Phagocytic cup squeezing as revealed by GFP-actin video microscopy

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RAW264 macrophage cells were transfected with pGFP-actin. IgG-opsonized sheep erythrocytes were fed to cells expressing GFP-actin. Fluorescence of GFP-actin in living cells during phagocytosis of sheep erythrocytes was observed by video microscopy using cooled CCD camera and MetaMorph Imaging System. Imaging of GFP-actin revealed focal F-actin assembly underneath plasma membrane where erythrocytes were bound. Such F-actin assembly made the pseudopod extension along the surface of erythrocytes to form phagocytic cups. During pseudopod extension erythrocytes in phagocytic cups were deformed in oval or dumbbell-like shape. It appeared that erythrocytes were tightly squeezed by phagocytic cups. This deformation of erythrocytes cannot be explained by the classical zipper closure model, but contractile activity of phagocytic cups. When the cells were treated with myosin inhibitors such as butanedione monoxirne and ML-7, phagocytic cup squeezing was not observed, while F-actin assembly and phagocytic cup formation occurred. These findings suggested that phagocytic cups circumferentially constricted erythirocytes by actomyosin-driven contractile activity. The phagocytic cup squeezing might be supplemental mechanism to facilitate sequential IgG-Fc and Fc-receptor binding (zipper closure) and/or to push extracellular solution out of phagosomes.
日本組織細胞化学会の論文