VPS13D recruitment by Miro GTPases. (A) Domain organization of human VPS13D and Miro1. TM, transmembrane. (B) Confocal images of COS7 cell expressing VPS13D^EGFP, showing that this protein decorates mitochondria, as shown by colocalization with mito-BFP (Box 1), in addition to being enriched in the Golgi area, visualized by the trans-Golgi marker ST-Halo (Box 2). (C) COS7 cell coexpressing VPS13D^EGFP and Halo-Miro1, showing dramatic increase in the localization of VPS13D^EGFP to mitochondria produced by Miro overexpression. The region within the white rectangle is shown at higher magnification next to the main field. Fig. 1, B and C, are also shown in Fig. S1, A and C. Scale bars, 10 µm in main panels, 3 µm in insets. (D) Recruitment of VPS13D to mitochondria can also be achieved by coexpression with Miro2. (E) VPS13D^Halo and EGFP-Miro1 colocalize in the cell body and processes of a mouse hippocampal neuron. (F) Optogenetic recruitment of the cytosolic domain of Miro1 to the OMM. Top panels: Schematic representation of the experiment, showing that Venus-iLID-Mito in the OMM recruits mCh-Miro1(ΔTM)-SspB upon blue light excitation. The recruitment of Miro1, in turn, triggers the recruitment of VPS13D. Middle and bottom panels: Confocal images of a COS7 cell show blue light–dependent recruitment and shedding of mCh-Miro1(ΔTM)-SspB and correspondingly of VPS13D^Halo. The illumination was started at time 0 s on a 5-µm² area shown in the leftmost panel. See also Video 1. Scale bars for D–F, 10 µm. (G) Time course of the recruitment and shedding of VPS13D^Halo and mCh-Miro1(ΔTM)-SSPB to the OMM. Top left panel: Snapshots of an isolated mitochondrion at the peak of recruitment. Top right panel: Example kymographs showing the increase and decrease in fluorescence along the stippled line shown in the left panels. Note that while the mCh-Miro1(ΔTM)-SSPB signal on mitochondria is lower than in the surrounding cytosol before illumination and after recovery, this is not the case for VPS13D^Halo, as a pool of this protein is bound to endogenous Miro. The kymographs start 10 s before illumination. Bottom panel: Graph showing the normalized fluorescence intensity (average ± SEM) along the length of kymographs of 18 independently illuminated mitochondria in 14 different cells. Scale bars, 1 µm. The decay in intensity of VPS13D^Halo and mCh-Miro1(ΔTM)-SspB on mitochondria was fitted to an exponential equation: For VPS13D^Halo, time constant τ = 86.51 s; 95% confidence interval, 77.64, 97.56; for mCh-Miro1(ΔTM)-SspB, time constant τ = 54.11 s; 95% confidence interval, 51.07, 57.54; adjusted R² of fits = 0.97 and 0.98, respectively.