Figure 5: Gβ2 has a regulatory role in mitochondrial morphology.
From: G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion
(a) HeLa cells were co-transfected with DsRed-Gβ2 shRNA (red) and mitochondrial matrix localized GFP (mito-GFP), and the expression of Gβ2 shRNA (box A) and mitochondrial morphology was visualized by confocal microscopy (left panel). Scale bar, 10 μm. (b) Gβ2 shRNA HeLa cells (red) were transfected with a mutant of Flag-Gβ2 (green) as described in Figure 2. The cells were stained with 10 nM Mitotracker Deep Red for visualizing mitochondria. Scale bar, 10 μm. (c) The Gβ2 protein levels in the cells were determined by western blot: HeLa cells (left lane), HeLa cells with either Gβ2 shRNA (middle lane) or Gβ2 shRNA with reintroduced mutant of Flag-Gβ2 (right lane). (d) Gβ2 RNAi block mitochondrial fusion. HeLa cells and Gβ2 shRNA stable cells were transfected with mito-PAGFP and followed by photoactivation. Images were collected every 10 min over 30 min. Representative images shown are pseudo-coloured projections of z-series to represent changes in the fluorescence intensities. The fluorescence intensity of mito-PAGFP is highlighted in the pseudocoloured images where fusion of photoactivated 'orange' mitochondria with nonactivated 'purple' organelles is followed by the formation of the 'yellow/blue' intermediates. (e) Normalized fluorescence intensity of mito-PAGFP in HeLa cells (black) and Gβ2 shRNA stable cells (red) at each time point was measured and plotted against time. Each experimental group contains at least 20 cells. (f) Values of percent mito-PAGFP fluorescence in HeLa cells and Gβ2 shRNA stable cells decrease after 10, 20, and 30 min in each experimental group, and were plotted against time. Filled box represents Gβ2 shRNA cells and open box represents normal HeLa cells. The error bars represent s.e.m., n=26 cell time-lapse measurements per group. Differences in the mean fluorescence decrease percentage between HeLa cells and HeLa cells with Gβ2 shRNA in 10 min and in 20 min were statistically significant *P<0.05 and **P<0.005, two-tailed t-test.
