Mitochondrial fusion events in FDB fibers. (A) FDB fiber expressing both mtDsRed and mtPA-GFP. During photoactivation of mtPA-GFP, mtDsRed is photobleached, allowing detection of the mixing of each fluorescent protein after mitochondrial fusion. Left image shows the photoactivation area (white dashed box) and its surrounding region 72 s after 2P photoactivation. Mitochondria shown in the white rectangular box undergo two sequential longitudinal fusion events at 76 s and 244 s, respectively. Event A: mitochondrion 1 donates fluorescent mtPA-GFP to mitochondrion 2 (acceptor) abruptly at 76 s. At the same time the green acceptor mitochondrion donates fluorescent mtDsRed to mitochondrion 1. Line scans also show the abrupt and complementary intermitochondrial transfer of the fluorescent proteins (F). Subsequently, 2.8 min later, mitochondrion 2 fuses with mitochondrion 3 (event B, 244 s). The line scan is only shown for the green color because of the relatively small gradient in the red fluorescence. Bottom plots show mtPA-GFP and mtDsRed mixing kinetics of each mitochondrion that undergoes fusion. Continuous line, PA-GFP accepting organelles; dashed lines, PA-GFP donating organelles. A single representative example is shown out of 333 events from more than 5 experiments. (B) Fusion event rate and orientation characteristics in different muscle cells. Left: representative images of mtPA-GFP diffusion within the first 8–24 s of photoactivation and further spreading after 8 min. Middle: mtPA-GFP fluorescence decay evaluated inside the photoactivation area. Gray curves, individual regions; black curves represent the mean curves. The fusion events rate is also indicated for each cell type (FDB fibers, n = 333 events, 86 cells; rat myotubes, n = 123 events, 5 cells; human differentiating myoblasts, n = 309, 15 cells; and H9c2 myoblasts, n = 209, 10 cells). Right: orientation distribution of the fusion events in each cell type. e-e, end to end; e-s, end to side; s-e, side to end; s-s, side to side.