Figure 7.

Depolarized mitochondria are rescued by ER-associated fusion. (A) Representative image of a U-2 OS cell expressing GFP-Mfn1 (yellow), mito-BFP (blue), and SNAP-Sec61β (green) and loaded with a dye that accumulates within the matrix of mitochondria in proportion to membrane potential [30 nm] TMRE (red). Note the occurrence of an Mfn1 punctum at an ER tubule crossing at the border of electrically uncoupled mitochondrial segments. (B) Another example as in A. (C) Table of mitochondria imaged as in A and B shows that ER MCSs and Mfn1 puncta mark the boundaries between the vast majority of electrically uncoupled segments. (D) Cells expressing an OMM marker (SNAP-OMP25) were loaded with [30 nM] TMRE to indicate membrane polarization. Individual mitochondria were irradiated with 405 nm (in dashed boxes) to photobleach the OMM marker and depolarize the membrane. Depolarized mitochondria were imaged for 8 min following irradiation. In this example, a photobleached and depolarized mitochondrion fuses ∼382 s after irradiation, and this fusion leads to an increase in OMM FI and a subsequent increase in TMRE signal (indicating rescue of membrane potential). (E) Plot of relative FI of SNAP-OMP25 and TMRE signal for experiment in D. (F) Representative time-lapse image series of irradiated mitochondria in cells expressing GFP-Drp1 (to label ER MCSs), SNAP-OMP25, and loaded with [30 nM] TMRE. Dashed box indicates irradiated area. Arrows indicate location of fusion event. (G) Relative FI plots of SNAP-OMP25 and TMRE signal from the time-lapse in F. Note that the TMRE signal recovers shortly after the time of OMM FI increase by fusion (arrow). (H) Two groups of mitochondria in (D; control and irradiated) were imaged over time following irradiation and were assigned to three categories: fission first, fusion first, and no event. The graph shows the distribution of categories between control and irradiated. *, P < 0.05 by χ² analysis (n = 78 mitochondria for control, n = 81 mitochondria for irradiated). (I) As in H except for the addition of transfected Drp1 (n = 91 mitochondria for control, n = 88 mitochondria for irradiated). *, P < 0.05 by χ² analysis. (J) Fusion rescues mitochondrial membrane potential. The mean normalized TMRE FI was measured for the 10 frames directly preceding fusion and the 10 frames immediately after fusion for individual irradiated mitochondria. The two measurements were connected by lines. Red lines indicate an increase in TMRE intensity after fusion (mean FI increase = 65%), and black lines indicate no change or a decrease in TMRE intensity. Events are categorized from irradiation experiments in U-2 OS cells expressing SNAP-OMP25 alone and SNAP-OMP25 with GFP-Drp1. Scale bars, 1 µm.