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First published online 11 May 2004
doi: 10.1242/jcs.01114

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Rab7 is required for the normal progression of the autophagic pathway in mammalian cells

Laboratorio de Biología Celular y Molecular-Instituto de Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo – CONICET, Mendoza 5500, Argentina

View larger version (66K): [in a new window]   Fig. 1. EGFP-Rab7 colocalizes with monodansylcadaverine (MDC) and monodansyl pentane (MDH), markers for autophagic vesicles. CHO cells stably transfected with pEGFP-Rab7 wt, the mutants Q67L and T22N or with the vector alone (pEGFP) were incubated in full nutrient medium (j-k) or in EBSS medium (starved cells; a-i and m-o) at 37°C for 2 hours. Afterwards, cells were labeled with MDC (A) or MDH (B) and analyzed by fluorescence microscopy as described under Materials and Methods. Arrows indicate colocalization between Rab7 decorated vesicles and MDC. (C) Stably transfected CHO cells overexpressing GFP-Rab7T22N were incubated in control medium (full nutrient) or under starvation conditions for 2 hours. Protein extracts (membranes or cytosol) were subjected to electrophoresis on a 10% SDS-PAGE. The GFP protein was detected by western blotting (top panel) using specific antibodies. The bands were visualized with the ECL system (Amersham, NJ) and quantified (bottom panel) by densitometry. Figure represents one of three independent experiments.

View larger version (63K): [in a new window]   Fig. 2. Targeting of the mutant Rab7T22N to autophagic vacuoles is prevented by inhibitors of autophagosome formation. CHO cells expressing EGFP-Rab7T22N were incubated in EBSS medium (starved cells) at 37°C for 2 hours in the presence of 50 µM vinblastine (Vb), 0.1 µM bafilomycin A1 (BfA1), 200 nM wortmannin (WM) or with 30 mM 3-methyl adenine (3-MA). In this last case, cells were also preincubated with the drug for 2 hours before the starvation period. For the N-ethylmaleimide treatment (NEM) cells were pre-incubated for 15 minutes with 50 µM NEM before induction of starvation. Cells were washed and incubated for a further 2 hours in the starvation medium. Cells were subsequently labeled with MDC and immediately analyzed by fluorescence microscopy as described in Fig. 1.

View larger version (53K): [in a new window]   Fig. 3. Rapamycin induces the localization of Rab7T22N to autophagic vacuoles. CHO cells expressing EGFP-Rab7T22N were incubated at 37°C for 2 hours in -MEM (control cells) or EBSS medium (starved cells) in the presence of 50 ng/µl rapamycin. Cells were briefly washed with PBS, labeled with MDC and immediately analyzed by fluorescence microscopy as described in Fig. 1.

View larger version (35K): [in a new window]   Fig. 4. Overexpression of Rab7T22N accumulates large autophagosomes. (A) The levels of overexpressed EGFP proteins were detected by western blot using an anti-EGFP antibody as indicated under Materials and Methods. (B) CHO cells expressing EGFP-Rab7 wt, the mutants Q67L or T22N, or the vector alone (EGFP) were incubated at 37°C for 2 hours in EBSS medium (starved cells). Following this incubation period, cells were incubated with 0.05 mM monodansylcadaverine (MDC) for 10 minutes at 37°C. Intracellular MDC was measured by fluorescence photometry as indicated under Materials and Methods. The data represent the mean ± s.e.m. of five independent experiments. (C,D) The number of MDC-labeled vacuoles per cell (C) and the size (D) were quantified by the Methamorph software, using the integrated morphometry analysis. Data represent the mean±s.e.m. (n=50 cells). **P<0.01.

View larger version (77K): [in a new window]   Fig. 5. The mutant Rab7T22N induces the accumulation of LC3-labeled autophagosomes. (A) CHO cells expressing EGFP-Rab7 wt or the mutant T22N were transiently transfected with the plasmid pCI-neo Myc-LC3. After 36 hours transfection, cells were subjected to indirect immunofluorescence as described under Materials and Methods and analyzed by confocal fluorescence microscopy. Briefly, cells were fixed, permeabilized and incubated with an antibody against the myc epitope. The corresponding Texas red-conjugated secondary antibody was used. Cells were mounted with Mowiol. (B,C) The number (B) and size (C) of LC3-labeled vacuoles per cell were quantified by the Methamorph software, using the integrated morphometry analysis. Data represent the mean±s.e.m. (n=25 cells). **P<0.01

View larger version (36K): [in a new window]   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.

View larger version (26K): [in a new window]   Fig. 7. Overexpression of Rab7 T22N suppresses the autophagic degradation of labeled long-lived proteins. CHO cells overexpressing EGFP-Rab7 wt, and the mutants Q67L or T22N, or the vector alone (EGFP) were labeled for 24 hours in medium containing 1 µCi/ml [3H]leucine (see Materials and Methods). Both the trichloroacetic acid (TCA)-precipitable radioactivity of the cell monolayers and the TCA-soluble radioactivity present in the medium were measured. Leucine released (in the TCA-soluble supernatant) was calculated as a percentage of the total cell radioactivity. Results were expressed as the mean±s.e.m. **Significantly different from the vector under starvation conditions P<0.001 (compared with ANOVA-1 and Turkey-Kramer test).

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