Fig. 1. Drosophila SVIP directly recruits VCP to tubular lysosomes in muscles.

a Distinct co-factors (shapes of different colors adjacent to arrows) spatially regulate VCP subcellular distribution and function. b Schematic of Drosophila CG32039 gene and protein organization (top) and sequence alignment with human, mouse, and rat SVIP orthologs (bottom). The region shaded in magenta corresponds to the predicted VCP-interaction motif (VIM), M indicates myristoylation site, asterisks indicate arginine residue mutated in part C, and serine highlighted in yellow indicates SVIP patient mutation identified (Fig. 9). c In vitro binding of recombinant MBP-VCP and GST-SVIP fusion proteins. d Co-localization of endogenously tagged SVIP-sfGFP and Spin-RFP (lysosomes) in Drosophila muscles. The dotted line indicates the nucleus. Boxes indicated insets, below at higher magnification. e Mander’s correlation coefficients for SVIP-sfGFP (GFP) and Spin-RFP (RFP) signal (n = 13 muscle cells examined over five independent animals). f Co-imaging of VCP-sfGFP and Spin-RFP in control and SVIP⁺ over-expression in muscles. Dotted lines as in d. g Co-imaging of endogenously tagged VCP-sfGFP and UAS-Spin-RFP (lysosomes) in control compared to SVIP⁺ and SVIP^RR over-expression in muscles. Dotted lines as in d and f. Yellow arrow (middle row) highlights signal co-localization. h Nuclear to cytoplasmic ratios of VCP-sfGFP signal was quantified for the genotypes indicated (top graph). Spin-RFP signal was used to create a masked ROI (white lines, “mask”) and plots represent GFP/RFP signal intensities within the masked region (bottom graph, see “overlay” in g). Data, artificial imaging units. (n = 10 muscle cells examined over five independent animals). See also Supplementary Fig. S1. Scale bars 5 μm. Data presented as mean and SEM. Student’s t-test for individual comparisons (*p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001).