Fig 2: WDFY2 controls MT1-MMP trafficking. a Confocal images showing localization of MT1-MMP and VAMP3 to GFP-WDFY2-positive endosome. Representative image from 10 cells. Scale bar: 10 µm, inset: 1 µm. b Confocal images showing localization of MT1-MMP and RAB4 to GFP-WDFY2-positive endosome. Representative image from 10 cells. Scale bar: 10 µm, inset: 1 µm. c TIRF micrograph of WT and WDFY2(−/−) cells transfected with pHluorin-MT1-MMP. Individual exocytic events (summed over two minutes) are indicated with a red circle. Representative image from 30 observed cells per condition. Scale bar: 10 µm, d Quantification of pHluorin-MT1-MMP exocytosis in WT and WDFY2(−/−) cells. Shown are events from three experiments, 10 cells per experiments per condition. Exocytosis events per minute are shown normalized to cell area. Student’s unpaired t test, p = 0.0026. Shown are the median, quartiles (boxes), and 1.5 times the interquartile range (whiskers). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not statistically significant. e Confocal micrographs of RPE1 (WT) and RPE1 cells stably expressing GFP-WDFY2. Cells overexpressing WDFY2 accumulates MT1-MMP in endosome positive for WDFY2. Scale bar: 10 µm, insets: 5 µm. f Quantification of MT1-MMP spots based on high-content microscopy show a significant increase in total intensity of MT1-MMP in hTERT-RPE1 GFP-WDFY2 cells when compared to hTERT-RPE1 parental cells. Shown are quantifications from three independent experiments, cells in total: hTERT-RPE1: 3570, hTERT-RPE1 GFP-WDFY2: 5250. Plotted are individual experiments and the mean ± 95% CI. p = 0.02007. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not statistically significant. g Quantification of pHlourin-MT1-MMP exocytosis in WDFY2(−/−) cells treated with siControl or siVAMP3. Shown are events from three experiments, ten cells per experiments per condition. Exocytosis events per minute are shown normalized to cell area. Student’s unpaired t-test, p = 0.0149. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not statistically significant. Source data are provided as a Source Data file

Fig 3: WDFY2 interacts and colocalizes with VAMP3. a Volcano plot identifying enriched proteins isolated from cells expressing GFP-WDFY2 by GFP-trap affinity purification of cell lysates in comparison to GFP-expressing control cells. Label-free quantification (LFQ) ratios of GFP-WDFY2/GFP pulldowns were plotted against the p-value, with the permutation-based FDR threshold (<0.01) indicated by the black lines. b GFP affinity purification of GFP-VAMP3 and mCherry-WDFY2. Affinity isolation of GFP-VAMP3 coprecipitates mCherry-WDFY2, whereas isolation of GFP alone does not coprecipitate mCherry-WDFY2. One representative blot from three experiments is shown. c Representative SIM image showing GFP-VAMP3 and mCherry-WDFY2 localization to endosomes and endosomal tubules. Scale bar overview image: 1 µm, scale bar of inset image: 0.5 µm. Representative image from 13 observed cells. d Sequential images showing GFP-WDFY2 and PA-mCherry-VAMP3 localization to endosomes and endosomal tubule. The tubule pinches off and remains positive for VAMP3. Shown are frames from a time-lapse movie with images acquired every second. Representative image from 20 cells. Scale bar: 1 µm. Source data are provided as a Source Data file

Fig 4: WDFY2 controls intracellular VAMP3 distribution. a Deconvolved widefield images of WT and WDFY2(-/-) cells transiently expressing GFP-VAMP3 and mCherry-CORTACTIN. Scale bar: 10 µm (representative image of 30 cells per condition. Hexagonal superpixel image showing VAMP3 distribution in the cells shown in the upper panel. Mean intensities of hexagonal ROIs were extracted and the ROI filled with the corresponding mean value, thereby generating superpixels. b Distribution of VAMP3 from the nucleus to the leading edge. Cells were transfected with VAMP3 and stained for CORTACTIN to identify leading edges. A line ROI from the nucleus to the leading edge was drawn, and evenly spaced ROIs were automatically generated. Mean intensity of VAMP3 in each ROI was extracted, normalized for each cell, and then plotted. 30 cells for each condition, shown are mean and 95% CI. c TIRF micrograph of WT and WDFY2(-/-) cells transfected with VAMP3-pHluorin. Individual secretion events (summed over a 2-min interval) are indicated with a red circle. Representative image from 30 cells per condition. Scale bar: 10 µm. d Quantification of VAMP3-pHluorin secretion in WT and WDFY2(-/-) cells, as shown in (c). Shown are events from three experiments, 10 cells per experiment per condition. Secretion events per minute are shown normalized to cell area. Student’s unpaired t test, p = 0.00205. Shown are the median, quartiles (boxes), and 1.5 times the interquartile range (whiskers). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not statistically significant. e Quantification of GFP-VAMP3 intensity in EEA1-positive endosomes in WT and WDFY2(-/-) cells. Plotted is the mean intensity within EEA1-positive endosomes per cell from four independent experiments (10, 12, 15, 10 cells (WT), 10, 10, 15, 10 cells (WDFY2(-/-)). Student’s unpaired t test, p = 0.00015. Shown are the median, quartiles (boxes), and 1.5 times the interquartile range (whiskers). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not statistically significant. f Quantification of GFP-VAMP3 intensity in LAMP1-positive vesicles in RPE1 (WT) and WDFY2(-/-) cells. Plotted is the mean intensity within LAMP1-positive vesicles per cell from four experiments (10, 12, 15, 10 cells (WT), 10, 10, 15, 10 cells (WDFY2(-/-)). Student’s unpaired t test, p = 0.00083. Shown are the median, quartiles (boxes), and 1.5 times the interquartile range (whiskers). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not statistically significant. Source data are provided as a Source Data file