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First published online 3 January 2006
doi: 10.1242/jcs.02735

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Functional specificity of the mammalian Beclin-Vps34 PI 3-kinase complex in macroautophagy versus endocytosis and lysosomal enzyme trafficking

Department of Biochemistry and Cancer Biology, Medical University of Ohio, Toledo, OH 43614, USA

View larger version (56K): [in a new window]   Fig. 1. siRNA-mediated suppression of Beclin expression in U-251 glioma cells. U-251 cells infected with retroviral vectors and surviving after 6 days in medium containing 1 µg/ml puromycin were pooled to generate stable cell lines. Expression levels of Beclin, LAMP1 and LDH were determined in whole-cell lysates from control and Beclin KD cells by immunoblot analysis as described in the Materials and Methods.

View larger version (43K): [in a new window]   Fig. 2. Immunoprecipitated Beclin complex from U-251 cells contains hVps34 but not Bcl-2 or Bcl-XL. (A) Cell lysates were prepared from parallel cultures as described in the Materials and Methods and 10% of each lysate was saved for immunoblot analysis. The remainder of each lysate was immunoprecipitated with either IgG against Beclin or a `control' IgG against an unrelated protein (SPARC, Santa Cruz Biotechnology). Equal aliquots of the immune complexes eluted from protein A Sepharose beads were probed by immunoblot analysis using the antibodies indicated at the left of each panel. The band appearing just below Beclin in the upper panel is a nonspecific crossreacting protein. (B) Immunoprecipitation was performed as described in panel A, except that a rabbit antibody against Bcl-2 was used for the pull-down. The immunoprecipitated protein complexes were probed with the antibodies indicated at the left of each panel.

View larger version (38K): [in a new window]   Fig. 3. Suppression of Beclin expression impairs the autophagy-associated post-translational processing of endogenous LC3-I to LC3-II. (A) Cells were incubated in DMEM + 10% FBS (normal) or HBSS (starved) for 4 hours. (B) Cells were treated with vehicle (–) or 10 µM C2-ceramide (+) for 24 hours. All cells were subjected to SDS-PAGE and immunoblot analysis to detect the endogenous cytosolic LC3-I and the PE-conjugated LC3-II. The total LC3-II and the ratios of LC3-II to LC3-I represented in the bar graphs were determined from scans of the blots performed with a Kodak 440CF Image Station.

View larger version (18K): [in a new window]   Fig. 4. Suppression of Beclin expression impairs macroautophagy as measured by sequestration of LDH. Control or Beclin KD cells were incubated in DMEM + 10% FBS (normal) or HBSS (starved) for 4 hours. Cells were lysed and fractionated as described in the Materials and Methods. LDH was assayed by immunoblotting aliquots of the cell lysate, and the pellet fraction was recovered after centrifugation through a metrizamide/sucrose cushion. LDH sequestered in the pellet fraction was expressed as a ratio to the total LDH in the cell lysate. The results are derived from separate fractionations of cells from three parallel cultures (mean ± s.e.).

View larger version (26K): [in a new window]   Fig. 5. Accumulation of acidic vesicular organelles (AVOs) induced by ceramide treatment is impaired in Beclin KD cells. (A) Control and Beclin KD cells were maintained for 24 hours in medium with or without 10 µM C2-ceramide, as indicated. Cells were then incubated with acridine orange (AO) and examined by fluorescence microscopy, using a filter combination to detect red fluorescence. All digital micrographs were taken at the same exposure setting. Bar, 50 µm. (B) Control and Beclin KD cells were treated with 0, 10 or 20 µM C2-ceramide for 24 hours and incubated with AO. The relative amount of AO trapped in vesicular compartments (red fluorescence; excitation at 488 nm, emission at 655 nm) was measured by fluorimetry and normalized to cellular DNA detected with ethidium bromide (EB). The data represent the mean ± s.e. of three determinations from parallel cultures.

View larger version (30K): [in a new window]   Fig. 6. Suppression of Beclin expression does not impede lysosomal enzyme sorting as measured by cathepsin D processing. (A) Immunoblot analysis was performed on endogenous cathepsin D in whole-cell lysates from control and Beclin KD cells. To demonstrate inhibition of cathepsin D processing, a separate control culture was incubated with 10 mM NH4Cl for 24 hours prior to harvest. Equal amounts of protein were loaded in each lane. The part of the blot above the dashed line was exposed seven times longer than the lower portion, to permit detection of the precursor forms of cathepsin D. The forms of cathepsin D are labeled as follows: P, proenzyme; I, endosomal intermediate; M, mature lysosomal enzyme. (B) Cells were labeled with 100 µCi/ml [35S]methionine, then harvested immediately or chased in medium with unlabeled methionine for 4 hours. A separate control culture was incubated with 15 mM NH4Cl during the 4 hour chase. Cathepsin D was immunoprecipitated and subjected to SDS-PAGE and fluorography. (C) Immunoprecipitations were performed on control and Beclin KD cells from three parallel cultures immediately after the pulse and after a 4 hour chase, and the total radioactivity recovered in the mature form of cathepsin D at 4 hours was expressed as a ratio to the total radiolabeled procathepsin D at 0 hours (mean ± s.e.). Quantification of radioactivity was performed with a Molecular Dynamics Storm Phosphorimager.

View larger version (87K): [in a new window]   Fig. 7. Morphology of endosomes, lysosomes and Golgi membranes is similar in Beclin KD cells compared with controls. Control or Beclin KD cells were seeded in parallel dishes at the same density and examined by immunofluorescence microscopy after two days, using the primary antibodies indicated at the left of the figure. Bar, 20 µm.

View larger version (13K): [in a new window]   Fig. 8. Suppression of Beclin expression does not interfere with endocytosis of the fluid phase marker horseradish peroxidase (HRP). Control () and Beclin KD () cells were seeded at the same density, grown for two days and then incubated for the indicated periods of time with 2 mg/ml HRP in DMEM + 1% BSA. Washed cells were lysed and HRP activity was determined as described in the Materials and Methods. Each point represents the mean ± s.e. from three determinations on parallel cultures. Similar results were obtained when the experiment was repeated with different batches of control and Beclin KD cells derived from separate retroviral infections and puromycin selections.

View larger version (58K): [in a new window]   Fig. 9. Suppression of Beclin expression does not disrupt EGF-stimulated endocytosis or post-endocytic degradation of the EGFR. (A) Control or Beclin KD cells were fixed and processed for immunofluorescence detection of EGFR after overnight growth in serum-free medium (No EGF), and after 30 minutes or 70 minutes incubation with 200 ng/ml EGF. Bar, 10 µm. (B) Cells were harvested at the indicated times after addition of EGF and subjected to immunoblot analysis for total EGFR (upper panel). The bar graph (lower panel) shows the data derived from Kodak 440CF Imager scans performed on blots from three cultures harvested at each time point. (C) Cells were harvested at the indicated times after addition of EGF and subjected to immunoblot analysis for total EGFR (upper panel) or EGFR phosphorylated on Tyr1068 (pEGFR). The bar graph depicts the ratio of pEGFR to total EGFR determined in three parallel control or Beclin KD cultures at 30 minutes after EGF stimulation (mean ± s.e.).

View larger version (13K): [in a new window]   Fig. 10. Suppression of Beclin expression does not affect the growth rate or clonogenicity of U-251 cells. (A) Control (solid line) and Beclin KD (dashed line) cells were seeded in 60 mm dishes on day 0 at an initial density of 200,000 cells per dish. On each of the indicated days, cells were harvested from three dishes in each group and counted with a Coulter Z1 particle counter (mean ± s.e.). (B) Control or Beclin KD cells were seeded in soft agar and assayed for colony formation after 14 days as described in the Materials and Methods. Results are means (± s.e.) of colony counts performed on three dishes.