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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 and HGF-R are present in DRMs. (A) Control and cholesterol-depleted L2071hEcad cells, preincubated or not with soluble internalin (InlA), were extracted with 0.5% Brij58. (B) L2071hEcad cells were extracted with 0.1% Triton X-100. (C) Lovo cells were extracted with 0.5% Triton X-100. After detergent extraction, cell lysates were subjected to ultracentrifugation on sucrose gradients. Fractions were collected from top to bottom and were processed for Western blotting analysis. Fractions 2–4/low density fractions (LD), and fractions 9 and 10/high density fractions (HD).
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fig7: E-cadherin and HGF-R are present in DRMs. (A) Control and cholesterol-depleted L2071hEcad cells, preincubated or not with soluble internalin (InlA), were extracted with 0.5% Brij58. (B) L2071hEcad cells were extracted with 0.1% Triton X-100. (C) Lovo cells were extracted with 0.5% Triton X-100. After detergent extraction, cell lysates were subjected to ultracentrifugation on sucrose gradients. Fractions were collected from top to bottom and were processed for Western blotting analysis. Fractions 2–4/low density fractions (LD), and fractions 9 and 10/high density fractions (HD).

Mentions: We performed cold detergent extraction of the L2071hEcad cells, using the nonionic detergent Brij 58. Cell lysates were fractionated according to density criteria by ultracentrifugation on sucrose gradients. Fractions were collected and subjected to Western blotting analysis. We used caveolin-2 as a control of our fractionation because it is well known that caveolin-2 is present within the low density DRM fractions (Mora et al., 1999). Interestingly, a significant proportion of E-cadherin molecules was present in DRMs suggesting an association of E-cadherin with lipid microdomains (Fig. 7 A, left). To further demonstrate this result, cholesterol was depleted before cell lysis. After cholesterol depletion E-cadherin molecules were not present in the DRMs confirming that a significant proportion of E-cadherin molecules was present in DRMs in a cholesterol-dependent manner. α-Catenin, the cytoskeletal molecule which links E-cadherin to the F-actin cytoskeleton was, similarly to E-cadherin, present in DRMs. We also performed cold Triton X-100 extraction of L2071hEcad cells (Fig. 7 B) and of Lovo cells (Fig. 7 C) and found that E-cadherin, as well as HGF-R, were present in the low density DRM fraction. Cells were then incubated with soluble internalin to determine if internalin would associate preferentially with the population of E-cadherin present in DRMs. Internalin was exclusively present to the low density fractions (Fig. 7 A, right). In contrast, after cholesterol depletion, internalin was associated to higher density fractions in which E-cadherin was present. These results show that internalin interacts preferentially with E-cadherin present within DRMs and that incubation of cells with soluble internalin does not affect the amount of E-cadherin present in the DRM fractions.


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 and HGF-R are present in DRMs. (A) Control and cholesterol-depleted L2071hEcad cells, preincubated or not with soluble internalin (InlA), were extracted with 0.5% Brij58. (B) L2071hEcad cells were extracted with 0.1% Triton X-100. (C) Lovo cells were extracted with 0.5% Triton X-100. After detergent extraction, cell lysates were subjected to ultracentrifugation on sucrose gradients. Fractions were collected from top to bottom and were processed for Western blotting analysis. Fractions 2–4/low density fractions (LD), and fractions 9 and 10/high density fractions (HD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172418&req=5

fig7: E-cadherin and HGF-R are present in DRMs. (A) Control and cholesterol-depleted L2071hEcad cells, preincubated or not with soluble internalin (InlA), were extracted with 0.5% Brij58. (B) L2071hEcad cells were extracted with 0.1% Triton X-100. (C) Lovo cells were extracted with 0.5% Triton X-100. After detergent extraction, cell lysates were subjected to ultracentrifugation on sucrose gradients. Fractions were collected from top to bottom and were processed for Western blotting analysis. Fractions 2–4/low density fractions (LD), and fractions 9 and 10/high density fractions (HD).
Mentions: We performed cold detergent extraction of the L2071hEcad cells, using the nonionic detergent Brij 58. Cell lysates were fractionated according to density criteria by ultracentrifugation on sucrose gradients. Fractions were collected and subjected to Western blotting analysis. We used caveolin-2 as a control of our fractionation because it is well known that caveolin-2 is present within the low density DRM fractions (Mora et al., 1999). Interestingly, a significant proportion of E-cadherin molecules was present in DRMs suggesting an association of E-cadherin with lipid microdomains (Fig. 7 A, left). To further demonstrate this result, cholesterol was depleted before cell lysis. After cholesterol depletion E-cadherin molecules were not present in the DRMs confirming that a significant proportion of E-cadherin molecules was present in DRMs in a cholesterol-dependent manner. α-Catenin, the cytoskeletal molecule which links E-cadherin to the F-actin cytoskeleton was, similarly to E-cadherin, present in DRMs. We also performed cold Triton X-100 extraction of L2071hEcad cells (Fig. 7 B) and of Lovo cells (Fig. 7 C) and found that E-cadherin, as well as HGF-R, were present in the low density DRM fraction. Cells were then incubated with soluble internalin to determine if internalin would associate preferentially with the population of E-cadherin present in DRMs. Internalin was exclusively present to the low density fractions (Fig. 7 A, right). In contrast, after cholesterol depletion, internalin was associated to higher density fractions in which E-cadherin was present. These results show that internalin interacts preferentially with E-cadherin present within DRMs and that incubation of cells with soluble internalin does not affect the amount of E-cadherin present in the DRM fractions.

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