Recruitment of VPS13D to mitochondria by Miro is unaffected by changes in cytosolic Ca²⁺. (A and B) COS7 cell expressing EGFP-Miro1, VPS13D^Halo and the RFP genetically encoded Ca²⁺ indicator for optical imaging (R-GECO). (A) Cytosolic Ca²⁺ levels before and after addition of the sarco/endoplasmic reticulum Ca²⁺-ATPase pump inhibitor thapsigargin. (B) Time-lapse confocal images showing snapshot from the time points indicated in A and demonstrating that the localization of VPS13D^Halo on mitochondria in the presence of overexpressed Miro1 is not affected by the Ca²⁺ concentration. (C) Time-lapse confocal images showing that the binding of VPS13D^Halo to mitochondria in the presence of coexpressed myc-Miro1 is unaffected by the addition of EGTA and BAPTA-AM to lower intracellular cytosolic Ca²⁺. Scale bars, 10 µm.