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Fig. 2. Overexpression of dynamitin (p50) induces mitochondrial collapse around the nucleus and MTOC. (A) HeLa cells were co-transfected with 0.5 µg mito.DsRed (a,b,e,f) and 1 µg pAdTrack-CMV (empty vector/control) (a,b) or p50.EGFP (e,f). Mitochondria were visualised with MitoTrackerRed^TM (c,d,g,h) in cells transfected with either pcDNA3 (empty vector/control) (c,d) or p50.pcDNA3 (g,h). Hatched boundaries in c and d indicate the cell periphery. Twelve hours after transfection, cells were either imaged on a Leica TCS-NT confocal microscope (a,b,e,f) or fixed (c,d,g,h) and were then immunostained with a mouse monoclonal anti-p50 antibody (1:250) and visualised with an Alexa Fluor 488 goat anti-mouse secondary antibody (1:500) before confocal imaging. p50-overexpressing cells were identified by exciting either EGFP or the secondary antibody at 488 nm. DsRed or MitoTrackerRed^TM fluorescence was visualised in the same cells by exciting at 568 nm. Typical confocal images of DsRed-labelled mitochondria in control or p50-expressing cells are shown in a and e, MitoTrackerRed^TM fluorescence in c and g, composite images b,d,f and h. Note the re-localisation of mitochondria close to the nucleus in p50-expressing cells (e versus a, g versus c). Bars, 10 µm. (B) Reconstruction of adjacent transmission electron microscopic fields showing a (B) control-HeLa cell with randomly distributed mitochondria throughout the cytosol and a (C) p50-transfected HeLa cell with mitochondria redistributed to the perinuclear region within 2-3 µm of the nuclear membrane. Mitochondria are largely absent at the cell periphery. (D) Same section as shown in C at higher magnification. Tight association of mitochondria with the ER is evident. ER, endoplasmic reticulum; m, mitochondria; nu, nucleus; pm, plasma membrane.

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