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First published online 2 March 2004
doi: 10.1242/jcs.01009

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Ganglioside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus

Department of Pathology and Cell Biology, Université de Montréal, Montreal, Quebec, Canada H3C 3J7

View larger version (26K): [in a new window]   Fig. 1. Expression of caveolin and GM1 in HeLa and CaCo-2 cells. (A) Lysates of CaCo-2 and HeLa cells were separated by 12% SDS-PAGE and subjected to immunoblotting with polyclonal caveolin and ß-actin antibodies. Caveolin expression was detected in HeLa cells using short (S) exposures but not in CaCo-2 cells even upon long (L) exposure of the film. (B) To detect ganglioside GM1, 2, 5 and 10 µg of total protein (in 2 µl) from lysates of HeLa and CaCo-2 cells were dot blotted onto nitrocellulose filter strips and then incubated with HRP-conjugated CTX B subunit and revealed by chemiluminescence. (C) Triton X-100 (TX-100) soluble (supernatant) and insoluble (pellet) fractions were prepared and caveolin was found to be present in the insoluble fraction of HeLa cells. (D) Dot blot analysis with HRP-CTX of dilutions of the TX-100 soluble (supernatant) and insoluble (pellet) fractions showed that ganglioside GM1 was found to be present in the TX-100 insoluble fraction in both HeLa and CaCo-2 cells.

View larger version (68K): [in a new window]   Fig. 2. Cell surface labeling of CTX-FITC. CaCo-2 (A-C) and HeLa (D-I) cells were incubated with CTX-FITC (A,D,G) for 30 minutes at 4°C prior to fixation. After fixation, the cells were labeled with either GM130 (B,E) or caveolin (H), as indicated. Merged confocal images present CTX-FITC in green (C,F,I), GM130 in blue (C,F) and caveolin in red (I). Arrows identify examples of HeLa cells that express low GM1 levels. Scale bars: 16 µm (A-F) and 20 µm (G-I).

View larger version (67K): [in a new window]   Fig. 3. Internalization of CTX in CaCo-2 and HeLa cells. CTX-FITC was internalized for 30 minutes at 37°C in CaCo-2 (A) and HeLa (F,J) cells and after fixation the Golgi apparatus was labeled for GM130 (B,G,K) and endosomes for TfR (C,H,L). Minimal internalization of CTX to either the Golgi apparatus or endosomes was detected in CaCo-2 cells (A-D). Internalized CTX was localized primarily to the Golgi in HeLa cells expressing high levels of GM1 (F-I). Increasing the sensitivity of confocal detection of cell-associated GM1 permitted the visualization of CTX in cells expressing low GM1 levels and overlap with the Golgi was not observed (J-M). The merged confocal images (D,I,M) present CTX-FITC in green, GM130 in red and TfR in blue with colocalization between CTX-FITC and GM130 in yellow and between CTX-FITC and TfR in cyan. (E) A DIC image of a CaCo-2 cell island. Arrows indicate colocalization of internalized CTX with TfR-positive endosomes. Scale bars: 8 µm (A-E) and 16 µm (F-M).

View larger version (51K): [in a new window]   Fig. 4. mßCD and genistein block CTX delivery to the Golgi but not to endosomes. HeLa cells were pretreated with either 5 mM mßCD for 30 minutes (A-D) or 100 µg/ml genistein for 30 minutes (E-H), then incubated with CTX-FITC (A,E) for 30 minutes at 37°C. The Golgi apparatus of a high-GM1-expressing HeLa cell is labeled with GM130 (B,F) and endosomes with TfR (C,G). Merged confocal images (D,H) present CTX-FITC in green, GM130 in red and TfR in blue with colocalization between CTX-FITC and GM130 in yellow and between CTX-FITC and TfR in cyan. Scale bar: 8 µm.

View larger version (21K): [in a new window]   Fig. 5. Quantification of CTX internalization in HeLa and CaCo-2 cells. CTX-FITC internalization to the GM130-positive Golgi (CTX-GM130 overlap; left panels) or to TfR-positive endosomes (CTX-TfR overlap; right panels) was quantified in untreated cells (black bars), and in the presence of mßCD (white bars) or genistein (grey bars) in high-GM1 HeLa cells (A), low-GM1 HeLa cells (B) and CaCo-2 cells (C), as indicated. Inhibition of CTX delivery to the Golgi with mßCD and genistein was significant (*P<0.05) in high-GM1 cells (A) but not in low GM1 (B) or CaCo-2 (C) cells.

View larger version (31K): [in a new window]   Fig. 6. Increased GM1 expression levels following addition of GM1 to the cell medium. (A) Dot blot analysis of GM1 expression in 0.25, 0.5, 1, 2 and 4 µg lysates from CaCo-2 and HeLa cells grown in the absence (GM1–) or presence (GM1+) of 5 mM GM1 in the medium for 4 hours followed by a 4-hour chase in regular medium. (B) The number of cells expressing high levels of GM1, as determined by CTX labeling, was counted in HeLa cells grown in regular medium (GM–) and GM1 supplemented medium (GM+).

View larger version (55K): [in a new window]   Fig. 7. mßCD and genistein block CTX delivery to the Golgi but not to endosomes in GM1-supplemented HeLa cells. HeLa cells grown in the presence of 5 mM GM1 for 4 hours followed by a 4-hour chase in regular medium were incubated with CTX-FITC for 30 minutes at 37°C (A,E,I) in regular medium (A-D) or in the presence of mßCD (E-H) or genistein (I-L). The Golgi apparatus was labeled with GM130 (B,F,J) and endosomes with TfR (C,G,K). Merged confocal images (D,H,L) show CTX-FITC in green, GM130 in red and TfR in blue with colocalization between CTX-FITC and GM130 in yellow and between CTX-FITC and TfR in cyan. Scale bar: 8 µm.

View larger version (65K): [in a new window]   Fig. 8. mßCD, genistein-sensitive uptake of CTX to the Golgi apparatus in CaCo-2 cells supplemented with GM1. CaCo-2 cells grown in the presence of 5 mM GM1 for 4 hours followed by a 4-hour chase in regular medium were incubated with CTX-FITC for 30 minutes at 37°C (A,E,I) in regular medium (A-D) or in the presence of mßCD (E-H) or genistein (I-L). The Golgi apparatus was labeled with GM130 (B,F,J) and endosomes with TfR (C,G,K). Merged confocal images (D,H,L) present CTX-FITC in green, GM130 in red and TfR in blue with colocalization between CTX-FITC and GM130 in yellow and between CTX-FITC and TfR in cyan. Distinct colocalization of internalized CTX with Golgi elements can be seen in untreated cells (A-D). Scale bar: 8 µm.

View larger version (17K): [in a new window]   Fig. 9. Quantification of CTX-FITC internalization in HeLa and CaCo-2 cells after incubation with GM1. CTX-FITC internalization to the GM130-positive Golgi (CTX-GM130 overlay; left panels) or to TfR-positive endosomes (CTX-TfR overlay; right panels) was quantified using a mask overlay assay in HeLa (A) and CaCo-2 (B) cells grown either in regular medium (GM–; speckled bars) or in the presence of 5 mM GM1 for 4 hours followed by a 4-hour chase in regular medium (GM+) and left untreated (black bars) or treated with mßCD (white bars) or genistein (grey bars), as indicated. Data for cells grown in regular medium (GM–) was taken from Fig. 5 for both the mßCD and genistein series of experiments. The increase in CTX uptake to the Golgi in GM1-supplemented HeLa and CaCo-2 cells and its inhibition by mßCD and genistein is significant as is the increased delivery of CTX to endosomes in mßCD treated CaCo-2 cells (*P<0.01; **P<0.05 relative to bar to the left).

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