Fig. 4. Effects of Rab22a overexpression on the uptake and intracellular distibution of Alexa-transferrin (Tfn). HeLa cells were infected with the Ankara strain Vaccinia T7 virus, followed by transfection of myc-tagged Rab22a wt, Q64L or S19N constructs in pGEM-1. Cell-surface binding and uptake (30 min) of Alexa-Tfn were then carried out as described in Materials and Methods, and the cells were processed for confocal immunofluorescence microscopy. Immunostaining for Rab22a is shown in A,C,F,I; Alexa-Tfn fluorescence in panels B,D,G,J; and overlays in E,H,K. In non-transfected cells (A,B), the Tfn was observed in characteristic endosomal structures. The volume or distribution of these structures was not significantly affected by expression of wt Rab22a (C-E). However, in cells expressing Rab22a Q64L (F-H) the Alexa-Tfn containing endosomes were redistributed to clusters near the leading edge. The Rab22a S19N mutant (I-K) had no detectable effect on the volume or distribution of internalized Alexa-Tfn. The wt Rab22a (E, arrowheads), but not the mutant proteins (H,K), showed a partial colocalization with Alexa-Tfn. Bar=10 µm.