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Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells.

Seveau S, Bierne H, Giroux S, Prévost MC, Cossart P - J. Cell Biol. (2004)

Bottom Line: We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry.In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent.Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

View Article: PubMed Central - PubMed

Affiliation: Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France.

ABSTRACT
Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

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E-cadherin recruitment at the entry site is cholesterol dependent. (A) Control and cholesterol-depleted L2071hEcad cells were fixed and immunofluorescently labeled for E-cadherin (clone HECD1; Alexa Fluor 546–conjugated secondary antibodies). Images were acquired with a 40× objective, and were processed for quantitative analysis using the Metamorph software. Results of three independent experiments were expressed relative to control cells. (B and C) Control and cholesterol-depleted L2071hEcad cells were incubated for 30 min with internalin-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for E-cadherin. (B) Phase-contrast and fluorescent images were acquired with a 100× objective. Two different plans acquired at different focus points were shown for the fluorescent images. (C) Quantitative analysis of the percentage of internalin-coated beads which recruited E-cadherin. Bars, 10 μm. Arrows indicate the internalin-coated beads, which recruited E-cadherin.
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fig6: E-cadherin recruitment at the entry site is cholesterol dependent. (A) Control and cholesterol-depleted L2071hEcad cells were fixed and immunofluorescently labeled for E-cadherin (clone HECD1; Alexa Fluor 546–conjugated secondary antibodies). Images were acquired with a 40× objective, and were processed for quantitative analysis using the Metamorph software. Results of three independent experiments were expressed relative to control cells. (B and C) Control and cholesterol-depleted L2071hEcad cells were incubated for 30 min with internalin-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for E-cadherin. (B) Phase-contrast and fluorescent images were acquired with a 100× objective. Two different plans acquired at different focus points were shown for the fluorescent images. (C) Quantitative analysis of the percentage of internalin-coated beads which recruited E-cadherin. Bars, 10 μm. Arrows indicate the internalin-coated beads, which recruited E-cadherin.

Mentions: To explain the decreased adherence of internalin-coated beads to L2071hEcad cells, we investigated if cholesterol depletion affected the amount of E-cadherin molecules present at the plasma membrane. By fluorescence microscopy, we quantified the level of E-cadherin expression at the plasma membrane (see Materials and methods). As shown in Fig. 6 A, similar amounts of E-cadherin were present at the surface of control and cholesterol-depleted L2071hEcad cells. This result demonstrated that the diminution in internalin-coated bead adherence to the plasma membrane was not due to a lower expression of E-cadherin but rather to a decrease in receptor affinity or avidity. We then assessed the ability of internalin-coated beads to recruit E-cadherin. Control and cholesterol-depleted cells were incubated for 30 min with internalin-coated beads, cells were then fixed and E-cadherin was labeled with fluorescent antibodies. As shown in control cells (Fig. 6 B), E-cadherin molecules were massively recruited at the entry site forming a fluorescent ring around the beads, whereas after cholesterol depletion E-cadherin was poorly recruited. We confirmed this result by showing that the percentage of beads associated to the cell surface and surrounded by a ring of fluorescent E-cadherin, severely decreased in cholesterol depleted cells (Fig. 6 C). In conclusion, cholesterol depletion affected the recruitment of E-cadherin at the cell surface. This result prompted us to analyze if E-cadherin had to be present in DRMs to promote internalin-dependent entry.


Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells.

Seveau S, Bierne H, Giroux S, Prévost MC, Cossart P - J. Cell Biol. (2004)

E-cadherin recruitment at the entry site is cholesterol dependent. (A) Control and cholesterol-depleted L2071hEcad cells were fixed and immunofluorescently labeled for E-cadherin (clone HECD1; Alexa Fluor 546–conjugated secondary antibodies). Images were acquired with a 40× objective, and were processed for quantitative analysis using the Metamorph software. Results of three independent experiments were expressed relative to control cells. (B and C) Control and cholesterol-depleted L2071hEcad cells were incubated for 30 min with internalin-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for E-cadherin. (B) Phase-contrast and fluorescent images were acquired with a 100× objective. Two different plans acquired at different focus points were shown for the fluorescent images. (C) Quantitative analysis of the percentage of internalin-coated beads which recruited E-cadherin. Bars, 10 μm. Arrows indicate the internalin-coated beads, which recruited E-cadherin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172418&req=5

fig6: E-cadherin recruitment at the entry site is cholesterol dependent. (A) Control and cholesterol-depleted L2071hEcad cells were fixed and immunofluorescently labeled for E-cadherin (clone HECD1; Alexa Fluor 546–conjugated secondary antibodies). Images were acquired with a 40× objective, and were processed for quantitative analysis using the Metamorph software. Results of three independent experiments were expressed relative to control cells. (B and C) Control and cholesterol-depleted L2071hEcad cells were incubated for 30 min with internalin-coated beads. Cells were washed three times, fixed, permeabilized, and labeled for E-cadherin. (B) Phase-contrast and fluorescent images were acquired with a 100× objective. Two different plans acquired at different focus points were shown for the fluorescent images. (C) Quantitative analysis of the percentage of internalin-coated beads which recruited E-cadherin. Bars, 10 μm. Arrows indicate the internalin-coated beads, which recruited E-cadherin.
Mentions: To explain the decreased adherence of internalin-coated beads to L2071hEcad cells, we investigated if cholesterol depletion affected the amount of E-cadherin molecules present at the plasma membrane. By fluorescence microscopy, we quantified the level of E-cadherin expression at the plasma membrane (see Materials and methods). As shown in Fig. 6 A, similar amounts of E-cadherin were present at the surface of control and cholesterol-depleted L2071hEcad cells. This result demonstrated that the diminution in internalin-coated bead adherence to the plasma membrane was not due to a lower expression of E-cadherin but rather to a decrease in receptor affinity or avidity. We then assessed the ability of internalin-coated beads to recruit E-cadherin. Control and cholesterol-depleted cells were incubated for 30 min with internalin-coated beads, cells were then fixed and E-cadherin was labeled with fluorescent antibodies. As shown in control cells (Fig. 6 B), E-cadherin molecules were massively recruited at the entry site forming a fluorescent ring around the beads, whereas after cholesterol depletion E-cadherin was poorly recruited. We confirmed this result by showing that the percentage of beads associated to the cell surface and surrounded by a ring of fluorescent E-cadherin, severely decreased in cholesterol depleted cells (Fig. 6 C). In conclusion, cholesterol depletion affected the recruitment of E-cadherin at the cell surface. This result prompted us to analyze if E-cadherin had to be present in DRMs to promote internalin-dependent entry.

Bottom Line: We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry.In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent.Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

View Article: PubMed Central - PubMed

Affiliation: Unité des Interactions Bactéries-Cellules, INSERM U604, Institut Pasteur, 75015 Paris Cedex 15, France.

ABSTRACT
Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

Show MeSH
Related in: MedlinePlus