Composition, structure, dynamics and function of c-type lectin receptor domains

DC-SIGN, a Ca2+-dependent C-type transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In these studies, DC-SIGN dynamics and distribution in microdomains have been explored with several fluorescence microscopy methods and compared with those for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence recovery after photobleaching (FRAP), line-scan fluorescence correlation spectroscopy and defined valency quantum dot single particle tracking measurements showed that full-length and cytoplasmically truncated DC-SIGN is essentially immobilized in microdomains, whereas HA is laterally mobile within and outside microdomains and exchanges between these two regions. By contrast, FRAP measurements indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. Wide-field fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. A super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another C-type lectin (CD206), and HA are all localized in small (∼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, while CD206 is often non-oligomerized. Two-color Blink determined that different C-type lectins rarely occupy the same nanodomain although they appear co-localized using widefield microscopy. Thus, a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts. These results contribute significantly to a young field directed at elucidation of the complex intradomain structural features underlying function.