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Qingxian Lu, Lixia Han, Yong Tang, Qian Liu, Jiayi Xie, Jiantao Feng, Dong Han, Qingjun Lu; Study Of The Mertk Mediated Phagocytosis Using Environmental Scanning Electron Microscope (esem) And Atomic Force Microscope (afm). Invest. Ophthalmol. Vis. Sci. 2012;53(14):1133.
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MerTK-mediated phagocytosis is crucial for professional phagocytes, e.g., macrophage, dendritic cell and retina pigment epithelium, to clear apoptotic and spent cell debris. This study was planned to test how MerTK affected phagosome uptaking; and whether the MerTK mutant phagocyte was able to uptake the large targets, if yes, by what mechanism.
The macrophage and RPE were isolated from wild-type and MerTK knockout mice and cultured in vitro for 24 hours before the assays. The time course of the FITC-labeled apoptotic lymphocytes was compared between two groups. The process of phagocytic cup formation was studied by environmental scanning electron microscope (eSEM) using the 7-µm beads pre-coated with the whole protein extract from apoptotic lymphocytes. The pulling strength generated by cytoskeletal and dynamic proteins during engulfment and phagosome formation was measured and analyzed by atomic force microscope (AFM).
The cultured MerTK mutant macrophages were able to uptake apoptotic lymphocytes, although at a significant low level compared to the wild-type counterparts. However, they adapted a macropinocytotic mechanism. In the wild-type phagocytes, the phagocytic cup formation was initiated by denting the membrane at the cell-cell contacting region and protruding the peripheral membrane to sheathe the target particle, during which process the inner membrane of the growing phagocytic cup was in close contact with the target surface. On the other hand, the MerTK mutant phagocytes ate the targets by membrane ruffling, and rolling the particles with membrane wave until engulfment was completed, a typical manifestation of macropinocytosis as described by Swanson et al., (Mol. Cell. Biol. 2008, 9:639). The AFM measurement of the particle-pulling strength suggested that the MerTK-mediated phagocytosis generated a vertical pulling force, which was necessary for up-taking the engulfed particles. Whereas the MerTK mutant cells decreased this type of vertical pulling power, but instead, generated additional horizontal pushing force, conceivably from the membrane ruffling when the particle was endocytosed.
Our study showed that the MerTK mutant cells were still able to uptake the targets although with inefficacy. The MerTK mutant cells preserved the macropinocytosis mechanism for enclosing the large bound-particles. While the wild-type phagocytes generated a vertical power for pulling the bound targets into the cytoplasm, the mutants were defective, but replace with a dominant horizontal pushing force to close-in the bound particles.
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