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

点击翻译按钮获取中文摘要

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.