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Internalization of cholera toxin by different endocytic mechanisms

¹ Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
² Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark

View larger version (14K): [in a new window]   Fig. 1. Rate of internalization of CT in HeLa K44A cells, with () and without () induction of mutant dynamin. The cells were washed, and TAG- and biotin-labeled CT (9 ng/ml) in 0.2 ml of Hepes-buffered MEM medium was added to each well containing approximately 40,000 cells. The cells were incubated at 37°C for 2-20 minutes, and bound and endocytosed CT were quantified as described in Materials and Methods, and are presented here as percent of total cell-associated toxin (mean ± s.d., n=6). The amount of total cell-associated CT at 2 and 20 minutes was 0.11 and 0.38 ng/40,000 cells, respectively. Equal amounts of CT were bound to non-induced and induced cells.

View larger version (11K): [in a new window]   Fig. 2. (A) Rate of internalization of CT in non-induced () and induced () BHK cells with inducible expression of antisense CHC, which inhibits clathrin-dependent endocytosis. The cells were washed, and TAG- and biotin-labeled CT (9 ng/ml) in 0.2 ml of Hepes-buffered MEM medium was added to each well containing approximately 35,000 cells. The cells were incubated at 37°C for 2-20 minutes, and bound and endocytosed CT were quantified as described in Materials and Methods, and is presented here as percent of total cell-associated toxin (mean ± s.d., n=6). The amount of total cell-associated CT after 2 and 20 minutes of endocytosis was 0.058 and 0.47 ng/35,000 cells, respectively. (B) Endocytosis of 125I-labeled transferrin in BHK cells with (open bar) and without (filled bar) induction of antisense CHC. Endocytosed transferrin was quantified after 5 minutes of internalization as described in Materials and Methods. The results are mean ± s.d. (n=6).

View larger version (14K): [in a new window]   Fig. 3. Endocytosis of CT after treatment of non-induced (filled bars) and induced (open bars) BHK antisense CHC cells with different inhibitors. The cells were preincubated with cytochalasin D (10 µg/ml), genistein (100 µg/ml) and filipin (5 µg/ml) in Hepes-buffered MEM medium for 30 minutes at 37°C, before TAG-and biotin-labeled CT (9 ng/ml) was added, and the cells were incubated at 37°C for 20 minutes in the presence of the different inhibitors. Endocytosed CT was quantified as described in Materials and Methods, and is presented here as percent of untreated control. The values are mean ± s.d. (n=4). The amount of CT associated with the cells was unaffected by the inhibitors added. Absolute values of controls for non-induced cells were in the range 31-48%, and for induced cells 8-19%.

View larger version (67K): [in a new window]   Fig. 4. Electron micrographs of HeLa K44A cells incubated with a CTB-HRP conjugate. In A-D the cells were incubated for 30 minutes at 0°C before fixation and processing for EM. The CTB-HRP conjugate labeling is shown on the cell surface and both in caveolae (A,C) and clathrin-coated pits (B,D). Incubation of the cells with CTB-HRP for 30 minutes at 37°C also labels both caveolae (E) and clathrin-coated pits (F), whereas the labeling of the non-specialized cell surface is strongly reduced. In G-J the cells were induced for mutant dynamin expression before incubation with CTB-HRP for 30 minutes at 37°C. Both caveolae (G,I) and particularly clathrin-coated pits, which were frequent (two- to threefold increase compared with controls), were distinctly labeled (H-J). Bar, 100 nm.

View larger version (107K): [in a new window]   Fig. 5. Electron micrographs of BHK antisense CHC cells incubated with a CTB-HRP conjugate. In A,B the non-induced control cells were incubated for 30 minutes at 0°C before fixation and processing for EM. The CT conjugate labeled the entire cell surface, including caveolae (A) and clathrin-coated pits (B). C,D show non-induced cells incubated with CTB-HRP for 30 minutes at 37°C, and both caveolae (C) and clathrin-coated pits (D) were labeled, whereas the rest of the cell surface appeared largely unlabeled. In E,F, the BHK cells were induced for antisense CHC expression before incubation with CTB-HRP for 30 minutes at 37°C. The toxin conjugate was distinctly present in caveolae (E) and in clathrin-coated pits (F), which were frequent (two to fivefold increase compared with non-induced control cells). Bar, 100 nm.

View larger version (73K): [in a new window]   Fig. 6. Electron micrographs of mutant dynamin-expressing HeLa K44A (A) and induced BHK antisense CHC cells (B,C) showing internalized CTB-HRP conjugate. In A, HeLa K44A cells were incubated with CTB-HRP conjugate for 30 minutes at 37°C before processing for EM. The CTB-HRP labeling was seen in an endosome (En) and surrounding tubulo-vesicular structures. In B,C, BHK antisense CHC cells were similarly incubated with CTB-HRP conjugate for 30 minutes at 37°C. Labeling was seen in an endosome (En) and surrounding tubulo-vesicular structures (B) as well as in a cistern (arrow) of a Golgi complex (Go) (C). Bars, 200 nm.

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