NME3 is enriched at the mitochondrial contact interface dependent on PLD6. (A) The enrichment of NME3-GFP at mitochondrial contact interface prior to fusion. The dynamic distribution of NME3-GFP in two contact mitochondria was observed in Cos-7 cells co-transfected with NME3-GFP and mito-BFP which labels the mitochondrial matrix. Arrows indicate the contact interface between two closely positioned mitochondria. Scale bar, 10 μm. Bar for inset images, 2 μm. (B) The enrichment of different mutant NME3-GFP at the mitochondrial contact interface. The GFP intensity of these constructs was converted to a heat map. Scale bar, 2 μm. (C) The intensity of GFP-tagged proteins at the mitochondrial contact interface was quantified, normalized with the non-contact area, compared to the control mitochondrial outer membrane protein, TOM20, and shown in C. (D) The following fate of two contact mitochondria after 1 min was analyzed and shown in D. (E) Knock-down efficiency of PLD6. HeLa cells were transfected with scrambled or PLD6 siRNA SMARTPool, and the knockdown efficiency was quantified by Western blotting and shown below the panel. (F) PLD6 depletion perturbs the enrichment of NME3-GFP at the mitochondrial contact interface. (G) NME3-GFP expressed in control or PLD6-depleted HeLa cells was imaged by time-lapse microscopy, quantified as described in B and shown in G. Scale bar, 2 μm. Data are expressed as mean ± SD of three independent experiments and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. Source data are available for this figure: SourceData F4.