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

Understanding lipid rafts and other related membrane domains

Evidence in support of the classical lipid raft hypothesis has remained elusive. Data suggests that transmembrane proteins and the actin-containing cortical cytoskeleton can organize lipids into short-lived nanoscale assemblies that can be assembled into larger domains under certain conditions. This supports an evolving view in which interactions between lipids, cholesterol, and proteins create and maintain lateral heterogeneity in the cell membrane

Editorial: Live cell imaging: Cell and developmental research bridging education, optical engineering, industry, software, shared facilities

Optical imaging is one of the original technological pillars of biomedical research, spanning centuries of discovery. Drive for biological understanding has fueled significant progress in imaging tool development, ranging from illumination sources, detectors, and mechanical stages, to novel optical components and analysis strategies. The number and variety of optical imaging tools is now so vast that in some cases cutting-edge technologies require specialized training across a wide set of skills. Considering the exponential growth in imaging tools, fruitful and conducive partnerships between academic researchers, commercial vendors, and instrument facilitators, such as core facility managers and staff, are more important than ever

Super-Resolution Imaging of C-Type Lectin and Influenza Hemagglutinin Nanodomains on Plasma Membranes Using Blink Microscopy

AbstractDendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, 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 CTL (CD206), and influenza hemagglutinin (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, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is 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

Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b

Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion

Low Copy Numbers of DC-SIGN in Cell Membrane Microdomains: Implications for Structure and Function: Quantification of DC-SIGN Copy Numbers in Microdomains

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1μm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (~50nm) pathogen, dengue virus, leading to infection of host cells

DC-SIGN and Influenza Hemagglutinin Dynamics in Plasma Membrane Microdomains Are Markedly Different

DC-SIGN, a Ca2+-dependent 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 this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. 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. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DC-SIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/or by protecting against rapid microdomain endocytosis