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A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation

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. Monodansycadaverine (MDC)-labeled vesicles are induced by starvation. (A) CHO cells were incubated in -MEM medium (control cells) or in EBSS medium (starved cells) at 37°C for two hours. Following this incubation period, both starved and control cells were incubated with MDC at 0.05 mM for 10 minutes at 37°C and then washed four times with PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy using an inverted microscope as described in Materials and Methods. Images were obtained with a CCD camera and processed using the Meta View program. (B) Cells were incubated in -MEM medium (control cells) or in EBSS medium (starved cells) at 37°C for two hours. Following this incubation period, both starved and control cells were incubated with MDC 0.05 mM for 10 minutes at 37°C, washed four times with PBS pH 7.4, and then lysed in 10 mM Tris-HCl, pH 8 containing 0.1% Triton X-100. Intracellular MDC was measured by fluorescence photometry as indicated in Experimental Procedures. The data represent the mean±s.e.m. of at least three independent experiments. (C) CHO cells were incubated in starvation medium for different periods of time. MDC labeling and measurement was performed as in B. Data are representative of at least three independent experiments.

View larger version (111K): [in a new window]   Fig. 2. Characterization of the MDC-labeled vacuoles. CHO cells were incubated in EBSS medium (starvation) at 37°C for two hours. Following this incubation period, cells were labeled with MDC as described in Fig. 1 and then incubated with 1 µM LysoTracker (LYS) for 10 seconds at RT. Cells were washed with PBS and immediately analyzed by fluorescence microscopy using the following filter system: excitation filter 510-560 nm, barrier filter 590 nm. MDC-labeled vacuoles are displayed in green and LYS in red. Arrows indicate some of the structures showing overlapping in the merged images (A-C). To label early endocytic compartments (EE), cells incubated under starvation conditions for two hours, were labeled with MDC and subsequently incubated with 0.5 mg/ml dextran tetramethylrhodamine for 5 minutes (D-F). To label late endosomes (LE), after the initial 5 minutes internalization of the endocytic marker, cells were washed and the probe was chased for 10 minutes before labeling with MDC. Cells were immediately analyzed by fluorescence microscopy (G-I). Endoplasmic reticulum (ER) was detected by indirect immunofluorescence (J-L). Cells were fixed, permeabilized and incubated with a rabbit antibody raised against ER membrane proteins (dilution 1:300) and with goat anti-rabbit Texas Red-conjugated secondary antibody. Cells were mounted with 50% glycerol in PBS and analyzed by fluorescence microscopy.

View larger version (68K): [in a new window]   Fig. 3. The accumulation of MDC is inhibited by inhibitors of the autophagic pathway. CHO cells were incubated in -MEM medium (control cells) or in EBSS medium (starved cells) for two hours in the absence or the presence of 10 mM 3-methyladenine (3-MA), 200 nM wortmannin (WM) or 2 µg/ml cycloheximide (CXM). In the case of 3-MA and CXM, cells were pre-incubated for 3 hours (3-MA) or 5 hours (CXM) with the corresponding inhibitor before the induction of autophagy. (A) Following the incubation period indicated above, cells were incubated with 0.05 mM MDC for 10 minutes at 37°C and then washed four times with PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy as indicated in Fig. 1. (B) Cells treated as indicated above were incubated with 0.05 mM MDC for 10 minutes at 37°C, washed four times with PBS pH 7.4, and then lysed in 10 mM Tris-HCl, pH 8 containing 0.1% Triton X-100. Intracellular MDC was measured by fluorescence photometry as indicated in Experimental Procedures. The data represent the mean±s.e.m. of two to four independent experiments.

View larger version (104K): [in a new window]   Fig. 4. N-ethylmaleimide (NEM) completely blocks the formation of MDC-labeled vacuoles. Cells were pre-incubated for 15 minutes in the presence or the absence of 50 µM NEM. Autophagy was subsequently induced by incubating the cells in starvation medium for 60 minutes. Cells were labeled with 0.05 mM MDC for 10 minutes at 37°C and then washed four times with cold PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy as indicated in Fig. 1.

View larger version (66K): [in a new window]   Fig. 5. Vinblastine-treatment causes a marked increase in the size of autophagic vacuoles. (A) CHO cells were incubated in full nutrient conditions (A,C,E) or in starvation medium (B,D,F) for 3 hours, in the presence (C,D,E,F) or absence (A,B) of 50 µM vinblastine. In E and F cells were pre-incubated for 3 hours with 10 mM 3-MA. Both starved and control cells were subsequently incubated with 0.05 mM MDC for 10 minutes at 37°C and then washed four times with cold PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy as indicated in Fig. 1. (G) The number and size of MDC-labeled vacuoles displayed in Fig. 5A-D were quantified by Methamorph software, using the integrated morphometry analysis. Data represent the mean±s.e.m.

View larger version (92K): [in a new window]   Fig. 6. Characterization of the MDC-labeled vacuoles generated in the presence of vinblastine. CHO cells were incubated in starvation medium for two hours at 37°C in the presence of 50 µM vinblastine. Following this incubation period, cells were labeled with MDC and then incubated with 1 µM LysoTracker (LYS) for 10 seconds at RT. Cells were washed with PBS and immediately analyzed by fluorescence microscopy using the following filter system: excitation filter 510-560 nm, barrier filter 590 nm. MDC-labeled vacuoles are displayed in green and LYS in red. Arrows indicate some of the structures showing overlapping (A-C). To label early endocytic compartments (EE), cells incubated under starvation conditions for two hours were labeled with MDC and subsequently incubated with 0.5 mg/ml dextran tetramethylrhodamine for 5 minutes (D-F). To label late endosomes (LE), after the initial 5 minutes internalization of the endocytic marker, cells were washed and the probe was chased for 10 minutes before labeling with MDC. Cells were immediately analyzed by fluorescence microscopy (G-I). Endoplasmic reticulum (ER) was detected by indirect immunofluorescence. Cells were fixed, permeabilized and incubated with a rabbit antibody raised against endoplasmic reticulum membrane proteins (dilution 1:300) and a goat anti-rabbit Texas Red-conjugated secondary antibody. Cells were mounted with 50% glycerol in PBS and analyzed by fluorescence microscopy (J-L).

View larger version (37K): [in a new window]   Fig. 7. MDC-vacuoles are labeled with LC3 a protein that associates with autophagosome membranes. CHO cells were transfected with pEGFP-LC3 as described in Materials and Methods. After 48 hours transfection cells were incubated for two hours at 37°C in -MEM medium (control), EBSS medium (starvation), or EBSS medium containing 50 µM vinblastine. Following this incubation period, cells were incubated with MDC at 0.05 mM for 10 minutes at 37°C and then washed four times with PBS pH 7.4. Cells were immediately analyzed by fluorescence microscopy. Typical images are shown. Insets: MDC-labeled vacuoles (red) are decorated with GFP-LC3 (green).