Recruitment of VPS13D by Miro GTPases requires their intact N-GTPase domain and EF-hand domains and the β-propeller region of VPS13D. (A) Domain organization of human Miro1 and mutations used for the experiments shown in B. TM, transmembrane. (B) Confocal images of COS7 cells showing that the recruitment of VPS13D^Halo by EGFP-Miro1 is impaired by mutations in the GTP binding site of its N-GTPase domain and in the Ca²⁺ binding sites of both its EF hands, but not by a mutation in the GTP binding site of its C-GTPase domain. Asterisks represent the point mutations in the Miro1 construct. Scale bar, 3 µm. (C) Top: Cartoons showing VPS13D constructs used for the experiments shown below. Bottom: Confocal images showing that removal of the C-terminal half of the protein or of the β-propeller region selectively, but not of the DH-PH domain, abolishes recruitment of VPS13D^EGFP by Halo-Miro1. All of several constructs encoding only the β-propeller region formed small aggregates, possibly due to misfolding. See also Fig. S4. Scale bar, 10 µm.