Figure 2.
MDCs form and stably persist at ER–mitochondria contacts.(A) Superresolution images and line scan analysis of yeast expressing Tom70-mCherry and Sec61-GFP treated with rapamycin. Asterisk (Tom70*) indicates that the fluorescence intensity was increased. Line marks fluorescence intensity profile position. Left and right y axes of the line scan graph correspond to Tom70-mCherry and Sec61-GFP fluorescence intensity, respectively. Bracket marks an MDC. Black arrows mark Sec61 associated with MDCs. Images show a single focal plane. Percent associated shows mean ± SEM of three replicates, n = 10 MDCs per replicate. Scale bar = 2 µm. (B) Superresolution images and line scan analyses of yeast expressing Tom70-mCherry and Mmm1-GFP, Mdm34-GFP or Gem1-263GFP treated with rapamycin. Gem1 was internally tagged with GFP after amino acid 262 to create Gem1-263GFP, a functional fusion protein (see Fig. S1, C–E, and Materials and methods for details). Asterisks (Tom70*, Mmm1*, Mdm34*, and Gem1*) indicate that the fluorescence intensity was increased. Lines mark fluorescence intensity profile position. The y axis of the line scan graph corresponds to Tom70-mCherry and Mmm1-GFP, Mdm34-GFP, or Gem1-263GFP fluorescence intensity. Brackets mark MDCs. White and black arrows mark Mmm1, Mdm34, or Gem1 associated with MDCs. Images show single focal planes. Percent associated shows mean ± SEM of three replicates, n = 10 MDCs per replicate. Scale bars = 2 µm. (C) Superresolution images and line scan analysis of yeast expressing Tom70-mCherry and Lam6-GFP treated with rapamycin. Asterisk (Tom70*) indicates that the fluorescence intensity was increased. Line marks fluorescence intensity profile position. The y axis of the line scan graph corresponds to Tom70-mCherry and Lam6-GFP fluorescence intensity. Bracket marks an MDC. White and black arrows mark Lam6 associated with MDC. Images show single focal plane. Percent associated shows mean ± SEM of three replicates, n = 10 MDCs per replicate. Scale bar = 2 µm. (D) Superresolution time-lapse images of yeast expressing Tom70-mCherry and Mdm34-GFP treated with rapamycin. Arrowheads mark Mdm34 associated with MDCs. Images show maximum intensity projections (MIP) and single focal planes (SFP). Percent associated shows mean ± SEM of three replicates, n = 23 MDCs total. Scale bar = 2 µm. See also Fig. S1 and Video 2. A.U., arbitrary units; Rap, rapamycin.

MDCs form and stably persist at ER–mitochondria contacts. (A) Superresolution images and line scan analysis of yeast expressing Tom70-mCherry and Sec61-GFP treated with rapamycin. Asterisk (Tom70*) indicates that the fluorescence intensity was increased. Line marks fluorescence intensity profile position. Left and right y axes of the line scan graph correspond to Tom70-mCherry and Sec61-GFP fluorescence intensity, respectively. Bracket marks an MDC. Black arrows mark Sec61 associated with MDCs. Images show a single focal plane. Percent associated shows mean ± SEM of three replicates, n = 10 MDCs per replicate. Scale bar = 2 µm. (B) Superresolution images and line scan analyses of yeast expressing Tom70-mCherry and Mmm1-GFP, Mdm34-GFP or Gem1-263GFP treated with rapamycin. Gem1 was internally tagged with GFP after amino acid 262 to create Gem1-263GFP, a functional fusion protein (see Fig. S1, C–E, and Materials and methods for details). Asterisks (Tom70*, Mmm1*, Mdm34*, and Gem1*) indicate that the fluorescence intensity was increased. Lines mark fluorescence intensity profile position. The y axis of the line scan graph corresponds to Tom70-mCherry and Mmm1-GFP, Mdm34-GFP, or Gem1-263GFP fluorescence intensity. Brackets mark MDCs. White and black arrows mark Mmm1, Mdm34, or Gem1 associated with MDCs. Images show single focal planes. Percent associated shows mean ± SEM of three replicates, n = 10 MDCs per replicate. Scale bars = 2 µm. (C) Superresolution images and line scan analysis of yeast expressing Tom70-mCherry and Lam6-GFP treated with rapamycin. Asterisk (Tom70*) indicates that the fluorescence intensity was increased. Line marks fluorescence intensity profile position. The y axis of the line scan graph corresponds to Tom70-mCherry and Lam6-GFP fluorescence intensity. Bracket marks an MDC. White and black arrows mark Lam6 associated with MDC. Images show single focal plane. Percent associated shows mean ± SEM of three replicates, n = 10 MDCs per replicate. Scale bar = 2 µm. (D) Superresolution time-lapse images of yeast expressing Tom70-mCherry and Mdm34-GFP treated with rapamycin. Arrowheads mark Mdm34 associated with MDCs. Images show maximum intensity projections (MIP) and single focal planes (SFP). Percent associated shows mean ± SEM of three replicates, n = 23 MDCs total. Scale bar = 2 µm. See also Fig. S1 and Video 2. A.U., arbitrary units; Rap, rapamycin.

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