(A) Immunofluorescence of Kermit cells transduced with shDRP1, using an anti-MFF antibody (green). Mitochondrial matrix (mtBFP) is shown in red. Insets on the right correspond to the framed areas on the left. Arrowheads point at naturally occuring constrictions on the mitochondria. Plots are linescans of the mitochondria (red) and MFF (green) signals around the constriction. X-axis is in µm. Y-axis is normalized fluorescence in arbitrary units. (B) as in A, but the cells were grown on gramophone records. Top left panel is a 3D projection of the same cell rotated by 90° around the x-axis. The white box on the left corresponds to the magnified area on the right. Arrowheads indicate mitochondrial constrictions at the groove’s edge. (C) U2OS cells were transduced with lentiviral vectors expressing mtBFP, RFP-Lifeact, GFP-MFF as well as shRNA targeting DRP1. Puromycin-resistant cells (shRNA-positive) were then infected with RFP-labeled S. flexneri, and imaged by time-lapse microscopy. MFF is recruited to sites of encounter with S. flexneri (white arrowheads). Right panels also show two examples of MFF enrichment at sites of mitochondria thinning (curly brackets), as indicated by reduction of matrix mtBFP signal, independent of Shigella encounter. (D) Line scan of mtBFP and MFF signal of the white dotted line in (C). Arrowhead and curly bracket correspond to same zones in (C). Normalized background-subtracted pixel values are plotted as arbitrary units. (E) Z-projected image of a transduced U2OS cell spanning the edge of vinyl groove expressing mtBFP, GFP-MFF as well as shRNA targeting DRP1. The groove’s edge is indicated by two facing arrowheads on top right panel. Two stabilized individual mitochondrial tubules span over the edge (white line), and show loss of matrix BFP signal and increased MFF signal. Another example of GFP-MFF enrichment to a constricted mitochondrial tubule outside of the edge area is indicated by a curly bracket. (F) Dotted white lines 1 and 2 in (E) are selected for line plots as in (D). (G) U2OS cells were transduced with lentiviral vectors expressing mtBFP, mCherry-Fis1TM (OMM), as well as shRNA targeting DRP1. Puromycin-resistant cells (shRNA-positive) were then transfected with GFP-MFF, and imaged by time-lapse microscopy. MFF spontaneously stabilizes thin mitochondrial section (curly brackets) that are devoid of matrix staining but retain continuous OMM signal. (H) Line scan of mtBFP, GFP-MFF and OMM signal of the curly bracket in (G). Scale bars, A-B, 5 µm, C-H 2 µm.