Mfn1 puncta mark sites of reversible membrane continuity. (A) Cartoon description of a FLiP experiment designed to measure OMM and matrix continuity at Mfn1 puncta. (B) The matrix (mito-EGFP) and OMM (SNAP-OMP25) fluorescent markers were simultaneously photobleached (in box 2), which is located on one side of the Mfn1 punctum (Mfn1 in green or magenta, respectively, at white arrow). The FIs in box 1 and box 2 are imaged over time following photobleaching (PB). In snapshots shown, after t = 80 s and t =148 s after PB, there is still no diffusion of FI past the Mfn1 punctum (white arrow) for either the matrix (top images) or OMM signal (bottom images), which demonstrates that the matrix and OMM are tethered at the Mfn1 punctum but not continuous. (C) Graph of matrix and OMM FI measurements in box 1 and box 2 measured over time confirm that FI does not diffuse past the Mfn1 punctum for either marker. (D–F) Cartoon (D) and corresponding FliP experiment (E and F) where, after PB, the matrix FI does not diffuse past the Mfn1 punctum (Mfn1 in magenta with matrix, or green with OMM at white arrow) but the OMM fluorescent signal does. Thus, in this example, the OMM is continuous across the Mfn1 punctum, but the matrix is not. (G–I) Cartoon (G) and corresponding Flip experiment (H and I) for an example where for the first 37 s following PB the membranes are tethered but not continuous at the Mfn1 puncta (Mfn1 in green with matrix or magenta with OMM, white arrow). Then, at t = 37 s after PB, a fusion event restores OMM and matrix continuity, and fluorescent signal from box 1 diffuses past the Mfn1 punctum into box 2.