Mitofusins form puncta that localize to ER tubules. (A) Representative image of a U-2 OS cell expressing mCh-Sec61β (ER, green) and immunostained with antibody against Mfn1/2 (magenta) and Tom20 (gray). Magnified merged image of insets show punctate distribution of Mfn1/2 (magenta) relative to mitochondria and ER (in right panels). (B) Graph of the MCC measured for Mfn1/2 relative to the ER, mitochondria, or a 90° rotated Mfn1/2 relative to the ER. Error bars represent SEM. ***, P < 0.0001 by two-tailed paired t test; normality was determined by Shapiro–Wilk test; n = 12 regions. (C) Representative live-cell image of a cell expressing GFP-Mfn1 (magenta), mCherry-Sec61β (green), and mito-BFP (gray). Magnified merged images of inset show GFP-Mfn1 puncta relative to mitochondria and ER (right panels). (D) Graph of the MCC of GFP-Mfn1 relative to ER, mitochondria, or a 90° rotated MFN1 relative to the ER. Significance determined as in B; ***, P = 0.0007, n = 10 regions. (E) Time-lapse images of live cells as in C reveals tracking of GFP-Mfn1 (magenta) puncta with ER tubules (green) over time. (F) Graph shows that the vast majority of Mfn1 puncta (84%) maintain their association with ER tubules during the entire 2-min video (n = 50 puncta from six cells). (G) Representative images of U-2 OS cells, GFP-Mfn1 or GFP-Mfn1-E209A. Insets and dashed lines correspond to the location of the linescans graphed below. (H) Linescan of Mfn1 signal on mitochondria showing the mean of five cells with seven linescans (as in Fig. 1 G) from each cell. Error bars represent SEM. Cartoon describes where measurements are taken and how signal is distributed on mitochondria. (I) Graph showing the difference in ratio of maximum punctate signal over the mean diffuse mitochondrial signal from GFP-Mfn1 and GFP-Mfn1-E209A. **, P = 0.0026 by two-tailed t test. Scale bars for whole cell image = 5 µm; insets = 1 µm. Error bars represent SEM.