Figure 3.
Mfn1 puncta and fusion events localize to bona fide ER contact sites. (A) Cartoon demonstrating the ddFP system: the monomers are targeted to the mitochondria (B) and the ER (RA), and an increase in fluorescent signal is indicative of dimerization at MCSs. (B) Histogram of ddFP spot values taken from regions of ER alone (monomer) and regions where ER crossed mitochondria (dimer) validates signal increase upon dimerization (n = 120 spots). (C) Representative image of cells expressing GA-MFF (green dimerization partner), B-MFF, and mito-mScarlet (magenta) shows that B-MFF is not punctate without an ER partner. (D) Representative image of a cell expressing the ddFP system (RA-Sec61β + B-MFF) with a marker for ER (green) and mitochondria (mito-BFP; gray). Note that a bright ddFP signal (magenta) accumulates at a position where an ER tubule crosses the mitochondria. Linescan analysis of dashed line along the mitochondria shows relative FI of ddFP domains relative to ER tubule crossing. (E) Most but not all ER tubule crossings (75%) label positive for ddFP dimerization, indicating that ∼75% of ER tubule crossings are bona fide MCSs. (F) Representative image of cells expressing the ddFP system (magenta), GFP-Mfn1 (green), and mito-BFP (gray). (G) Magnified merged images and linescan analysis of F show the position of ddFP-positive domains relative to GFP-Mfn1 puncta along mitochondria. (H) Graph of MCC for GFP-Mfn1 puncta relative to ddFP-positive domains, mitochondria, or 90° rotated GFP-Mfn1 puncta shows that Mfn1 puncta and ddFP ER MCSs strongly overlap. (I) Representative merged time-lapse images of a ddFP-positive fusion event in a cell expressing the ddFP system (magenta) and mito-BFP (gray). (J) Linescan analysis of dashed line in I shows the relative FI of ddFP-positive domains along the length of a mitochondrion at the location of fusion (white arrow). (K) Percentage of fusion events that are ddFP positive for ER contact (90%) as in I relative to the mean coverage of ddFP (13%) on mitochondria. Scale bar: 5 µm in whole cell images; insets, 1 µm.

Mfn1 puncta and fusion events localize to bona fide ER contact sites. (A) Cartoon demonstrating the ddFP system: the monomers are targeted to the mitochondria (B) and the ER (RA), and an increase in fluorescent signal is indicative of dimerization at MCSs. (B) Histogram of ddFP spot values taken from regions of ER alone (monomer) and regions where ER crossed mitochondria (dimer) validates signal increase upon dimerization (n = 120 spots). (C) Representative image of cells expressing GA-MFF (green dimerization partner), B-MFF, and mito-mScarlet (magenta) shows that B-MFF is not punctate without an ER partner. (D) Representative image of a cell expressing the ddFP system (RA-Sec61β + B-MFF) with a marker for ER (green) and mitochondria (mito-BFP; gray). Note that a bright ddFP signal (magenta) accumulates at a position where an ER tubule crosses the mitochondria. Linescan analysis of dashed line along the mitochondria shows relative FI of ddFP domains relative to ER tubule crossing. (E) Most but not all ER tubule crossings (75%) label positive for ddFP dimerization, indicating that ∼75% of ER tubule crossings are bona fide MCSs. (F) Representative image of cells expressing the ddFP system (magenta), GFP-Mfn1 (green), and mito-BFP (gray). (G) Magnified merged images and linescan analysis of F show the position of ddFP-positive domains relative to GFP-Mfn1 puncta along mitochondria. (H) Graph of MCC for GFP-Mfn1 puncta relative to ddFP-positive domains, mitochondria, or 90° rotated GFP-Mfn1 puncta shows that Mfn1 puncta and ddFP ER MCSs strongly overlap. (I) Representative merged time-lapse images of a ddFP-positive fusion event in a cell expressing the ddFP system (magenta) and mito-BFP (gray). (J) Linescan analysis of dashed line in I shows the relative FI of ddFP-positive domains along the length of a mitochondrion at the location of fusion (white arrow). (K) Percentage of fusion events that are ddFP positive for ER contact (90%) as in I relative to the mean coverage of ddFP (13%) on mitochondria. Scale bar: 5 µm in whole cell images; insets, 1 µm.

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