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Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

Cambi A, de Lange F, van Maarseveen NM, Nijhuis M, Joosten B, van Dijk EM, de Bakker BI, Fransen JA, Bovee-Geurts PH, van Leeuwen FN, Van Hulst NF, Figdor CG - J. Cell Biol. (2004)

Bottom Line: The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood.During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm.Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands.

ABSTRACT
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

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DC-SIGN colocalizes with lipid rafts on K-DC-SIGN. (A) To investigate the effect of cholesterol depletion on DC-SIGN–mediated adhesion, K-DC-SIGN cells were incubated in serum-free medium with or without 20 mM MCD for 30 min at 37°C. Subsequently, gp120-coated fluorescent beads (1-μm diam) were added and the mixture was incubated for an additional 30 min at 37°C. Binding was measured by flow cytometry. After MCD treatment, cell viability was assessed by trypan blue staining. The values represent the mean of three independent experiments ±SD. (B) K-DC-SIGN were solubilized with 1% Triton X-100, subjected to sucrose gradient centrifugation and analyzed by Western blotting for the indicated molecules. The numbers indicate the gradient fractions. Fractions 9 and 10 are low density fractions containing DRM and are referred to as raft fractions. (C) Confocal microscopy analysis of copatching of DC-SIGN and GM1. K-DC-SIGN cells were stained at 4°C with 10 μg/ml anti–DC-SIGN (or anti-CD55 or anti-CD46) and 10 μg/ml FITC-CTxB. Co-patching was induced by adding secondary Ab (Materials and methods), and, after fixation in PFA, cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representatives of multiple cells in three independent experiments. Bar, 5 μm.
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fig2: DC-SIGN colocalizes with lipid rafts on K-DC-SIGN. (A) To investigate the effect of cholesterol depletion on DC-SIGN–mediated adhesion, K-DC-SIGN cells were incubated in serum-free medium with or without 20 mM MCD for 30 min at 37°C. Subsequently, gp120-coated fluorescent beads (1-μm diam) were added and the mixture was incubated for an additional 30 min at 37°C. Binding was measured by flow cytometry. After MCD treatment, cell viability was assessed by trypan blue staining. The values represent the mean of three independent experiments ±SD. (B) K-DC-SIGN were solubilized with 1% Triton X-100, subjected to sucrose gradient centrifugation and analyzed by Western blotting for the indicated molecules. The numbers indicate the gradient fractions. Fractions 9 and 10 are low density fractions containing DRM and are referred to as raft fractions. (C) Confocal microscopy analysis of copatching of DC-SIGN and GM1. K-DC-SIGN cells were stained at 4°C with 10 μg/ml anti–DC-SIGN (or anti-CD55 or anti-CD46) and 10 μg/ml FITC-CTxB. Co-patching was induced by adding secondary Ab (Materials and methods), and, after fixation in PFA, cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representatives of multiple cells in three independent experiments. Bar, 5 μm.

Mentions: To determine whether lipid rafts are important for DC-SIGN function, K-DC-SIGN cells were treated with methyl-β-cyclodextrin (MCD) to extract membrane cholesterol, and its effect on DC-SIGN–mediated ligand binding was tested by the fluorescent beads adhesion assay. As shown in Fig. 2 A, MCD treatment partially inhibits binding to gp120-coated beads, indicating that cholesterol extraction partially affected DC-SIGN ligand binding capacity.


Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells.

Cambi A, de Lange F, van Maarseveen NM, Nijhuis M, Joosten B, van Dijk EM, de Bakker BI, Fransen JA, Bovee-Geurts PH, van Leeuwen FN, Van Hulst NF, Figdor CG - J. Cell Biol. (2004)

DC-SIGN colocalizes with lipid rafts on K-DC-SIGN. (A) To investigate the effect of cholesterol depletion on DC-SIGN–mediated adhesion, K-DC-SIGN cells were incubated in serum-free medium with or without 20 mM MCD for 30 min at 37°C. Subsequently, gp120-coated fluorescent beads (1-μm diam) were added and the mixture was incubated for an additional 30 min at 37°C. Binding was measured by flow cytometry. After MCD treatment, cell viability was assessed by trypan blue staining. The values represent the mean of three independent experiments ±SD. (B) K-DC-SIGN were solubilized with 1% Triton X-100, subjected to sucrose gradient centrifugation and analyzed by Western blotting for the indicated molecules. The numbers indicate the gradient fractions. Fractions 9 and 10 are low density fractions containing DRM and are referred to as raft fractions. (C) Confocal microscopy analysis of copatching of DC-SIGN and GM1. K-DC-SIGN cells were stained at 4°C with 10 μg/ml anti–DC-SIGN (or anti-CD55 or anti-CD46) and 10 μg/ml FITC-CTxB. Co-patching was induced by adding secondary Ab (Materials and methods), and, after fixation in PFA, cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representatives of multiple cells in three independent experiments. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171967&req=5

fig2: DC-SIGN colocalizes with lipid rafts on K-DC-SIGN. (A) To investigate the effect of cholesterol depletion on DC-SIGN–mediated adhesion, K-DC-SIGN cells were incubated in serum-free medium with or without 20 mM MCD for 30 min at 37°C. Subsequently, gp120-coated fluorescent beads (1-μm diam) were added and the mixture was incubated for an additional 30 min at 37°C. Binding was measured by flow cytometry. After MCD treatment, cell viability was assessed by trypan blue staining. The values represent the mean of three independent experiments ±SD. (B) K-DC-SIGN were solubilized with 1% Triton X-100, subjected to sucrose gradient centrifugation and analyzed by Western blotting for the indicated molecules. The numbers indicate the gradient fractions. Fractions 9 and 10 are low density fractions containing DRM and are referred to as raft fractions. (C) Confocal microscopy analysis of copatching of DC-SIGN and GM1. K-DC-SIGN cells were stained at 4°C with 10 μg/ml anti–DC-SIGN (or anti-CD55 or anti-CD46) and 10 μg/ml FITC-CTxB. Co-patching was induced by adding secondary Ab (Materials and methods), and, after fixation in PFA, cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representatives of multiple cells in three independent experiments. Bar, 5 μm.
Mentions: To determine whether lipid rafts are important for DC-SIGN function, K-DC-SIGN cells were treated with methyl-β-cyclodextrin (MCD) to extract membrane cholesterol, and its effect on DC-SIGN–mediated ligand binding was tested by the fluorescent beads adhesion assay. As shown in Fig. 2 A, MCD treatment partially inhibits binding to gp120-coated beads, indicating that cholesterol extraction partially affected DC-SIGN ligand binding capacity.

Bottom Line: The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood.During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm.Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Tumor Immunology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, Netherlands.

ABSTRACT
The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host.

Show MeSH
Related in: MedlinePlus