Figure 2A Representative microscopy images (z-projections) of the mitochondrial network stained with TOMM20 in SH-SY5Y cells stably expressing a green fluorescent protein (GFP)-tagged wild-type form of tau protein (wtTau-GFP), a GFP-tagged P301L mutant form of tau protein (P301L-GFP) or cell expression the GFP only (control cells = WT-GFP). GFP signal is displayed in gray on the images, TOMM20 signal is in red, and the nuclei are delimited by white dotted lines. B-C Mitochondrial length (B) and percentage of mitochondria in the perinuclear area (C) in WT-GFP, wtTau-GFP and P301L-GFP expressing cells. On average 1,000-2,500 mitochondrial organelles were analyzed per group (n = 20-25 images per group, 3 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm B-C * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. TOMM20: translocase of the outer mitochondrial membrane complex subunit 20Visualization of the IP3R-VDAC (D), IP3R-GRP75 (E) interactions by in situ proximity ligation assay (PLA) WT-GFP, wtTau-GFP and P301L-GFP expressing cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. Data information: Data are presented as mean ±SEM. White scale bars: 20 µm GRP75: glucose-regulated protein 75 IP3R: inositol trisphosphate receptor VDAC: voltage-dependent anion channel.G-I Quantitative analysis of the PLA signal represented as number of contact points between IP3R­VDAC (G), IP3R-GRP75 (H) and VAPB-PTPIP51 (I) per cell in percentage of the WT-GFP cells. In total, 60-75 cells were analyzed per group (5 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm G-I * P<0.05, ** P<0.01, ***P<0.001; One-way ANOVA + Tukey's post hoc test. GRP75: glucose-regulated protein 75 PTPIP51: protein tyrosine phosphatase-interacting protein 51 VDAC: voltage-dependent anion channel.K-L Mitochondrial length (K) and percentage of mitochondria in the perinuclear area (L) in WT cells and Tau KO cells. On average 1,000-2,500 mitochondrial organelles were analyzed per group (n = 20-25 images per group, 3 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm * P<0.05, ***P<0.001; Student unpaired t-test.M-O Quantitative analysis of the PLA signal represented as number of contact points between IP3R-VDAC (M), IP3R-GRP75 (N) and VAPB-PTPIP51 (O) per cell in percentage of the WT cells. In total, 60-75 cells were analyzed per group (3 independent experiments). Data information: Data are presented as mean ±SEM. White scale bars: 20 µm * P<0.05, ***P<0.001; Student unpaired t-test. IP3R: inositol trisphosphate receptor; PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B; VDAC: voltage-dependent anion channel.P-R Visualization of the IP3R-VDAC (P), IP3R-GRP75 (Q) and VAPB-PTPIP51 (R) interactions by in situ PLA in WT and Tau KO cells. Interactions between the two targeted proteins are depicted in red (63X magnification) and cell surfaces are delimited by a white line. Data information: Data are presented as mean ±SEM. White scale bars: 20 µm * P<0.05, ***P<0.001; Student unpaired t-test. GRP75: glucose-regulated protein 75; IP3R: inositol trisphosphate receptor; PTPIP51: protein tyrosine phosphatase-interacting protein 51 VAPB: vesicle-associated membrane protein-associated protein B VDAC: voltage-dependent anion channel.