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Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG - J. Cell Biol. (2003)

Bottom Line: The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm.Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts).We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

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The anthrax toxin enters preferentially via a clathrin-mediated pathway. (A) HeLa cells were transiently transfected with the dominant-negative caveolin-1 mutant (GFP-Cav1), incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting for the presence of heptameric PA63. (B) CHO cells were transiently transfected with caveolin-1-GFP (which has a wild-type phenotype), then incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 30 min at 37°C, and cells were then fixed and visualized using a fluorescent microscope. A blow-up of a region of the plasma membrane is shown. Bar, 10 μm. (C) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 0, 5, 15, or 30 min at 37°C, cells were then submitted to a cold acid wash, fixed, and visualized. Bar, 10 μm. (D) CHO cells were incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by SDS-PAGE and Western blotting for the presence of SDS-resistant heptameric PA63. The band intensity was quantified by densitometry (expressed in arbitrary units, a.u.), and is shown as a histogram to clearly illustrate the appearance and the degradation of the heptamer. (E) Cells were treated as in C, then incubated with protein A coupled to 10 nm gold, fixed, and processed for embedding in Epon and sectioning. Three examples are shown on which the clathrin coat is clearly visible (a–c, arrows); d is an example showing PASNKE in an invagination with no apparent coat (arrowhead). Bar, 200 nm. (F) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich (X-link+37°C). Internalization was allowed to proceed either for 5 min at 37°C in the presence of FITC-transferrin (Tf) or for 15 min in the presence of FITC-dextran. Cells were then submitted to a cold acid wash, fixed, and visualized. Examples of colocalization are indicated by arrowheads. Bar, 10 μm. (G) HeLa cells induced to express either wild-type or K44A dominant-negative dynamin-1 were treated and analyzed as in A. Numbers below lanes represent the incubation times at 37°C in min. The band intensities were quantified and plotted as in C. (H) HeLa cells transiently transfected with wild-type or dominant-negative EΔ95/295 Eps15 mutant were treated and analyzed as in G.
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fig5: The anthrax toxin enters preferentially via a clathrin-mediated pathway. (A) HeLa cells were transiently transfected with the dominant-negative caveolin-1 mutant (GFP-Cav1), incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting for the presence of heptameric PA63. (B) CHO cells were transiently transfected with caveolin-1-GFP (which has a wild-type phenotype), then incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 30 min at 37°C, and cells were then fixed and visualized using a fluorescent microscope. A blow-up of a region of the plasma membrane is shown. Bar, 10 μm. (C) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 0, 5, 15, or 30 min at 37°C, cells were then submitted to a cold acid wash, fixed, and visualized. Bar, 10 μm. (D) CHO cells were incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by SDS-PAGE and Western blotting for the presence of SDS-resistant heptameric PA63. The band intensity was quantified by densitometry (expressed in arbitrary units, a.u.), and is shown as a histogram to clearly illustrate the appearance and the degradation of the heptamer. (E) Cells were treated as in C, then incubated with protein A coupled to 10 nm gold, fixed, and processed for embedding in Epon and sectioning. Three examples are shown on which the clathrin coat is clearly visible (a–c, arrows); d is an example showing PASNKE in an invagination with no apparent coat (arrowhead). Bar, 200 nm. (F) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich (X-link+37°C). Internalization was allowed to proceed either for 5 min at 37°C in the presence of FITC-transferrin (Tf) or for 15 min in the presence of FITC-dextran. Cells were then submitted to a cold acid wash, fixed, and visualized. Examples of colocalization are indicated by arrowheads. Bar, 10 μm. (G) HeLa cells induced to express either wild-type or K44A dominant-negative dynamin-1 were treated and analyzed as in A. Numbers below lanes represent the incubation times at 37°C in min. The band intensities were quantified and plotted as in C. (H) HeLa cells transiently transfected with wild-type or dominant-negative EΔ95/295 Eps15 mutant were treated and analyzed as in G.

Mentions: The fact that internalization of the ATR was both ligand-triggered and raft-dependent raised the possibility that caveolin-dependent endocytosis might be involved (Pelkmans and Helenius, 2002). However, the observation that PA63 and caveolin-1 showed different behaviors on cells at 4°C (Fig. 1 B and Fig. 2 F) and the fact that caveosomes are neutral and do not appear to communicate with any acidic organelle (Pelkmans and Helenius, 2002; knowing that the PA63 heptamer requires an acidic pH for membrane insertion) argue against a caveolar mediated uptake. To clarify this issue, we investigated whether expression of a dominant-negative mutant of caveolin-1, namely caveolin-1 with the GFP fused to its NH2 terminus (GFP-Cav1; Pelkmans et al., 2001), would affect anthrax toxin entry. The kinetics of appearance of the SDS-resistant PA63 heptamer (Fig. 5 A) as well as the kinetics of MEK1 cleavage (unpublished data) were very similar in control and GFP-Cav1–expressing cells. Also, we found little colocalization between 30-min internalized antibody-clustered PA83 and caveolin-1-GFP, used here as a marker of caveolae and caveosomes (large green structures seen in Fig. 5 B, which were never positive for PA; note that fusion of GFP to the COOH terminus of caveolin-1 does not lead to a dominant-negative phenotype; Pelkmans et al., 2001). Finally, to further rule out a caveolin-mediated entry pathway and transport to caveosomes, we found that intracellular SDS-resistant PA63 heptamers also form in macrophages RAW264.7 and FRT cells; two cell types that do not express caveolin-1 (Zurzolo et al., 1994; Kiss et al., 2000; unpublished data), in agreement with the fact that macrophages are sensitive to LF (Tang and Leppla, 1999).


Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process.

Abrami L, Liu S, Cosson P, Leppla SH, van der Goot FG - J. Cell Biol. (2003)

The anthrax toxin enters preferentially via a clathrin-mediated pathway. (A) HeLa cells were transiently transfected with the dominant-negative caveolin-1 mutant (GFP-Cav1), incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting for the presence of heptameric PA63. (B) CHO cells were transiently transfected with caveolin-1-GFP (which has a wild-type phenotype), then incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 30 min at 37°C, and cells were then fixed and visualized using a fluorescent microscope. A blow-up of a region of the plasma membrane is shown. Bar, 10 μm. (C) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 0, 5, 15, or 30 min at 37°C, cells were then submitted to a cold acid wash, fixed, and visualized. Bar, 10 μm. (D) CHO cells were incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by SDS-PAGE and Western blotting for the presence of SDS-resistant heptameric PA63. The band intensity was quantified by densitometry (expressed in arbitrary units, a.u.), and is shown as a histogram to clearly illustrate the appearance and the degradation of the heptamer. (E) Cells were treated as in C, then incubated with protein A coupled to 10 nm gold, fixed, and processed for embedding in Epon and sectioning. Three examples are shown on which the clathrin coat is clearly visible (a–c, arrows); d is an example showing PASNKE in an invagination with no apparent coat (arrowhead). Bar, 200 nm. (F) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich (X-link+37°C). Internalization was allowed to proceed either for 5 min at 37°C in the presence of FITC-transferrin (Tf) or for 15 min in the presence of FITC-dextran. Cells were then submitted to a cold acid wash, fixed, and visualized. Examples of colocalization are indicated by arrowheads. Bar, 10 μm. (G) HeLa cells induced to express either wild-type or K44A dominant-negative dynamin-1 were treated and analyzed as in A. Numbers below lanes represent the incubation times at 37°C in min. The band intensities were quantified and plotted as in C. (H) HeLa cells transiently transfected with wild-type or dominant-negative EΔ95/295 Eps15 mutant were treated and analyzed as in G.
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fig5: The anthrax toxin enters preferentially via a clathrin-mediated pathway. (A) HeLa cells were transiently transfected with the dominant-negative caveolin-1 mutant (GFP-Cav1), incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, and transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by Western blotting for the presence of heptameric PA63. (B) CHO cells were transiently transfected with caveolin-1-GFP (which has a wild-type phenotype), then incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 30 min at 37°C, and cells were then fixed and visualized using a fluorescent microscope. A blow-up of a region of the plasma membrane is shown. Bar, 10 μm. (C) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich as in Fig. 3 (X-link+37°C). Internalization was allowed to proceed for 0, 5, 15, or 30 min at 37°C, cells were then submitted to a cold acid wash, fixed, and visualized. Bar, 10 μm. (D) CHO cells were incubated for 1 h at 4°C with1 μg/ml trypsin-nicked PA83, transferred to 37°C for different periods of time. 40 μg of total cell extracts were analyzed by SDS-PAGE and Western blotting for the presence of SDS-resistant heptameric PA63. The band intensity was quantified by densitometry (expressed in arbitrary units, a.u.), and is shown as a histogram to clearly illustrate the appearance and the degradation of the heptamer. (E) Cells were treated as in C, then incubated with protein A coupled to 10 nm gold, fixed, and processed for embedding in Epon and sectioning. Three examples are shown on which the clathrin coat is clearly visible (a–c, arrows); d is an example showing PASNKE in an invagination with no apparent coat (arrowhead). Bar, 200 nm. (F) CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE followed by an antibody sandwich (X-link+37°C). Internalization was allowed to proceed either for 5 min at 37°C in the presence of FITC-transferrin (Tf) or for 15 min in the presence of FITC-dextran. Cells were then submitted to a cold acid wash, fixed, and visualized. Examples of colocalization are indicated by arrowheads. Bar, 10 μm. (G) HeLa cells induced to express either wild-type or K44A dominant-negative dynamin-1 were treated and analyzed as in A. Numbers below lanes represent the incubation times at 37°C in min. The band intensities were quantified and plotted as in C. (H) HeLa cells transiently transfected with wild-type or dominant-negative EΔ95/295 Eps15 mutant were treated and analyzed as in G.
Mentions: The fact that internalization of the ATR was both ligand-triggered and raft-dependent raised the possibility that caveolin-dependent endocytosis might be involved (Pelkmans and Helenius, 2002). However, the observation that PA63 and caveolin-1 showed different behaviors on cells at 4°C (Fig. 1 B and Fig. 2 F) and the fact that caveosomes are neutral and do not appear to communicate with any acidic organelle (Pelkmans and Helenius, 2002; knowing that the PA63 heptamer requires an acidic pH for membrane insertion) argue against a caveolar mediated uptake. To clarify this issue, we investigated whether expression of a dominant-negative mutant of caveolin-1, namely caveolin-1 with the GFP fused to its NH2 terminus (GFP-Cav1; Pelkmans et al., 2001), would affect anthrax toxin entry. The kinetics of appearance of the SDS-resistant PA63 heptamer (Fig. 5 A) as well as the kinetics of MEK1 cleavage (unpublished data) were very similar in control and GFP-Cav1–expressing cells. Also, we found little colocalization between 30-min internalized antibody-clustered PA83 and caveolin-1-GFP, used here as a marker of caveolae and caveosomes (large green structures seen in Fig. 5 B, which were never positive for PA; note that fusion of GFP to the COOH terminus of caveolin-1 does not lead to a dominant-negative phenotype; Pelkmans et al., 2001). Finally, to further rule out a caveolin-mediated entry pathway and transport to caveosomes, we found that intracellular SDS-resistant PA63 heptamers also form in macrophages RAW264.7 and FRT cells; two cell types that do not express caveolin-1 (Zurzolo et al., 1994; Kiss et al., 2000; unpublished data), in agreement with the fact that macrophages are sensitive to LF (Tang and Leppla, 1999).

Bottom Line: The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm.Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts).We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

Show MeSH
Related in: MedlinePlus