Rfp booster

hTERT-RPE1 cells stably expressing GFP-WDFY2 were transiently transfected with mCherry-RAB5/RAB4/RAB11, mCherry-VAMP3 or stained with anti-RAB7/VPS26/VPS35. Cells were fixed with 4% PFA and 0.1% glutaraldehyde and stained with mouse anti-GFP antibody (Roche) and RFP-Booster (Chromotek) to visualize tagged proteins. Alternatively, anti-GFP antibody (Roche) and antibodies targeting RAB7, VPS26, and VPS35 and fitting secondary antibodies were used to visualize endogenous proteins. Samples were mounted in ProLongGold Antifade Reagent (Life Technologies). Three dimensional SIM imaging was performed on Deltavision OMX V4 microscope with an Olympus ×60 NA 1.42 objective and three PCO.edge sCMOS cameras and 488nm and 568nm laser lines. Cells were illuminated with a grid pattern and for each image plane, 15 raw images (5 phases and 3 rotations) were acquired. Super-resolution images were reconstructed from the raw image files, aligned and projected using Softworx software (Applied Precision, GE Healthcare). Images were processed in ImageJ/Fiji^{26 (link)}.

hTERT-RPE1 WT and WDFY2(–/–) cells were transiently transfected with GFP-VAMP3 and mCherry-CORTACTIN overnight and fixed in 3% PFA. Cells were labeled with anti-GFP (Roche) and RFP-booster (Chromotek) Images were acquired on LSM710 confocal microscope using a Plan-Apochromat 63×/1.40 oil DIC III (Carl Zeiss) objective. The leading edges or cells were selected in the red channel (CORTACTIN). Images were processed in Fiji using a custom Python script, a schematic of the image processing is shown in Supplementary Fig. 9a. For the generation of superpixels to visualize Vamp3 distribution, cells were outlined using ImageJ and an ImageJ macro (https://gist.github.com/mutterer/035ade419bf9c96475ce) was used to generate hexagonal ROIs. Mean intensity values for each hexagon were measured and the corresponding hexagon filled with the mean value (Supplementary Fig. 9b).