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Plasma membrane microdomains act as concentration platforms to facilitate intoxication by aerolysin.

Abrami L, van Der Goot FG - J. Cell Biol. (1999)

Bottom Line: Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore.We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution.Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

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Antibody cross-linking of receptor bound proaerolysin at the cell surface. BHK cells were treated or not with β-MCD (10 mM in IM at 37°C for 1 h) or saponin (0.4% at 4°C for 1 h), then incubated consecutively at 4°C with proaerolysin (2 nM) for 1 h, with antiproaerolysin mAbs for 1 h, and finally with FITC-labeled secondary antibodies for 1 h. Cells were then fixed first in 3% paraformaldehyde in PBS for 4 min at 8°C and then in methanol for 5 min at −20°C. The punctate pattern observed in β-MCD cells (b) was similar to that observed in control cells (a), but could not be found in any of the saponin-treated cells (c). Similar patterns were observed when fixing cells with paraformaldehyde (5 min at 4°C, followed by 20 min at room temperature) only, even after treatment with 0.2% or 0.4% saponin for 30 min at 4°C. The staining observed in the nuclear region in saponin-treated cells is due to background staining of the antibody and can also be seen when cells have not been treated with the toxin (results not shown). This staining is absent in control and β-MCD–treated cells because the cells are not permeabilized during antibody treatment. Bar, 10.5 μm.
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Figure 5: Antibody cross-linking of receptor bound proaerolysin at the cell surface. BHK cells were treated or not with β-MCD (10 mM in IM at 37°C for 1 h) or saponin (0.4% at 4°C for 1 h), then incubated consecutively at 4°C with proaerolysin (2 nM) for 1 h, with antiproaerolysin mAbs for 1 h, and finally with FITC-labeled secondary antibodies for 1 h. Cells were then fixed first in 3% paraformaldehyde in PBS for 4 min at 8°C and then in methanol for 5 min at −20°C. The punctate pattern observed in β-MCD cells (b) was similar to that observed in control cells (a), but could not be found in any of the saponin-treated cells (c). Similar patterns were observed when fixing cells with paraformaldehyde (5 min at 4°C, followed by 20 min at room temperature) only, even after treatment with 0.2% or 0.4% saponin for 30 min at 4°C. The staining observed in the nuclear region in saponin-treated cells is due to background staining of the antibody and can also be seen when cells have not been treated with the toxin (results not shown). This staining is absent in control and β-MCD–treated cells because the cells are not permeabilized during antibody treatment. Bar, 10.5 μm.

Mentions: Previously, we have shown that receptor bound proaerolysin can be clustered using antitoxin antibodies, and that clustering leads to a characteristic punctate distribution by immunofluorescence (Abrami et al. 1998b). Within these clusters, the toxin was found to fully colocalize with antibody cross-linked alkaline phosphatase and partially with caveolin-1 (Abrami et al. 1998b). Similar antibody-induced clustering has been observed for a variety of GPI-anchored proteins (for review see Brown and London 1998). We have investigated whether treatments with β-MCD or saponin would affect this antibody-induced clustering capacity. The staining pattern found on β-MCD–treated cells (Fig. 5 b) was similar to that observed on control cells. In contrast, a diffuse staining was observed when cells were treated with saponin (Fig. 5 c) indicating that, under these conditions, even a sandwich of primary and secondary antibodies, before cell fixation, could not induce clustering of proaerolysin bound to its receptor.


Plasma membrane microdomains act as concentration platforms to facilitate intoxication by aerolysin.

Abrami L, van Der Goot FG - J. Cell Biol. (1999)

Antibody cross-linking of receptor bound proaerolysin at the cell surface. BHK cells were treated or not with β-MCD (10 mM in IM at 37°C for 1 h) or saponin (0.4% at 4°C for 1 h), then incubated consecutively at 4°C with proaerolysin (2 nM) for 1 h, with antiproaerolysin mAbs for 1 h, and finally with FITC-labeled secondary antibodies for 1 h. Cells were then fixed first in 3% paraformaldehyde in PBS for 4 min at 8°C and then in methanol for 5 min at −20°C. The punctate pattern observed in β-MCD cells (b) was similar to that observed in control cells (a), but could not be found in any of the saponin-treated cells (c). Similar patterns were observed when fixing cells with paraformaldehyde (5 min at 4°C, followed by 20 min at room temperature) only, even after treatment with 0.2% or 0.4% saponin for 30 min at 4°C. The staining observed in the nuclear region in saponin-treated cells is due to background staining of the antibody and can also be seen when cells have not been treated with the toxin (results not shown). This staining is absent in control and β-MCD–treated cells because the cells are not permeabilized during antibody treatment. Bar, 10.5 μm.
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Related In: Results  -  Collection

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Figure 5: Antibody cross-linking of receptor bound proaerolysin at the cell surface. BHK cells were treated or not with β-MCD (10 mM in IM at 37°C for 1 h) or saponin (0.4% at 4°C for 1 h), then incubated consecutively at 4°C with proaerolysin (2 nM) for 1 h, with antiproaerolysin mAbs for 1 h, and finally with FITC-labeled secondary antibodies for 1 h. Cells were then fixed first in 3% paraformaldehyde in PBS for 4 min at 8°C and then in methanol for 5 min at −20°C. The punctate pattern observed in β-MCD cells (b) was similar to that observed in control cells (a), but could not be found in any of the saponin-treated cells (c). Similar patterns were observed when fixing cells with paraformaldehyde (5 min at 4°C, followed by 20 min at room temperature) only, even after treatment with 0.2% or 0.4% saponin for 30 min at 4°C. The staining observed in the nuclear region in saponin-treated cells is due to background staining of the antibody and can also be seen when cells have not been treated with the toxin (results not shown). This staining is absent in control and β-MCD–treated cells because the cells are not permeabilized during antibody treatment. Bar, 10.5 μm.
Mentions: Previously, we have shown that receptor bound proaerolysin can be clustered using antitoxin antibodies, and that clustering leads to a characteristic punctate distribution by immunofluorescence (Abrami et al. 1998b). Within these clusters, the toxin was found to fully colocalize with antibody cross-linked alkaline phosphatase and partially with caveolin-1 (Abrami et al. 1998b). Similar antibody-induced clustering has been observed for a variety of GPI-anchored proteins (for review see Brown and London 1998). We have investigated whether treatments with β-MCD or saponin would affect this antibody-induced clustering capacity. The staining pattern found on β-MCD–treated cells (Fig. 5 b) was similar to that observed on control cells. In contrast, a diffuse staining was observed when cells were treated with saponin (Fig. 5 c) indicating that, under these conditions, even a sandwich of primary and secondary antibodies, before cell fixation, could not induce clustering of proaerolysin bound to its receptor.

Bottom Line: Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore.We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution.Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland.

ABSTRACT
It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.

Show MeSH
Related in: MedlinePlus