Rapid and efficient engulfment of apoptotic cells is usually an essential

Rapid and efficient engulfment of apoptotic cells is usually an essential property of phagocytes for removal of the large number of apoptotic cells generated in multicellular organisms. the engulfment of apoptotic cells. Although anion transfer and nucleotide binding buy 9007-28-7 mutations did not impact the mitochondrial membrane potential, they exerted a dominant-negative effect on Ucp2-mediated engulfment. Furthermore, none buy 9007-28-7 of our Ucp2 mutations increased the phagocytic capacity. We determine that dissipation of the proton gradient by Ucp2 is usually not the only determinant of the phagocytic capacity and that anion transfer or nucleotide binding by Ucp2 is usually also essential for Ucp2-mediated engulfment of apoptotic cells. for 10 min and mitochondria were collected from the supernatant by centrifugation at 10,000 for 20 min. The isolated mitochondria were lysed in RIPA buffer and subjected to immunoblotting. To stain PLXNC1 cells for microscopy, LR73 cells were plated and transfected with FLAG-tagged Ucp2. One day after transfection, the cells were incubated with Mitotracker Deep Red (100 nM, Molecular Probes) in the culture medium for 20 min. The cells were then fixed with 3% paraformaldehyde (Sigma) in PBS for 30 min, permeabilized with 0.1% Triton Times-100 (Sigma) and blocked with 5% clarified milk. Antibody staining was then performed using antibodies to FLAG (Clone M5, Sigma) and AIF (Deb39D2, Cell Signaling Technologies). The stained cells were analyzed by Axio Imager Deb2 (Zeiss). Phagocytosis assay The 1 105 LR73 cells were plated on a 24-well culture plate and transfected with GFP alone or GFP and the indicated plasmids using Lipofectamine 2000. One day after transfection, the cells were incubated with 1 l of carboxylate-modified reddish fluorescent beads (2 m in diameter) (Invitrogen) or 2 106 TAMRA stained apoptotic thymocytes for 2 h, trypsinized, and analyzed by circulation cytometry (Canto II, BD). GFP and red-fluorescent double-positive cells were classified as phagocytes engulfing carboxylate beads or apoptotic thymocytes. To make TAMRA-stained apoptotic thymocytes, thymocytes were prepared from buy 9007-28-7 thymi of 5 to 8 week-old C57/W6 wild type mice, and stained with 50 M TAMRA. To induce apoptosis of thymocytes, cells cultured in RPMI 1640 medium (made up of 10% FBS and 1% penicillin-streptomycin-glutamine) were stimulated with 50 M dexamethasone (Calbiochem) in a 5% CO2 incubator for 4 h, washed with phagocyte culture medium twice, and resuspended with phagocyte culture medium at a concentration of 2 106 cells/300 l. Measuring mitochondrial membrane potential To detect mitochondrial membrane potential, LR73 cells were stained with Mitotracker Deep Red FM (Invitrogen) according to the manufacturers instructions. For TMRE staining, cells were stained with 50 nM TMRE in culture medium in a 5% CO2 incubator for 30 min and fluorescence intensity was assessed by circulation cytometry (Canto II, BD). Mean fluorescence intensity was compared to that of a control experiment and mitochondrial membrane potential was displayed as a buy 9007-28-7 value comparative to that of a control experiment. RESULTS Ucp2 promotes the engulfment of apoptotic cells but not of indigestible surrogate targets Ucp2 is usually a mitochondrial inner membrane protein that dissipates the mitochondrial membrane potential (Arsenijevic et al., 2000; Fleury et al., 1997; Flier and Lowell, 1997). Our first task was to determine whether exogenously expressed Ucp2 was localized appropriately in mitochondria. Therefore, Ucp2-FLAG was expressed in LR73 cells, which were used as phagocytes in this study, and was detected only in the mitochondrial portion, not in the cytosolic portion (Fig. 1A). The co-localization of Ucp2-FLAG and apoptosis-inducing factor (AIF), a protein that localizes to the mitochondrial inner membrane, indicated that Ucp2 was localized properly, despite being exogenously expressed in LR73 cells (Fig. 1B). Next, we tested whether Ucp2 reduces the mitochondrial membrane potential. For this purpose, Ucp2-FLAG was expressed in LR73 cells, and these cells were stained with MitoTracker, a dye whose accumulation in mitochondria is usually proportional to the mitochondrial membrane potential. Ucp2-positive cells incorporated much less MitoTracker than Ucp2-unfavorable cells (Fig. 1C), indicating that the mitochondrial membrane potential was reduced in the former cells. This observation was not limited to LR73 cells, because NIH/3T3 cells conveying Ucp2 were also less able to incorporate MitoTracker (Fig. 1D), indicating that overexpression of Ucp2 in mammalian cells reduces the mitochondrial membrane potential. An alternate method to detect disruption of.

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