Video 1.
Mitochondrial networking is measured using a mitochondria-targeted, photoactivatable green fluorescent protein (mito-PA-GFP). Mitochondrial networking was evaluated by transfecting pulmonary artery smooth muscle cells with a Mito-PA-GFP plasmid (FuGENE HD Transfection Reagent, 1 ng/2 ml, 72 h). Cells were also incubated with tetramethylrhodamine (10 nM, 37°C, 30 min incubation), a potentiometric dye, which is taken up by mitochondria in proportion to the negativity of their membrane potential (red). PA-GFP was photoactivated in a defined area (rectangular boxes). We then calculated the mitochondrial networking factor, as previously described. In brief, the number of green pixels outside the activation box was measured at the first image (5 s) after photoactivation and divided by the number of green pixels (activated PA-GFP) inside the boxed activation area. Scale bar = 1 μm. The movie duration is 2 minutes. The mitochondrial network was imaged with a Leica STED super-resolution confocal microscope (Leica, Wetzlar, Germany) at ×63 magnification and ×4 digital zoom.