Fig. 6. Rab7 T22N hampers fusion between autophagosomes and late endosome/lysosomal compartment. (PA) Outline of the method used to label the late endocytic/lysosomal compartment with Rhodamine-dextran. (B) CHO cells overexpressing EGFP-Rab7 wt or T22N were allowed to internalize for 20 minutes at 37°C Rhodamine-dextran (0.5 mg/ml) by fluid phase endocytosis. The marker was chased for 20 minutes at 37°C to label the late endosome/lysosomal compartment. Subsequently, cells were placed for 2 hours in the starvation medium (EBSS) to induce autophagy and then were labeled with MDC as indicated in Fig. 1. Cells were immediately analyzed by fluorescence microscopy. In cells transfected with Rab7 wt arrows indicate Rab7 decorated vesicles that colocalized with both MDC and Rhodamine-dextran. In contrast, in cells overexpressing the mutant Rab7 T22N the MDC-labeled vesicles are not labeled with the endocytic probe (arrow). (C) CHO cells overexpressing EGFP-Rab7 wt or the mutants Q67L and T22N, were incubated under starvation conditions for 2 hours and afterwards subjected to indirect immunofluorescence to detect the lysosomal enzyme cathepsin D, as described under Materials and Methods. The corresponding Texas Red-conjugated secondary antibody was used. Cells were mounted and analyzed by confocal fluorescence microscopy.