Fig 2: Parkin-mediated Miro degradation correlates with the dissociation of VPS13D from mitochondria. COS7 or HeLa cells were cotransfected with myc-Miro1, VPS13D, and EGFP-Parkin. (A and D) Western blots showing the decrease of myc-Miro1 level upon 10 µM valinomycin treatment of COS7 (A) or HeLa cells (D). Scarlet, a modified RFP, was visualized by anti-RFP antibodies. Sizes in kD are indicated next to each blot. (B, C, and E) Confocal time lapses of COS7 (B and C) or HeLa cells (E) coexpressing EGFP-Parkin, VPS13D^Halo, and myc-Miro1. VPS13D, initially recruited to mitochondria by Miro, is shed from mitochondria and relocated to the cytosol upon treatment with valinomycin, which induces the recruitment of EGFP-Parkin. The boxed regions in B are shown in higher magnification in C. Scale bars, 30 µm in the main panels and 3 µm in insets.

Fig 3: Localization of the constructs comprising the ß-propeller region of VPS13D only. (A) Cartoon depicting full-length VPS13D and the VPS13D fragments used in the imaging experiment shown in B and C. (B and C) Confocal images of representative examples of the heterogeneous localization of two mCherry-tagged VPS13D fragments in COS7 cells coexpressing EGFP-Miro1. The first column shows a cytosolic localization; the second column shows the presence of small aggregates; and the third and fourth columns show the colocalization of VPS13D with Miro on mitochondria whose localization is disrupted. Scale bars, 10 µm. (D) List of VPS13D fragments containing the ß-propeller region that were tested for Miro-induced recruitment to mitochondria. All of them behaved similarly. The constructs used for fields in B and C are highlighted in green.

Fig 4: Miro is required for VPS13D recruitment to mitochondria. (A) Confocal images of HeLa cells showing the moderate recruitment of VPS13D^EGFP to mitochondria in control cells (left panel) and the loss of such signal upon knockdown of the two Miro genes via RNAi (right panel). Areas enclosed by stippled white rectangles are shown at higher magnification next to the main field, and they also show the localization of the mitochondrial marker mito-BFP. Scale bars, 30 µm in the main panel and 3 µm in the insets. (B) Immunoblot (IB) of mitochondrial fractions from HeLa cells showing the decrease of Miro1 and Miro2 levels upon RNAi treatment. Sizes in kD are indicated next to the blot. The black arrowhead indicates the Miro1 band. (C) Quantification of VPS13D^EGFP enrichment at mitochondria in control conditions and upon Miro knockdown or overexpression. The signal from mito-BFP was first used to generate a mitochondrial mask and a mask profiling a thin (1-pixel wide) cytosolic area surrounding mitochondria; the intensity from EGFP was then measured within each of these masks, and, for each cell analyzed, the ratio between these two measurements was plotted on the graph. Number of cells analyzed for scrambled (Scr) RNAi, Miro RNAi, and mCh-Miro1 conditions are 204, 204, and 96, respectively. ****, P < 0.0001 (Welch’s corrected ANOVA with Games-Howell's post hoc test).

Fig 5: 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.