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Fig. 4. The effect of p50-overexpression on mitochondrial distribution is mimicked by anti-dynein antibody. (A) Cells were transfected with 0.5 µg mito.DsRed for 16 hours then microinjected together with either 1 mg ml–1 control IgG (a,b) or a mouse monoclonal anti-dynein intermediate chain (DIC) antibody (c,d) and 1 mg ml–1 Oregon Green BAPTA dextran. Two to four hours after injection, antibody-treated cells were identified by exciting Oregon Green at 488 nm and using fluorescein isothiocyanate filters for fluorescence emission on the Leica confocal microscope. Typical 568 nm in vivo confocal images of mitochondria in control IgG (a) and anti-DIC antibody (c) injected cells; b and d composite images. Bars, 10 µm. (B) The collapsed mitochondria phenotype was scored in cells transfected with p50 or microinjected with the anti-DIC antibody. Data are presented as the number of cells exhibiting this phenotype as a fraction of the total number of cells transfected or microinjected. Two-hundred p50-expressing and ten microinjected cells were analysed in five independent experiments. (C) Effects of p50 overexpression on endocytic organelles. HeLa cells were transiently transfected with p50.EGFP (as described above), and fixed and stained 12 hours later. Immunocytochemistry was performed using a (a,b) monoclonal anti-TGN38 antibody (Lee and Banting, 2002) and a (c,d) monoclonal anti-lysosome-associated membrane protein LAMP-1 antibody (a marker of late endosomes and lysosomes (Ihrke et al., 1998). Fluorescence of an Alexa 568 goat anti-mouse secondary antibody was then visualised in a and c at 568 nm. Intrinsic EGFP fluorescence was visualized at 488 nm in b and d, which are composite images also showing TGN38 and LAMP1 immunostaining, respectively .Arrowheads in a) indicate the position of p50-expressing cells; arrowheads in c) indicate the extreme shift of LAMP-positive late endosomes and lysosomes into the periphery after p50 overexpression. Asterisks indicate control cells. Bars, 10 µm. (D) Overexpression of p50 has no effect on mitochondrial membrane potential (
mit). Cells were incubated with 1 µM TMRE for 30 minutes and confocal images were subsequently acquired. Using identical confocal settings, TMRE fluorescence was 28±3.5 (n=6) and 30±3.1 (n=14) arbitrary units in control (a) and p50-expressing (b) cells, respectively. Bars=10 µm (A-E). Hatched boundaries in A, C and D, obtained from an overlay with the transmitted image of the cell, indicate the cell periphery.
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