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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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Raft association of PA triggers cellular uptake. (A) Antibody cross-linking leads to the appearance of intracellular PA83. As schematized in the right panel, CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE. Cells were then either (1) submitted to antibody cross-linking at 4°C and then further incubated at 4°C (condition: X-link+4°C) or 37°C (condition: X-link+37°C) for 30 min or (2) incubated at 37°C for 30 min and then submitted to the antibody sandwich at 4°C (condition: 37°C+X-link). For all conditions, cells were submitted or not to an acid wash before fixation. Bar, 10 μm. (B) Cholesterol depletion inhibits intracellular accumulation of PA63. CHO cells were treated or not with β-MCD, then 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 for 30 min at 37°C and cells were then submitted to a cold acid wash, fixed, and visualized using a fluorescent microscope.
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fig3: Raft association of PA triggers cellular uptake. (A) Antibody cross-linking leads to the appearance of intracellular PA83. As schematized in the right panel, CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE. Cells were then either (1) submitted to antibody cross-linking at 4°C and then further incubated at 4°C (condition: X-link+4°C) or 37°C (condition: X-link+37°C) for 30 min or (2) incubated at 37°C for 30 min and then submitted to the antibody sandwich at 4°C (condition: 37°C+X-link). For all conditions, cells were submitted or not to an acid wash before fixation. Bar, 10 μm. (B) Cholesterol depletion inhibits intracellular accumulation of PA63. CHO cells were treated or not with β-MCD, then 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 for 30 min at 37°C and cells were then submitted to a cold acid wash, fixed, and visualized using a fluorescent microscope.

Mentions: Next, we investigated whether ATR clustering is also a trigger for internalization. Again, we made use of the PA variant that cannot be processed by furin because such mutants remain at the cell surface (Beauregard et al., 2000), and investigated whether antibody clustering would promote internalization. Cells were incubated with PASNKE at 4°C, and a sandwich of primary and secondary antibodies was then added (Fig. 3 A, middle and bottom rows) or not (Fig. 3 A, top row) at 4°C. Cells were then shifted to 37°C to allow internalization (Fig. 3 A, top and middle rows) or kept on ice (Fig. 3 A, bottom row). Control cells (PASNKE with no antibodies; Fig. 3 A, top row) were shifted back to 4°C and were then treated with primary and secondary antibodies. Finally, cells were submitted or not to an acid wash in order to remove surface-bound antibodies and to reveal only the intracellular PASNKE. Intracellular PASNKE could only be detected when antibody cross-linking was performed before the internalization step (Fig. 3 A, middle row), demonstrating that clustering of PA83 is necessary and sufficient to promote its cellular uptake. When cells were treated with β-MCD before PA and antibody addition, intracellular accumulation was significantly reduced (Fig. 3 B).


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)

Raft association of PA triggers cellular uptake. (A) Antibody cross-linking leads to the appearance of intracellular PA83. As schematized in the right panel, CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE. Cells were then either (1) submitted to antibody cross-linking at 4°C and then further incubated at 4°C (condition: X-link+4°C) or 37°C (condition: X-link+37°C) for 30 min or (2) incubated at 37°C for 30 min and then submitted to the antibody sandwich at 4°C (condition: 37°C+X-link). For all conditions, cells were submitted or not to an acid wash before fixation. Bar, 10 μm. (B) Cholesterol depletion inhibits intracellular accumulation of PA63. CHO cells were treated or not with β-MCD, then 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 for 30 min at 37°C and cells were then submitted to a cold acid wash, fixed, and visualized using a fluorescent microscope.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172673&req=5

fig3: Raft association of PA triggers cellular uptake. (A) Antibody cross-linking leads to the appearance of intracellular PA83. As schematized in the right panel, CHO cells were incubated for 20 min at 4°C with 500 ng/ml PASNKE. Cells were then either (1) submitted to antibody cross-linking at 4°C and then further incubated at 4°C (condition: X-link+4°C) or 37°C (condition: X-link+37°C) for 30 min or (2) incubated at 37°C for 30 min and then submitted to the antibody sandwich at 4°C (condition: 37°C+X-link). For all conditions, cells were submitted or not to an acid wash before fixation. Bar, 10 μm. (B) Cholesterol depletion inhibits intracellular accumulation of PA63. CHO cells were treated or not with β-MCD, then 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 for 30 min at 37°C and cells were then submitted to a cold acid wash, fixed, and visualized using a fluorescent microscope.
Mentions: Next, we investigated whether ATR clustering is also a trigger for internalization. Again, we made use of the PA variant that cannot be processed by furin because such mutants remain at the cell surface (Beauregard et al., 2000), and investigated whether antibody clustering would promote internalization. Cells were incubated with PASNKE at 4°C, and a sandwich of primary and secondary antibodies was then added (Fig. 3 A, middle and bottom rows) or not (Fig. 3 A, top row) at 4°C. Cells were then shifted to 37°C to allow internalization (Fig. 3 A, top and middle rows) or kept on ice (Fig. 3 A, bottom row). Control cells (PASNKE with no antibodies; Fig. 3 A, top row) were shifted back to 4°C and were then treated with primary and secondary antibodies. Finally, cells were submitted or not to an acid wash in order to remove surface-bound antibodies and to reveal only the intracellular PASNKE. Intracellular PASNKE could only be detected when antibody cross-linking was performed before the internalization step (Fig. 3 A, middle row), demonstrating that clustering of PA83 is necessary and sufficient to promote its cellular uptake. When cells were treated with β-MCD before PA and antibody addition, intracellular accumulation was significantly reduced (Fig. 3 B).

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