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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 resides in lipid rafts on immature DCs. (A) DC-SIGN–mediated adhesion to ligand-coated fluorescent beads (1-μm diam) on immature DCs was measured after cholesterol depletion by 20 mM MCD. The assay was performed as described in Fig. 2 A. Data shown are means ± SD of one representative experiment performed in triplicate out of three. One representative experiment out of three is shown. (B) Confocal microscopy analysis of copatching of DC-SIGN and GM1 on immature DCs. Staining was performed as described in Fig. 2 C. Cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representative of multiple cells in two independent experiments. Bar, 5 μm. (C) Whole-mount samples of immature DCs were double labeled for DC-SIGN (5 nm gold) and the raft marker GM1 (10 nm gold) and analyzed by TEM. Thin white arrows indicate GM1 colocalizing in DC-SIGN microdomains. Thick white arrows indicate DC-SIGN microdomains with no GM1. Black arrow indicates GM1 alone. Results are representatives of multiple cells in two independent experiments. Bar, 50 nm.
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fig5: DC-SIGN resides in lipid rafts on immature DCs. (A) DC-SIGN–mediated adhesion to ligand-coated fluorescent beads (1-μm diam) on immature DCs was measured after cholesterol depletion by 20 mM MCD. The assay was performed as described in Fig. 2 A. Data shown are means ± SD of one representative experiment performed in triplicate out of three. One representative experiment out of three is shown. (B) Confocal microscopy analysis of copatching of DC-SIGN and GM1 on immature DCs. Staining was performed as described in Fig. 2 C. Cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representative of multiple cells in two independent experiments. Bar, 5 μm. (C) Whole-mount samples of immature DCs were double labeled for DC-SIGN (5 nm gold) and the raft marker GM1 (10 nm gold) and analyzed by TEM. Thin white arrows indicate GM1 colocalizing in DC-SIGN microdomains. Thick white arrows indicate DC-SIGN microdomains with no GM1. Black arrow indicates GM1 alone. Results are representatives of multiple cells in two independent experiments. Bar, 50 nm.

Mentions: Because, on K-DC-SIGN, we found a clear association of DC-SIGN with lipid rafts, we investigated whether DC-SIGN also colocalized with lipid rafts on immature DCs. Extraction of cholesterol by MCD resulted in a significant block in binding of both ICAM-3– and gp120-coated beads (Fig. 5 A). In contrast, DC-SIGN–mediated binding on intermediate DCs was not affected by cholesterol depletion (unpublished data).


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 resides in lipid rafts on immature DCs. (A) DC-SIGN–mediated adhesion to ligand-coated fluorescent beads (1-μm diam) on immature DCs was measured after cholesterol depletion by 20 mM MCD. The assay was performed as described in Fig. 2 A. Data shown are means ± SD of one representative experiment performed in triplicate out of three. One representative experiment out of three is shown. (B) Confocal microscopy analysis of copatching of DC-SIGN and GM1 on immature DCs. Staining was performed as described in Fig. 2 C. Cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representative of multiple cells in two independent experiments. Bar, 5 μm. (C) Whole-mount samples of immature DCs were double labeled for DC-SIGN (5 nm gold) and the raft marker GM1 (10 nm gold) and analyzed by TEM. Thin white arrows indicate GM1 colocalizing in DC-SIGN microdomains. Thick white arrows indicate DC-SIGN microdomains with no GM1. Black arrow indicates GM1 alone. Results are representatives of multiple cells in two independent experiments. Bar, 50 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171967&req=5

fig5: DC-SIGN resides in lipid rafts on immature DCs. (A) DC-SIGN–mediated adhesion to ligand-coated fluorescent beads (1-μm diam) on immature DCs was measured after cholesterol depletion by 20 mM MCD. The assay was performed as described in Fig. 2 A. Data shown are means ± SD of one representative experiment performed in triplicate out of three. One representative experiment out of three is shown. (B) Confocal microscopy analysis of copatching of DC-SIGN and GM1 on immature DCs. Staining was performed as described in Fig. 2 C. Cells were analyzed by confocal microscopy. Merged images are shown in the right panel. Results are representative of multiple cells in two independent experiments. Bar, 5 μm. (C) Whole-mount samples of immature DCs were double labeled for DC-SIGN (5 nm gold) and the raft marker GM1 (10 nm gold) and analyzed by TEM. Thin white arrows indicate GM1 colocalizing in DC-SIGN microdomains. Thick white arrows indicate DC-SIGN microdomains with no GM1. Black arrow indicates GM1 alone. Results are representatives of multiple cells in two independent experiments. Bar, 50 nm.
Mentions: Because, on K-DC-SIGN, we found a clear association of DC-SIGN with lipid rafts, we investigated whether DC-SIGN also colocalized with lipid rafts on immature DCs. Extraction of cholesterol by MCD resulted in a significant block in binding of both ICAM-3– and gp120-coated beads (Fig. 5 A). In contrast, DC-SIGN–mediated binding on intermediate DCs was not affected by cholesterol depletion (unpublished data).

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