Fig. 2. Endocytosis and recycling of VSG and separation of VSG and fluid-phase cargo. Digitally deconvolved 3D-fluorescence images of representative cells at different time points are shown. (A) Kinetics of endocytosis of VSG_biotin at 37°C detected with Alexa Fluor 488 Streptavidin (yellow). Control refers to a surface-biotinylated cell before endocytosis. In the other samples, surface-associated biotin label has been removed prior to immuno-labelling. The lysosome (L) is marked by an anti-p67 monoclonal antibody and Alexa Fluor 594-labelled goat anti-mouse IgG (red); the kinetoplast (K) and nucleus (N) are visualised with DAPI (blue). (B) Kinetics of recycling of VSG_biotin (yellow). Biotinylated cells were allowed to endocytose VSG_biotin for 3 minutes at 37°C, then stripped of surface label at 0°C and finally incubated at 37°C for the indicated times. The flagellar pocket and the cell surface are marked by open and closed arrows, respectively. The kinetoplast (K) and nucleus (N) are visualised with DAPI (blue). (C) Kinetics of segregation of VSG_biotin and a fluid-phase marker. Cells were labelled at the surface with sulfo-NHS-SS-biotin and AMCA-sulfo-NHS (blue). Endocytosis was allowed in the presence of Alexa Fluor 594 dextran (red) at 37°C, the biotin label at the surface was stripped at 0°C and the cells were then processed for staining with Alexa Fluor 488 Streptavidin (green). The flagellar pocket is marked by open arrows. Blue size bars: 3 µm.