Ping Liu

DC-SIGN and influenza hemagglutinin dynamics in plasma membrane microdomains are markedly different

DC-SIGN, a Ca(2+)-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.

Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites

Dendritic cells express DC-SIGN and CD206, C-type lectins (CTLs) that bind a variety of pathogens and may facilitate pathogen uptake for subsequent antigen presentation. Both proteins form punctate membrane nanodomains (∼80 nm) on naïve cells. We analyzed the spatiotemporal distribution of CTLs following host-fungal particle contact using confocal microscopy and three distinct methods of cluster identification and measurement of receptor clusters in super-resolution datasets: DBSCAN, Pair Correlation and a custom implementation of the Getis spatial statistic. Quantitative analysis of confocal and super-resolution images demonstrated that CTL nanodomains become concentrated in the contact site relative to non-contact membrane after the first hour of exposure and established that this recruitment is sustained out to 4 h. DC-SIGN nanodomains in fungal contact sites exhibit a 70% area increase and a 38% decrease in interdomain separation. Contact site CD206 nanodomains possess 90% greater area and 42% lower interdomain separation relative to non-contact regions. Contact site CTL clusters appear as disk-shaped domains of approximately 150-175 nm in diameter. The increase in length scale of CTL nanostructure in contact sites suggests that the smaller nanodomains on resting membranes may merge during fungal recognition, or that they become packed closely enough to achieve sub-resolution inter-domain edge separations of <30 nm. This study provides evidence of local receptor spatial rearrangements on the nanoscale that occur in the plasma membrane upon pathogen binding and may direct important signaling interactions required to recognize and respond to the presence of a relatively large pathogen.

Low copy numbers of DC-SIGN in cell membrane microdomains: implications for structure and function.

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 > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 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 (∼50 nm) pathogen, dengue virus, leading to infection of host cells.

The Formation and Stability of DC-SIGN Microdomains Require its Extracellular Moiety

Dendritic cell‐specific intercellular adhesion molecule (ICAM)‐3‐grabbing non‐integrin (DC‐SIGN) is a Ca2+‐dependent transmembrane lectin that binds a large variety of pathogens and facilitates their uptake for subsequent antigen presentation. This receptor is present in cell surface microdomains, but factors involved in microdomain formation and their exceptional stability are not clear. To determine which domain/motif of DC‐SIGN facilitates its presence in microdomains, we studied mutations at key locations including truncation of the cytoplasmic tail, and ectodomain mutations that resulted in the removal of the N‐linked glycosylation site, the tandem repeats and the carbohydrate recognition domain (CRD), as well as modification of the calcium sites in the CRD required for carbohydrate binding. Confocal imaging and fluorescence recovery after photobleaching measurements showed that the cytoplasmic domain and the N‐linked glycosylation site do not affect the ability of DC‐SIGN to form stable microdomains. However, truncation of the CRD results in complete loss of visible microdomains and subsequent lateral diffusion of the mutants. Apart from cell adhesions, membrane domains are thought to be localized primarily via the cytoskeleton. By contrast, we propose that interactions between the CRD of DC‐SIGN and the extracellular matrix and/or cis interactions with transmembrane scaffolding protein(s) play an essential role in organizing these microdomains.

Beyond attachment: Roles of DC-SIGN in dengue virus infection

Dendritic cell‐specific intercellular adhesion molecule‐3‐grabbing non‐integrin (DC‐SIGN), a C‐type lectin expressed on the plasma membrane by human immature dendritic cells, is a receptor for numerous viruses including Ebola, SARS and dengue. A controversial question has been whether DC‐SIGN functions as a complete receptor for both binding and internalization of dengue virus (DENV) or whether it is solely a cell surface attachment factor, requiring either hand‐off to another receptor or a co‐receptor for internalization. To examine this question, we used 4 cell types: human immature dendritic cells and NIH3T3 cells expressing either wild‐type DC‐SIGN or 2 internalization‐deficient DC‐SIGN mutants, in which either the 3 cytoplasmic internalization motifs are silenced by alanine substitutions or the cytoplasmic region is truncated. Using confocal and super‐resolution imaging and high content single particle tracking, we investigated DENV binding, DC‐SIGN surface transport, endocytosis, as well as cell infectivity. DC‐SIGN was found colocalized with DENV inside cells suggesting hand‐off at the plasma membrane to another receptor did not occur. Moreover, all 3 DC‐SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co‐receptor because cells expressing the internalization‐deficient mutants could still be infected.

Complexity Revealed: A Hierarchy of Clustered Membrane Proteins.

One relatively unexplored aspect of membranes, dating back to the Singer Nicolson Fluid-Mosaic model (1), is the lateral heterogeneity that exists in the plane of the membrane. This is an intrinsic feature of membranes and therefore must be a key to functionality. Indeed, the plasma membrane is generally accepted as being compartmentalized, permitting lipids and proteins to be organized in specific domains whose size and composition vary (2,3). Such compartmentalization may rely predominantly on the membrane-apposed cytoskeleton, termed the “membrane skeleton fence model” (4).

Rapid, directed transport of DC-SIGN clusters in the plasma membrane

Very rapid, directed movement of pathogen receptors in the plasma membrane is associated with MT close to the inner leaflet. C-type lectins, including dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor–driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions.

Plasma Membrane DC-SIGN Clusters and Their Lateral Transport: Role in the Cellular Entry of Dengue Virus

DC-SIGN (a single-pass transmembrane protein and C-type lectin) is a major receptor for a variety of pathogens on human dendritic cells including dengue virus (DENV), which has become a global health threat. DENV binds to cell-surface DC-SIGN and the virus/receptor complexes migrate to clathrin-coated pits where the complexes are endocytosed; during subsequent processing, the viral genome is released for replication. DC-SIGN exists on cellular plasma membranes in nanoclusters that may themselves be clustered on longer length scales that appear as microdomains in wide-field and confocal fluorescence microscopy. We have investigated the dynamic structure of these clusters using fluorescence and super-resolution imaging in addition to large-scale single particle tracking. While clusters themselves can be laterally mobile there appears to be little mobility of DC-SIGN within clusters or exchange of DC-SIGN between the clusters and the surroundings. We end this account with some outstanding issues that remain to be addressed with respect to the composition and architecture of DC-SIGN domains and some highly unusual aspects of their lateral mobility on the cell surface that may accompany and perhaps facilitate DENV infection.