Figure 6.
Overexpression of VAPB or PTPIP51 Increases IP3 Receptor3-VDAC1 Interactions and ER-Mitochondria Ca2+ Exchange
(A) HeLa cells were transfected with control EGFP vector, CFP-VAPB, or EGFP-PTPIP51 and proximity ligation assays (PLAs) for IP3 receptor3-VDAC1 interactions then performed. Representative images with PLA signals in the different transfected cells are shown. The scale bar represents 10 μm. The bar chart shows quantification of PLA signals. Data were analyzed by one-way ANOVA and Tukey’s post hoc test. n = 77–142 cells per condition from three independent experiments. Error bars are SEM; ∗∗∗p ≤ 0.001.
(B) Cytosolic (upper) and mitochondrial (lower) Ca2+ levels following oxotremorine-M (OxoM)-induced Ca2+ release from ER stores. HEK293 cells were co-transfected with M3R and either control empty vector (CTRL), Myc-VAPB, or HA-PTPIP51 and treated with oxotremorine-M. Representative traces of Fluo4 (cytosolic) and Rhod2 (mitochondrial) fluorescence are shown on the left, and normalized peak values are shown on the right. Fluo4 and Rhod2 fluorescence shows transient increases in cytosolic and mitochondrial Ca2+ levels upon OxoM-induced Ca2+ release from ER stores. Compared to control, VAPB and PTPIP51 expression decreased peak cytosolic and increased peak mitochondrial Ca2+ levels. Data were analyzed by one-way ANOVA and Tukey’s post hoc test. n = 48–81 cells from three independent experiments. Error bars are SEM; ∗p ≤ 0.05; ∗∗∗p ≤ 0.001.
See also Figure S3.