Figure S1. Overexpression of the ERMES... | Rockefeller University Press

Figure S1.

$Overexpression of the ERMES complex does not extend nucleus–mitochondria contacts. (A) Quantitation of the distances (nanometers) between the nuclear and mitochondrial membranes in a SEY6210.1 strain as determined by three different tomograms. The boxes represent the interquartile range of distance measurements per tomogram (tomogram 1: n = 27,743; tomogram 2: n = 40,401; tomogram 3: n = 9,509); bars mark 0.95 and 0.05 percentiles. The line at the center of the box represents the median. The dotted line represents the mean distance of all three samples. (B) 3D segmentation of the nucleus–mitochondria contact in yeast based on the tomogram in Fig. 1 B. M, mitochondrion; N, nucleus; PM, plasma membrane. (C) The nucleus–mitochondria reporter Nsg1-VC/Tom70-VN correctly identifies proximities between the two organelles. Nuclei are visualized by the red fluorophore (tdTomato) fused to a nuclear localization signal (NLS-TFP) . Mitochondria are visualized by a BFP fused to a mitochondrial targeting sequence (MTS-BFP). The fluorescent signal of the reporter is only localized to areas of proximity between mitochondria and the nucleus. Scale bar, 5 µm. (D) Quantitation of either the nucleus–mitochondria reporter (Nsg1-VN/Tom70-VC) or the ER–mitochondria reporter (Tom70-VN/Pho88-VC) sizes in control strains or those overexpressing Mdm34 (OE Mdm34) N terminally tagged with mCherry. The reporter sizes were determined by the number of pixels of the reporter signal using ScanR Olympus soft imaging solutions version 3.2. While Mdm34 overexpression affects the ER–mitochondria reporter, it does not alter the nucleus–mitochondria one. (E) An example of the effect of overexpressing Mdm34 N terminally tagged with mCherry on the background of the two reporters in D. Scale bar, 5 µm. (F) Statistical analysis of colocalization between the nucleus–mitochondria contact reporter and the ERMES subunit Mmm1 tagged with mKate on its C terminus. Cells were imaged in stationary phase, and colocalization events were counted manually using a cell counter plugin in ImageJ (Schindelin et al., 2012). Full colocalization was denoted in cases where both punctate signals were completely overlapping (see top image), partial colocalization was designated if the Mmm1 signal only colocalized with a small fraction of the reporter signal (see middle image), whereas no colocalization was scored when the reporter did not overlap any Mmm1 signal whatsoever (see bottom image). Scale bar, 5 µm; n = 400.$

Overexpression of the ERMES complex does not extend nucleus–mitochondria contacts. (A) Quantitation of the distances (nanometers) between the nuclear and mitochondrial membranes in a SEY6210.1 strain as determined by three different tomograms. The boxes represent the interquartile range of distance measurements per tomogram (tomogram 1: n = 27,743; tomogram 2: n = 40,401; tomogram 3: n = 9,509); bars mark 0.95 and 0.05 percentiles. The line at the center of the box represents the median. The dotted line represents the mean distance of all three samples. (B) 3D segmentation of the nucleus–mitochondria contact in yeast based on the tomogram in Fig. 1 B. M, mitochondrion; N, nucleus; PM, plasma membrane. (C) The nucleus–mitochondria reporter Nsg1-VC/Tom70-VN correctly identifies proximities between the two organelles. Nuclei are visualized by the red fluorophore (tdTomato) fused to a nuclear localization signal (NLS-TFP) . Mitochondria are visualized by a BFP fused to a mitochondrial targeting sequence (MTS-BFP). The fluorescent signal of the reporter is only localized to areas of proximity between mitochondria and the nucleus. Scale bar, 5 µm. (D) Quantitation of either the nucleus–mitochondria reporter (Nsg1-VN/Tom70-VC) or the ER–mitochondria reporter (Tom70-VN/Pho88-VC) sizes in control strains or those overexpressing Mdm34 (OE Mdm34) N terminally tagged with mCherry. The reporter sizes were determined by the number of pixels of the reporter signal using ScanR Olympus soft imaging solutions version 3.2. While Mdm34 overexpression affects the ER–mitochondria reporter, it does not alter the nucleus–mitochondria one. (E) An example of the effect of overexpressing Mdm34 N terminally tagged with mCherry on the background of the two reporters in D. Scale bar, 5 µm. (F) Statistical analysis of colocalization between the nucleus–mitochondria contact reporter and the ERMES subunit Mmm1 tagged with mKate on its C terminus. Cells were imaged in stationary phase, and colocalization events were counted manually using a cell counter plugin in ImageJ (Schindelin et al., 2012). Full colocalization was denoted in cases where both punctate signals were completely overlapping (see top image), partial colocalization was designated if the Mmm1 signal only colocalized with a small fraction of the reporter signal (see middle image), whereas no colocalization was scored when the reporter did not overlap any Mmm1 signal whatsoever (see bottom image). Scale bar, 5 µm; n = 400.