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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01081

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Efficient endosome-to-Golgi transport of Shiga toxin is dependent on dynamin and clathrin

Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

View larger version (26K): [in a new window]   Fig. 6. Endosome-to-Golgi transport of Shiga toxin in BHK cells. BHK cells were grown with (control) and without [antisense (As) CHC] tetracycline for 48 hours and with and without 2 mM butyric acid (ba) for the last 24 hours. (A) Sulfation of STxB-Sulf2 was analysed by incubating the cells with radioactive sulfate for 3 hours at 37°C before STxB-Sulf2 (2.8 µg ml–1) was added for 15 minutes or 1 hour. The cells were subsequently washed, lysed and immunoprecipitated with rabbit anti-Shiga-toxin antibodies. The adsorbed material was analysed by 12% SDS-PAGE before autoradiography. Quantified average signal intensities from three independent experiments are shown in the lower panel of (A) (as the percentage of the value in control cells). As shown, butyric acid increased the sulfation strongly, and expression of CHC antisense RNA gave a strong reduction in butyric-acid-treated cells but not in untreated cells. (B) In parallel, the internalization of Shiga toxin for the different conditions in (A) was analysed by incubating the cells with TAG- and biotin-labelled Shiga toxin (5 ng ml–1) for 15 minutes or 1 hour. The SS-linked biotin on the cell-surface-bound toxin was then removed by incubating the cells with 0.1 M MeSNa for 1 hour at 0°C. Subsequently, the cells were washed and lysed, and the amounts of TAG- and biotin-labelled Shiga toxin in the lysates were measured using streptavidin beads and an Origen Analyzer. Average values from these three experiments are shown as the percentage of the value in control cells. As shown, butyric acid increased the proportion of Shiga toxin endocytosed in a clathrin-dependent manner. (C) The effect of expressing CHC antisense RNA on the endosome-to-Golgi transport of Shiga toxin (shown as the percentage of the value in control cells) was calculated by correcting the average signal intensities in (A) for the amount of Shiga toxin that was internalized in (B). The error bars show the standard error of the mean from the three experiments.

View larger version (21K): [in a new window]   Fig. 1. Shiga toxin and transferrin endocytosis in HeLa and BHK cells. HeLa and BHK cells grown with (control) and without (dynK44A for HeLa cells or antisense CHC for BHK cells) tetracycline for 48 hours were incubated with TAG- and biotin-labelled (A) Shiga toxin (5 ng ml–1) or (B) transferrin (50 ng ml–1) for 5 or 15 minutes. Half of the cells were then incubated with 0.1 M MESNa for 1 hour at 0°C to remove the SS-linked biotin on the cell surface-bound toxin, before the cells were washed and lysed. The amount of TAG- and biotin-labelled toxin in the lysates was then measured using streptavidin beads and Origen Analyzer, and the degree of endocytosis (as percentage of total cell-associated protein) was calculated. The error bars show the standard deviation between three to eight independent experiments.

View larger version (17K): [in a new window]   Fig. 2. Butyric acid sensitizes HeLa and BHK cells to Shiga toxin. HeLa (A) and BHK (B) control cells were grown with tetracycline for 48 hours and with or without 2 mM butyric acid (ba) for the last 24 hours. The cells were then incubated with increasing concentrations of Shiga toxin for 2 hours and the protein synthesis measured. The error bars represent deviations between duplicates.

View larger version (36K): [in a new window]   Fig. 3. Effect of butyric acid on transport of Shiga toxin to the Golgi apparatus. HeLa (A-C) and BHK (D-F) control cells grown with tetracycline for 48 hours and with (B,C,E,F) or without (A,D) 2 mM butyric acid for the last 24 hours were incubated with 1 µg ml–1 Alexa Fluor 488-labelled Shiga toxin for 20 minutes. The Golgi apparatus was labelled with mouse anti-GM130 antibodies followed by CY-5 labelled goat anti-mouse IgG (C,F).

View larger version (24K): [in a new window]   Fig. 4. Endosome-to-Golgi transport of Shiga toxin in HeLa cells. HeLa cells were grown with (control) and without (dynK44A) tetracycline for 48 hours and with and without 2 mM butyric acid (ba) for the last 24 hours. (A) Sulfation of STxB-Sulf2 was analysed by incubating the cells with radioactive sulfate for 3 hours at 37°C before STxB-Sulf2 (2.8 µg ml–1) was added for 15 minutes or 1 hour. The cells were subsequently washed, lysed and immunoprecipitated with rabbit anti-Shiga toxin antibodies. The adsorbed material was analysed by 12% SDS-PAGE before autoradiography. Quantified average signal intensities (as percentage of the value in control cells) from two independent experiments are shown (A, bottom). As shown, butyric acid strongly increased the sulfation and expression of mutant dynamin gave a strong reduction. (B) In parallel, the internalization of Shiga toxin for the different conditions in (A) was analysed by incubating the cells with TAG- and biotin-labelled Shiga toxin (5 ng ml–1) for 15 minutes or 1 hour. The SS-linked biotin on the cell-surface-bound toxin was then removed by incubating the cells with 0.1 M MESNa for 1 hour at 0°C. Subsequently, the cells were washed and lysed, and the amounts of TAG- and biotin-labelled Shiga toxin in the lysates were measured using streptavidin beads and an Origen Analyzer. Average values from these two experiments are shown as the percentage of the value in control cells. As shown, butyric-acid treatment almost doubled the amount of endocytosed toxin. (C) The effect of expressing mutant dynamin on the endosome-to-Golgi transport of Shiga toxin (shown as percentage of the value in control cells) was calculated by correcting the average signal intensities in (A) for the amount of Shiga toxin that was internalized in (B). The error bars show the standard error of the mean from the two experiments.

View larger version (30K): [in a new window]   Fig. 5. Butyric acid increases the proportion of Shiga toxin endocytosed in a dynamin- and clathrin-dependent manner. HeLa and BHK control cells were grown with tetracycline for 48 hours and with and without 2 mM butyric acid (ba) for the last 24 hours. The cells were then incubated with TAG- and biotin-labelled Shiga toxin (5 ng ml–1) (A) or transferrin (50 ng ml–1) (B) for 15 minutes. Half of the cells were then incubated with 0.1 M MESNa for 1 hour at 0°C to remove the SS-linked biotin on the cell-surface-bound toxin before the cells we re washed and lysed. The amounts of TAG- and biotin-labelled toxin in the lysates were then measured using streptavidin beads and an Origen Analyzer, and the degree of endocytosis (as the percentage of total cell-associated protein) was calculated. The error bars represent deviations between two independent experiments.

View larger version (36K): [in a new window]   Fig. 7. Localization of Shiga toxin and clathrin heavy chain in K44A and BHK control cells. HeLa (A) and BHK (B) control cells grown with tetracycline for 48 hours and with (+ ba) or without (–ba) 2 mM butyric acid for the last 24 hours were incubated with 1 µg ml–1 Alexa Fluor 488-labelled Shiga toxin (STx) for 20 minutes. CHC staining was performed by using goat anti-CHC antibodies followed by rhodamine-labelled donkey anti-goat IgG. Quantification of the extent of colocalization between STx and CHC (as the percentage of the total amount of toxin) from eight or nine cells is shown in (A,B, bottom). The error bars represent the standard error of the mean.

View larger version (49K): [in a new window]   Fig. 8. Localization of Shiga toxin and CHC in Myc-Rab5Q79L-transfected cells. HeLa (A) and BHK (B) control cells grown in the presence of tetracycline were transfected with a GTPase-deficient mutant form of Rab5 (Myc-Rab5Q79L) to induce the formation of enlarged early endosomes (EE). The cells were further grown with (+ ba) or without (–ba) butyric acid for 24 hours before incubation with 1 µg ml–1 Alexa Fluor 488-labelled Shiga toxin (STx) for 20 minutes. CHC staining was performed by using goat anti-CHC antibodies followed by rhodamine-labelled donkey anti-goat IgG. The enlarged endosomes in transfected cells were identified with mouse anti-Myc antibodies followed by CY5-labelled donkey anti-mouse IgG. The white squares to the left are shown in enlarged versions to the right (green and red channels alone and the merged picture). The graphs show quantification of the extent of colocalization between STx and CHC on EE (as the percentage of the total amount of toxin) of 6-11 endosomes from four to six cells. The error bars represent the standard error of the mean.

View larger version (23K): [in a new window]   Fig. 9. Cytotoxicity of Shiga toxin in HeLa and BHK cells. HeLa (A) and BHK (B) cells were grown with (control) and without (dynK44A for HeLa cells or antisense CHC for BHK cells) tetracycline for 48 hours and with and without 2 mM butyric acid (ba) for the last 24 hours. The cells were then incubated with increasing concentrations of Shiga toxin for 2 hours and protein synthesis measured. The error bars represent deviations between duplicates.