Fig. 7. The effect of Rab22a on the endocytic trafficking of internalized TRITC-dextran. BHK-21 cells were transfected (for 24 hours) with the mycRab22a wt, Q64L or S19N cDNAs (indicated on the left) in pcDNA3.1 or the plain vector plasmid (Mock), followed by 1 hour internalization of TRITC-dextran and a 3 hour chase in the absence of the compound. The cells were then immunostained for EEA1 and LAMP-1 and viewed under a confocal microscope. TRITC-dextran fluorescence is shown in panels A,F,K,P, staining for EEA1 in B,G,L, LAMP-1 staining in C,H,M,R, and Rab22a staining in Q. Pairwise overlays of the channels are shown in D,E,I,J,N,O,S and T. In mock-transfected cells the TRITC-dextran was transported to structures that contained LAMP-1 and showed no overlap with the early endosome marker EEA1 during the chase (A-E). In cells expressing Rab22a wt, the transport of TRITC-dextran proceeded in a similar fashion, despite the presence of abnormally large EEA1-positive vacuolar endosomes induced by the GTPase (F-J). In a majority of cells expressing Rab22a Q64L, however, the fluorescent dextran ended up in large vacuolar endosomes which now contained markers of both early (EEA1) and late (LAMP-1) endocytic compartments (K-O). Expression of the S19N mutant had no detectable effect on the trafficking of TRITC-dextran (P-T). In Q, staining for the Rab is shown instead of EEA1, as S19N-expressing cells cannot be identified on the basis of their endosome morphology. Bar=10 µm.