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Nora B. Caberoy, Gabriela Alvarado, Wei Li; Tubby Stimulates Microglial Phagocytosis Through Mertk Receptor. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3343.
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Mutation in tubby causes retinal degeneration with undefined mechanism. We recently identified tubby as a MerTK-specific eat-me signal for retinal pigment epithelium (RPE) and macrophage phagocytosis. Microglia are important professional phagocytes to remove up to ~50% of unwanted excess neurons during retinogenesis and share many functional and molecular similarities with RPE and macrophages. The purpose of this study is to characterize the functional role of tubby as a new eat-me signal in microglial phagocytosis.
Microglial phagocytosis was analyzed using florescence-labeled apoptotic cells or membrane vesicles in the presence or absence of purified recombinant tubby. Phagocytosed fluorescent cargos were analyzed by confocal microscopy and quantified for their fluorescence intensity. MerTK activation was characterized by Western blot to detect receptor autophosphorylation using anti-phospho-MerTK antibody. Mutant tubby associated with retinal degeneration was expressed, purified and analyzed for its capacity to stimulate microglial phagocytosis. MerTK-dependent rearrangement of cytoskeletal protein non-muscle myosin II (NMMII) during phagocytosis and phagosome co-localization with ingested cargos were analyzed by immunocytochemistry.
Tubby stimulated microglial phagocytosis of apoptotic cells or membrane vesicles in a concentration-dependent manner. Tubby at 0.1 nM significantly stimulate microglial phagocytosis with a plateau at 10 nM. Tubby failed to induce microglial phagocytosis of healthy cells. Mutation deletion of tubby C-terminal 44 amino acids abolished tubby stimulation of microglial phagocytosis. Tubby induced MerTK activation with receptor phosphorylation at 0.1 nM and reached to maximal receptor activation at ~10 nM. Correlation of tubby concentration curves between microglial phagocytosis and MerTK activation implicated the important role of MerTK in tubby-mediated microglial phagocytosis. Immunocytochemical studies showed that phagocytosed cargos were co-localized with phagosome biomarkers and rearranged NMMII.
These data indicated that tubby is a MerTK-specific ligand for microglial phagocytosis. These findings suggest that tubby may play an important role for the clearance of unwanted excess neurons in retinogenesis and neurogenesis during which tubby with unconventional secretion is highly expressed in the central nervous system.
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