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A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum.

Abrami L, Fivaz M, Glauser PE, Parton RG, van der Goot FG - J. Cell Biol. (1998)

Bottom Line: Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases.Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments.Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

View Article: PubMed Central - PubMed

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

ABSTRACT
In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

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Effects of proaerolysin on the viability and morphology  of BHK cells. (a) Cells were incubated with 0.38 nM proaerolysin  at 37°C for 75 min, and then submitted to the DEAD/LIVE viability assay (see text). At this time point, all cells excluded ethidium dimer–1 and all retained cellular esterases as witnessed by the  conversion of Calcein-AM to fluorescent calcein. Phase contrast  image of control cells (b), cells incubated with 0.34 nM proaerolysin (c) for 1 h at 37°C. In d, cells were incubated with 6 U/ml of  PI-PLC for 1 h at 37°C in the presence of 10 μg/ml cycloheximide,  and then incubated with 0.38 nM proaerolysin for 1 h at 4°C,  washed and incubated in a toxin-free medium for 1 h at 37°C.  Bar, 10.5 μm.
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Figure 8: Effects of proaerolysin on the viability and morphology of BHK cells. (a) Cells were incubated with 0.38 nM proaerolysin at 37°C for 75 min, and then submitted to the DEAD/LIVE viability assay (see text). At this time point, all cells excluded ethidium dimer–1 and all retained cellular esterases as witnessed by the conversion of Calcein-AM to fluorescent calcein. Phase contrast image of control cells (b), cells incubated with 0.34 nM proaerolysin (c) for 1 h at 37°C. In d, cells were incubated with 6 U/ml of PI-PLC for 1 h at 37°C in the presence of 10 μg/ml cycloheximide, and then incubated with 0.38 nM proaerolysin for 1 h at 4°C, washed and incubated in a toxin-free medium for 1 h at 37°C. Bar, 10.5 μm.

Mentions: To investigate whether the overall integrity of the plasma membrane was maintained, toxin-treated cells were subjected to a viability assay (LIVE/DEAD assay; Molecular Probes). The principle of this assay is to incubate cells with calcein-AM, a membrane permeable reagent that can be cleaved by esterases in the living cells thereby yielding green fluorescence. Simultaneously, cells are incubated with ethidium homodimer–1, which only labels the nucleic acids of membrane-compromised cells because the dye is membrane impermeant, yet small enough to diffuse through the nuclear pore (red fluorescence). At various times, the number of cells, respectively, green or red were counted (Table I). Cells retained their plasma membrane integrity during several hours of toxin treatment since cellular esterases were not released and ethidium homodimer–1 was excluded (Table I; and Fig. 8 a). Similarly, propidium iodide and trypan blue were excluded from these cells.


A pore-forming toxin interacts with a GPI-anchored protein and causes vacuolation of the endoplasmic reticulum.

Abrami L, Fivaz M, Glauser PE, Parton RG, van der Goot FG - J. Cell Biol. (1998)

Effects of proaerolysin on the viability and morphology  of BHK cells. (a) Cells were incubated with 0.38 nM proaerolysin  at 37°C for 75 min, and then submitted to the DEAD/LIVE viability assay (see text). At this time point, all cells excluded ethidium dimer–1 and all retained cellular esterases as witnessed by the  conversion of Calcein-AM to fluorescent calcein. Phase contrast  image of control cells (b), cells incubated with 0.34 nM proaerolysin (c) for 1 h at 37°C. In d, cells were incubated with 6 U/ml of  PI-PLC for 1 h at 37°C in the presence of 10 μg/ml cycloheximide,  and then incubated with 0.38 nM proaerolysin for 1 h at 4°C,  washed and incubated in a toxin-free medium for 1 h at 37°C.  Bar, 10.5 μm.
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Related In: Results  -  Collection

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Figure 8: Effects of proaerolysin on the viability and morphology of BHK cells. (a) Cells were incubated with 0.38 nM proaerolysin at 37°C for 75 min, and then submitted to the DEAD/LIVE viability assay (see text). At this time point, all cells excluded ethidium dimer–1 and all retained cellular esterases as witnessed by the conversion of Calcein-AM to fluorescent calcein. Phase contrast image of control cells (b), cells incubated with 0.34 nM proaerolysin (c) for 1 h at 37°C. In d, cells were incubated with 6 U/ml of PI-PLC for 1 h at 37°C in the presence of 10 μg/ml cycloheximide, and then incubated with 0.38 nM proaerolysin for 1 h at 4°C, washed and incubated in a toxin-free medium for 1 h at 37°C. Bar, 10.5 μm.
Mentions: To investigate whether the overall integrity of the plasma membrane was maintained, toxin-treated cells were subjected to a viability assay (LIVE/DEAD assay; Molecular Probes). The principle of this assay is to incubate cells with calcein-AM, a membrane permeable reagent that can be cleaved by esterases in the living cells thereby yielding green fluorescence. Simultaneously, cells are incubated with ethidium homodimer–1, which only labels the nucleic acids of membrane-compromised cells because the dye is membrane impermeant, yet small enough to diffuse through the nuclear pore (red fluorescence). At various times, the number of cells, respectively, green or red were counted (Table I). Cells retained their plasma membrane integrity during several hours of toxin treatment since cellular esterases were not released and ethidium homodimer–1 was excluded (Table I; and Fig. 8 a). Similarly, propidium iodide and trypan blue were excluded from these cells.

Bottom Line: Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases.Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments.Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

View Article: PubMed Central - PubMed

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

ABSTRACT
In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid "rafts." We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.

Show MeSH
Related in: MedlinePlus