Fig. 7: Dual reporter reveals MVB transport before fusion and endosome acidification after uptake.

Live confocal microscopy was used to image pHluo_M153R-CD63-mScarlet-expressing HT1080 cells. a Representative of 35 cells from four independent experiments. Top: Images were captured at 10 s intervals for 60 min (see Supplementary Movie 6). Time-series images with 10 min intervals show frames at the beginning and end of the movie, with the rectangle across the leading edge in the 0 min frame indicating location from which the kymograph (middle) was derived. pHluo_M153R-CD63 and CD63-mScarlet are shown in green and magenta, respectively. Scale bar, 50 µm. Middle: Kymograph shows events over time across the indicated rectangle. Two regions (i and ii) selected from kymograph (middle) are enlarged at the bottom of the panel. Magenta arrows in kymograph zooms indicate CD63-mScarlet-positive MVB trafficking and docking to protrusions while white arrows indicate MVB fusion and exosome secretion. b Time-series images from live confocal microscopy of pHluo_M153R-CD63-mScarlet-expressing HT1080 cells with 1 min intervals from Supplementary Movie 7. White arrows indicate examples where filopodia contact exosome deposits. Magenta arrows indicate acidified exosome-containing endosomal compartments after exosome uptake. pHluo_M153R-CD63 and CD63-mScarlet are shown in green and magenta, respectively. n = 58 exosome uptake events from ten cells from five independent experiments. Scale bar, 25 µm. c Quantitation of internalization time of exosome deposits after initial filopodial contact from (b). Median time from contact to uptake is marked by a dotted line. d Quantitation of time from exosome endocytosis to acidification as shown in (b). Median time of acidification is marked by a dotted line. Source data are provided as a Source Data file.