FIG 3.

VP3 displays a discontinuous distribution along the membrane of PtdIns(3)P-enriched compartments. (A) Spinning-disc confocal microscopy analysis of VP3 protein distribution phenotype along the membrane of enlarged Rab5Q79L/PtdIns(3)P compartments. QM7-VP3 cells were cotransfected with RFP-2FYVE and EGFP-Rab5-Q79L for 12 h, and the cells were fixed, permeabilized, and stained with antibodies against VP3 (white) prior to analysis by spinning-disc confocal microscopy. (Left) Merged image showing a single confocal plane. (Middle) Magnified single plane from the framed region showing a giant endosome positive for VP3-, RFP-2FYVE-, and EGFP-Rab5-Q79L-derived signals. (Right) Single channels and surface intensity plots corresponding to the endosome in the middle. The images are representative of three independent experiments. Bars, 10 μm. (B) Improved-resolution analysis of VP3 distribution in enlarged EEs. QM7-VP3 cells where transfected with EGFP-Rab5-Q79L and processed as described for panel A, and the cells were analyzed using enhanced resolution imaging, acquired with a Hyvolution microscopy system as described in Materials and Methods. (Top left) Merged image showing a single confocal plane; (top right) Merged image of a magnified single plane from the framed region on the left showing enlarged endosomes positive for VP3- and EGFP-Rab5-Q79L-derived signals. (Bottom) Single planes of the framed region above. Bars, 10 μm. The images are representative of three independent experiments.