Rho GTPases and cytoskeletal rearrangements involved in particle capture and phagocytosis by macrophages

我们计划阐明是否存在两种不同的吞噬模式：Fc γ受体介导的吞噬杯形成与补体受体介导的下沉。我们的初步工作提出了一个工作模型（在天然巨噬细胞），其中补体受体介导的吞噬作用涉及吞噬杯形成（膜达到），而不是颗粒下沉，我们现在推测有一个共同的ITAM/Syk介导的吞噬杯途径。我们将使用延时3D旋转圆盘共聚焦显微镜来成像不同包被（例如未包被或IgG ± C3 b包被）的人红细胞如何被野生型、NOTAM、FcR-γ-链-/-、Syk cKO、Cdc 42 cKO、Hem 1 KO、WASP KO或Myo 10-/-巨噬细胞（± Rho或其他靶标的抑制剂）吞噬。这些实验将补充相结合的激光扫描共聚焦和原子力显微镜，以测量颗粒吞噬细胞相互作用的机械性能。除了补体介导的吞噬作用，我们将扩大我们的研究到丝状伪足的捕获和摄取颗粒的作用。使用来自野生型、Lifeact-EGFP和Cdc 42 cKO小鼠的巨噬细胞的大量初步数据表明了一种模型，其中Toll样受体刺激诱导丝状伪足形成，从而引发巨噬细胞通过丝状伪足收缩、冲浪、清扫或这些机制的组合来捕获细菌颗粒。与此同时，我们将完成Myo 18 a和Myo 10基因敲除小鼠模型的表型分析，并特别探索丝状伪足诱导Myo 10在捕获和吞噬颗粒中的作用。

We plan to clarify whether or not there are two distinct modes of phagocytosis: Fcgamma-receptor- mediated phagocytic cup formation versus complement receptor-mediated sinking in. Our preliminary work suggests a working model (in native macrophages) in which complement receptor-mediated phagocytosis involves phagocytic cup formation (the membrane reaching up), rather than particle sinking, and we now speculate that there is a common ITAM/Syk-mediated phagocytic cup pathway. We will use time-lapse 3D spinning disk confocal microscopy to image how variously coated (e.g. uncoated or IgG ± C3b coated) human red blood cells are engulfed by wild-type, NOTAM, FcR-gamma-chain-/-, Syk cKO, Cdc42 cKO, Hem1 KO, WASP KO or Myo10-/- macrophages (± inhibitors of Rho or other targets). These experiments will be complemented by combined laser scanning confocal and atomic force microscopy to measure the mechanical properties of particle-phagocyte interactions. In addition to complement-mediated phagocytosis, we will extend our study into the roles of filopodia in the capture and ingestion of particles. Extensive preliminary data using macrophages from wild-type, Lifeact-EGFP and Cdc42 cKO mice suggest a model in which Toll-like receptor stimulation induces filopodia formation, thereby priming macrophages for the capture of bacterial particles via filopodial retraction, surfing, sweeping or combinations of these mechanisms. In parallel, we will complete phenotypic analyses of Myo18a- and Myo10-knockout mouse models, and specifically explore the roles of filopodia-inducing Myo10 in the capture and engulfment of particles.