Both VPS13D and VPS13A interact with mitochondria, but only the interaction of VPS13D is mediated by Miro. (A and B) Confocal images of COS7 cells coexpressing either VPS13D^EGFP (A) or VPS13A^Halo (B) and the mitochondrial marker mito-BFP, but not Miro. The fluorescence of both VPS13 paralogues decorates mitochondria, with the fluorescence of VPS13A showing the typical discontinuous pattern that was shown (Kumar et al., 2018) to reflect its selective concentration at mitochondria–ER contact sites even without VAP overexpression. (C and D) Upon coexpression with Miro1, the localization of VPS13D^EGFP (C) but not VPS13A^Halo (D) at mitochondria is drastically enhanced. Note that VPS13D^EGFP colocalizes with Miro1 along the entire mitochondrial surface, while the punctate localization of VPS13A^Halo along mitochondria is unaffected by the overexpression of Miro1. (E) COS7 cell coexpressing VPS13A^Halo and VPS13D^EGFP as well as myc-Miro1 (not shown), showing the different localization of the two proteins on mitochondria: VPS13D^EGFP is enriched throughout the entire mitochondrial surface, while VPS13A^Halo localizes only to hot spots. Higher magnifications of the areas enclosed by stippled white rectangles are shown for all fields. Fig. S1, A and C, are also shown in Fig. 1, B and C. Scale bars, 10 µm in the main panels and 3 µm in insets.