物被破坏的小鼠系来研究其在分枝杆菌中的作用。 感染哺乳动物模型。总的来说，这些研究都将利用斑马鱼系统， 在真实的时间提供动态肌动蛋白细胞骨架的见解，并询问WASH复合体在 巨噬细胞迁移拟议工作的完成将标志着我们在这方面的一个重大进展。 了解肌动蛋白细胞骨架如何在感染的背景下改变，以及WASH复合体如何在感染后的细胞中发挥作用。 调节巨噬细胞生物学，而不仅仅是内吞运输。调查结果将强调以下方面的重要性： 研究宿主-病原体相互作用以阐明宿主因子在感染和体内平衡中的功能。

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.