Abstract
DC-SIGN, a dendritic cell (DC)-specific C-type lectin that binds many different pathogens, is a receptor for HIV-1 and promotes subsequent infection of T cells. Our previous study demonstrated that DC-SIGN forms sub-micron scale domains on the surface of immature DCs as well as cell lines that ectopically express DC-SIGN. In this study, we investigated the occupancy and dynamics of DC-SIGN surface domains. First, we developed a single molecule approach, based on total internal reflection fluorescence (TIRF) microscopy, to examine the number of DC-SIGN molecules in a single domain. By comparing the brightness of a single fluorophore to the total brightness of a fluorescently-labeled DC-SIGN domain, we show that the number of DC-SIGN proteins in a domain ranges from a few to over hundred molecules. The size of each domain, as measured from the full-width half-maximum (FWHM) of a Gaussian fit of the emission profile of a domain, varies from the diffraction limit to micron scale. Second, scanning fluorescence correlation spectroscopy (sFCS) and fluorescence recovery after photobleaching (FRAP) were carried out to investigate the mobility of DC-SIGN in a domain, both of which showed that DC-SIGN is highly immobile. This was corroborated by single particle tracking of quantum dots attached to DC-SIGN molecules within a domain. By contrast, photobleaching of a lipid modified-fluorescent protein (mRFP) in a DC-SIGN domain area showed full recovery similar to that outside of the domain, indicating that lipids inside the DC-SIGN domain are highly mobile and and can freely exchange with the surrounding membrane. Finally, a deletion mutation study of DC-SIGN was carried out to further investigate which moiety of DC-SIGN facilitates surface domain formation. Supported by NIH GM41402.
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