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First published online 7 March 2006
doi: 10.1242/jcs.02833

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Gene silencing reveals a specific function of hVps34 phosphatidylinositol 3-kinase in late versus early endosomes

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

View larger version (66K): [in a new window]   Fig. 1. siRNA-mediated suppression of hVps34 expression in U-251 glioma cells induces accumulation of LAMP1-positive cytoplasmic vacuoles. (A) U-251 cells infected with retrovirus carrying the control or hVps34 KD siRNA sequences surviving after 5 days of puromycin selection were subjected to immunoblot analysis with a polyclonal anti-hVps34 IgG as described in the Materials and Methods. Nuclear lamin B2 served as one of several controls for nonspecific effects of the siRNA. (B) Phase contrast images of the live control and hVps34 KD cells. Bar, 10 µm. Based on counting 100 cells in multiple phase micrographs, 73% of the hVps34 KD cells exhibited the vacuolar phenotype, defined as multiple small-sized to intermediate-sized vacuoles (0.5-1.0 µ), with at least two vacuoles per cell exceeding a diameter of 2 µ. (C) Control and hVps34 KD cells were seeded at 100,000 cells/dish in 35 mm dishes. 24 hours later, cells were examined by immunofluorescence microscopy using a primary antibody against the lysosomal and late endosomal membrane protein LAMP1. Bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 2. Accumulation of LAMP1-positive vacuoles is specific to suppression of hVps34 expression. (A) U-251 cells infected with retrovirus harboring the control or two different hVps34-specific RNAi sequences (KD and KD2) were subjected to immunoblot analysis as described previously. (B) Control, hVps34 KD and hVps34 KD2 cells were seeded at 100,000 cells/dish in 35 mm dishes. 48 hours later, cells were examined by immunofluorescence microscopy using an antibody against LAMP1. Bar, 10 µm.

View larger version (94K): [in a new window]   Fig. 3. Vacuoles in the hVps34 KD cells are membrane-bound structures with occasional internal vesicles and electron-dense material. Control and hVps34 KD cells were examined by electron microscopy. (A) Control and KD cells magnified 3,900x. Bar, 5 µm. (B) Highlighted region of KD cell from panel A at 21,000x. Bar, 1 µm. Labeled structures: m, mitochondria; n, nucleus; v, vacuole. The arrowhead points to a single membrane surrounding the vacuole. Arrows point to occasional small internal vesicles and electron-dense material.

View larger version (62K): [in a new window]   Fig. 4. Vacuoles in the hVps34 KD cells exhibit characteristics of late endosomes or lysosomes. Control and hVps34 KD cells were seeded at 100,000 cells/dish in 35 mm dishes. 24 hours later, cells were examined by immunofluorescence microscopy using the primary antibodies indicated at the left of the figure. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 5. Vacuoles in the hVps34 KD cells are acidic and receive traffic from the endocytic compartment. (A) Control and hVps34 KD cells were incubated with 2.5 µg/ml Acridine Orange (AO) for 30 minutes. The cells were then examined by fluorescence microscopy using green (excitation wavelength: 465-495 nm; emission wavelength: 515-555 nm) and red (excitation wavelength: 528-553 nm; emission wavelength 600-660 nm) filters. Red fluorescence emanates from AO in acidic compartments. Bar, 10 µm. (B) Control and KD cells were incubated with 500 µg/ml Texas Red (TxR)-dextran for 16 hours. Following incubation for 2 hours, cells were fixed and co-stained with a monoclonal antibody against LAMP1. Arrows indicate vacuoles containing both LAMP1 and TxR-dextran. Arrowheads point to vacuoles lacking TxR-dextran. Bar, 10 µm.

View larger version (51K): [in a new window]   Fig. 6. Suppression of hVps34 expression does not prevent membrane association of the early endosome marker EEA1. (A) Control and hVps34 KD cells were seeded at 100,000 cells/dish in 35 mm dishes. 24 hours later, cells were examined by immunofluorescence microscopy using an anti-EEA1 antibody. Control cells treated with 0.1 µM or 1 µM wortmannin for 1 hour are also shown. (B) Cytosol (S) and particulate (P) fractions were prepared from control cells, hVps34 KD cells, or control cells treated for 1 hour with the indicated concentrations of wortmannin (0.1 µM or 1 µM), as described in the Materials and Methods. Immunoblot analysis was performed with an antibody against EEA1 and the relative amount of EEA1 recovered in the P fraction is expressed as a ratio to the amount in the S fraction. The results are means (±s.e.m.) of three determinations performed on separate cultures. The asterisk indicates that the decrease relative to the control and KD cells was significant at P< 0.05 (Student's t test). Essentially the same results were obtained when the amount of EEA1 recovered in the P fraction was normalized to the amount of a membrane marker (calreticulin) recovered in the same fraction (not shown).

View larger version (83K): [in a new window]   Fig. 7. LAMP1-positive vacuoles formed in hVps34 KD cells are depleted of PtdIns(3)P, as measured by localization of a GFP-2xFYVE probe. Control or hVps34 KD cells were transfected with a vector encoding GFP-2xFYVE. After 16 hours, the cells were processed for immunofluorescence, using primary antibodies against GFP and LAMP1. Bar, 10 µm.

View larger version (20K): [in a new window]   Fig. 8. (A) Suppression of hVps34 expression does not interfere with the endocytosis of a fluid phase marker, horseradish peroxidase (HRP). (A) Control (black) and hVps34 KD (gray) cells were 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.m. from triplicate dishes of each cell line. (B) Suppression of hVps34 expression does not impair recycling of transferrin. Control (black) and hVps34 KD (gray) cells were allowed to internalize biotinylated transferrin for 30 minutes, then were chased with an excess of unlabeled transferrin for 15 or 30 minutes. The amounts of biotinylated transferrin in the cells and medium were determined as described in the Materials and Methods. The recycled transferrin (released into the medium) is expressed as percentage of total biotinylated transferrin. The bar graph in the inset shows the relative amount of biotinylated transferrin (normalized to calreticulin) taken up by the control (black bar) and KD (gray bar) cells after the 30 minutes pre-loading period. Each point represents the mean±s.e.m. from triplicate dishes of each cell line.

View larger version (48K): [in a new window]   Fig. 9. Suppression of hVps34 expression does not impede internalization of the EGFR, but slows initial receptor degradation. Control and hVps34 KD cells were stimulated with EGF after overnight incubation with serum-free medium. (A) KD cells were fixed and co-stained with EGFR and LGP85 antibodies before addition of EGF (0 min) and at different time points (30 min, 60 min) after addition of EGF. Arrows indicate vacuoles positive for both the EGFR and LGP85. The brightness of the EGFR image at 60 minutes was enhanced by approximately 50% to compensate for its weaker EGFR immunofluorescence compared with the brightness of the image at 30 minutes. Bar, 10 µm. (B) To measure EGFR degradation, triplicate cultures of control and KD cells were harvested at the indicated times after the addition of EGF and subjected to immunoblot analysis for total EGFR. The graph illustrates the data (mean±s.e.m.) generated from densitometer scans of ECL signals on film.

View larger version (31K): [in a new window]   Fig. 10. Suppression of hVps34 expression potentiates EGFR signaling. To measure EGFR phosphorylation and signaling, the control and KD cells were harvested at the indicated times after the addition of EGF and subjected to immunoblot analysis for (A) phospho-EGFR (pEGFR) and total EGFR, or (B) phospho-ERK1/2 (pERK) and total ERK1/2. The bar graphs illustrate the data generated from Kodak Imager scans of blots from triplicate cultures of each cell line.

View larger version (51K): [in a new window]   Fig. 11. The late endosomal intermediate form of cathepsin D accumulates in the absence of hVps34, but there is no inhibition of the early step of procathepsin D processing. (A) Control and hVps34 KD cells were seeded at 350,000 cells/dish in 10 cm dishes. Cells were labeled with 100 µCi/ml [35S]methionine, then harvested after 30 minutes 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. Pro, newly synthesized 51-53 kDa procathepsin D containing a propeptide; Intermediate, cathepsin D after removal of the propeptide to generate an intermediate form that migrates at 46-48 kDa; Mature, cathepsin D after cleavage to generate the mature form that contains two non-covalently linked chains of 31 kDa and 14 kDa. (B) Immunoblot analysis of endogenous cathepsin D in whole cell lysates from control and hVps34 KD cells. (C) The hVps34 KD cells were subjected to immunogold labeling with an antibody against cathepsin D. The left panel shows a lysosome heavily labeled for cathepsin D (arrow) adjacent to a larger electron-lucent vacuole. The right panel shows cathepsin D delivered to the lumen of an enlarged vacuole (black arrowhead), with a lysosome adjacent to the vacuole (white arrowhead). Bar, 0.5 µm.

View larger version (15K): [in a new window]   Fig. 12. hVps34 KD cells exhibit a marked reduction in growth rate. (A) Following 2 days of selection, control (black) and hVps34 KD (gray) cells were seeded in 35 mm dishes at an equal density of 50,000 cells/dish. At the indicated time points, triplicate dishes from each cell line were harvested and counted with a Coulter Z1 particle counter (mean±s.e.m.). (B) Control (black) and KD (gray) cells were seeded in 25 cm2 flasks at 150,000 cells/flask. On the indicated days, triplicate flasks of each cell line were incubated with [3H]thymidine (1.0 µCi/ml) for 5 hours. Radioactivity incorporated into TCA-precipitable material was counted and normalized to total cellular protein (mean±s.e.m.). (C) Control (black) and KD (gray) cells were seeded in 60 mm dishes at 200,000 cells/dish. On the indicated days, duplicate dishes from each cell line were harvested and stained with annexin-V. Annexin-positive cells were counted using a Guava personal cytometer. Cells treated overnight with TNF- (white) served as a positive control for apoptosis.