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Fig. 4. Activated RhoB inhibits transport of receptor-mediated and fluid-phase endocytosed cargo to the juxtanuclear area and impairs endosome motility. The effect of RhoB^G14V on receptor-mediated endocytosis was studied by incubating cells with 50 µg ml^–1 Texas Red-labelled human transferrin for 15 minutes. Myc-RhoB^G14V was detected with 9E10 and FITC-labelled secondary antibodies (a). Fluid-phase endocytosis was analysed by 15 minutes incubation with the fluid-phase endocytic marker sulforhodamine 101 (SR101), followed by a 60 minutes chase (b). The effect of RhoB^G14V on endosome motility was analysed on live cells transfected with pEGFP-FYVE (c). Myc-RhoB^G14V cDNA was microinjected together with a 70 kDa Texas-Red-dextrane to mark the injected cells. Endosome dynamics was observed by time-lapse fluorescence confocal microscopy and analysed by projection of the time-lapse images (36 images with a time interval of 15 seconds) where vesicle movement is seen as green tracks. The projected image was then combined with a red-coloured image corresponding to t=0. In the RhoB^G14V expressing cell (asterisk) vesicles form much shorter tracks (c, arrows) than in control non-injected cells (c, arrowheads). Nuclei of cells microinjected with myc-RhoB^G14V are marked with an asterisk. Nuclei of control non-injected cells are marked with a circle. Bars: 10 µm.

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