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Selective regulation of the Rab9-independent transport of ricin to the Golgi apparatus by calcium

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

View larger version (47K): [in a new window]   Fig. 1. Effect of thapsigargin on the sulfation of (A) ricin sulf-2 and (B) STxB-Sulf2. MDCK II cells were incubated with radioactive sulfate for 3 hours at 37°C, and during the last 30 minutes thapsigargin was added. Then ricin sulf-2 (200 ng/ml) (A) or STxB-Sulf2 (2.8 µg/ml) (B) was added to the medium and the incubation was continued for 2 hours. The cells were subsequently washed, lysed and immunoprecipitated with rabbit anti-ricin antibodies (A) or rabbit anti-Shiga toxin antibodies (B). The adsorbed material was analyzed by SDS-PAGE (12%) before autoradiography (details in Materials and Methods).

View larger version (20K): [in a new window]   Fig. 2. Effect of thapsigargin on the endocytosis of ricin. MDCK II cells were preincubated with or without the indicated concentrations of thapsigargin for 30 minutes at 37°C before TAG- and biotin-labeled ricin (30 ng/ml) was added to the medium. The incubation was continued for either 20 minutes or 2 hours before the cells were washed with 0.1 M lactose and then lysed. The amount of TAG- and biotin-labeled ricin in the lysates was measured using streptavidin beads and Origen Analyzer. Deviations between duplicates are represented by error bars.

View larger version (35K): [in a new window]   Fig. 3. Effect of the calcium ionophore A23187 on the sulfation of ricin sulf-2. MDCK II cells were incubated with radioactive sulfate for 3 hours at 37°C, and 10 µM A23187 was added for the last 30 minutes. Then ricin sulf-2 (200 ng/ml) was added to the medium, and the incubation was continued for 2 hours. The cells were subsequently washed with 0.1 M lactose, lysed and immunoprecipitated with rabbit anti-ricin antibodies. The adsorbed material was analyzed by SDS-PAGE (12%) before autoradiography.

View larger version (32K): [in a new window]   Fig. 4. Effect of thapsigargin (Thaps) on the sulfation of apically or basolaterally internalized ricin sulf-2. MDCK II cells grown on polycarbonate filters were incubated with radioactive sulfate for 3 hours at 37°C, and thapsigargin (1 µg/ml) was added for the last 30 minutes. Then ricin sulf-2 (200 ng/ml) was added either to the apical (A) or basolateral pole (B), and the cells were further treated as described in the legend to Fig. 3.

View larger version (61K): [in a new window]   Fig. 5. Effect of thapsigargin on the localization of ricin B-chain. MDCK II cells grown on coverslips were incubated for 30 minutes at 37°C in the absence (A-C) or presence of 1 µg/ml thapsigargin (D-F). Then CY3-labeled ricin B-chain (1 µg/ml) (red) was added, and the incubation continued for 2 hours. The Golgi apparatus was labeled with sheep anti-human TGN46 antibodies followed by donkey anti-sheep/goat IgG FITC (green). C and F represent the merged images.

View larger version (18K): [in a new window]   Fig. 6. Effect of Rab9S21N and thapsigargin (Thaps) on the sulfation of M6PR46-HMY and ricin sulf-2. (A,B) MDCK II cells transfected with M6PR46-HMY or contransfected with M6PR46-HMY and Rab9S21N were preincubated for 30 minutes at 37°C with or without 0.1 µg/ml thapsigargin. Then radioactive sulfate was added, and the incubation was continued for 3 hours. The cells were subsequently washed, lysed and immunoprecipitated using Ni-agarose beads. The adsorbed material was eluted with 25 mM EDTA and analyzed by SDS-PAGE (12%) before autoradiography. (C) Graphic illustration of the signal intensities of the bands in B representing sulfated M6PR46-HMY. The band intensities were determined by densitometric quantification using Image-Quant 5.0. (D) MDCK II cells untransfected or transfected with Rab9S21N were incubated with radioactive sulfate for 3 hours at 37°C, and thapsigargin (0.1 µg/ml) was added for the last 30 minutes. Then ricin sulf-2 (200 ng/ml) was added to the medium, and the cells were further treated as described in the legend to Fig. 3.

View larger version (46K): [in a new window]   Fig. 7. Effect of the PI 3-kinase inhibitors wortmannin and LY294002 on the thapsigargin-stimulated transport of ricin to the Golgi apparatus. MDCK II cells were incubated with radioactive sulfate for 3 hours at 37°C, and after 2 hours increasing concentrations of wortmannin (A) or LY294002 (B) were added to the medium. During the last 30 minutes the cells were incubated in the presence of 0.1 µg/ml thapsigargin. Then ricin sulf-2 (200 ng/ml) was added, and the incubation was continued for 2 hours. The cells were further treated as described in the legend to Fig. 3. Because of the instability of wortmannin, equal amounts were added every 45 minutes during the incubation time (3 hours) so that the final concentrations were 100 nM, 1 µM or 10 µM.