Aaron K. Neumann

Distribution and lateral mobility of DC-SIGN on immature dendritic cells - Implications for pathogen uptake

The receptor C-type lectin DC-SIGN (CD209) is expressed by immature dendritic cells, functioning as an antigen capture receptor and cell adhesion molecule. Various microbes, including HIV-1, can exploit binding to DC-SIGN to gain entry to dendritic cells. DC-SIGN forms discrete nanoscale clusters on immature dendritic cells that are thought to be important for viral binding. We confirmed that these DC-SIGN clusters also exist both in live dendritic cells and in cell lines that ectopically express DC-SIGN. Moreover, DC-SIGN has an unusual polarized lateral distribution in the plasma membrane of dendritic cells and other cells: the receptor is preferentially localized to the leading edge of the dendritic cell lamellipod and largely excluded from the ventral plasma membrane. Colocalization of DC-SIGN clusters with endocytic activity demonstrated that surface DC-SIGN clusters are enriched near the leading edge, whereas endocytosis of these clusters occurred preferentially at lamellar sites posterior to the leading edge. Therefore, we predicted that DC-SIGN clusters move from the leading edge to zones of internalization. Two modes of lateral mobility were evident from the trajectories of DC-SIGN clusters at the leading edge, directed and non-directed mobility. Clusters with directed mobility moved in a highly linear fashion from the leading edge to rearward locations in the lamella at remarkably high velocity (1420±260 nm/second). Based on these data, we propose that DC-SIGN clusters move from the leading edge–where the dendritic cell is likely to encounter pathogens in tissue–to a medial lamellar site where clusters enter the cell via endocytosis. Immature dendritic cells may acquire and internalize HIV and other pathogens by this process.

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.

A novel pseudopodial component of the dendritic cell anti-fungal response: the fungipod.

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion.

Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues

Trafficking through a variety of cellular structures and organelles is essential for the interaction between gene-delivery vectors (i.e., adeno-associated virus (AAV) and liposomes) and host cells/tissues. Here, we present a method of computer-assisted quantitative 3D biodistribution microscopy that samples the whole population of fluorescently-labeled vectors and document their trafficking routes. Using AAV as a working model, we first experimentally defined numerical parameters for the singularity of Cy5-labeled particles by combining confocal microscopy and atomic force microscopy (AFM). We then developed a robust approach that integrates single-particle fluorescence imaging with 3D deconvolution and isosurface rendering to quantitate viral distribution and trafficking in human cells as well as animal tissues at the single-particle level. Using this quantitative method, we uncovered an as yet uncharacterized rate-limiting step during viral cell entry, while delineating nuclear accumulation of virions during the first 8 hours postinfection. Further, our studies revealed for the first time that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. Such 3D resolution and quantitative dissection of vector-host interactions at the subcellular level should significantly improve our ability to resolve trafficking mechanisms of gene-delivery particles and facilitate the development of enhanced viral vectors.

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.

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.

Nanoscopic cell-wall architecture of an immunogenic ligand in Candida albicans during antifungal drug treatment.

Candida albicans evades immunity by limiting cell-wall β-glucan exposure. dSTORM imaging reveals that “unmasking”of glucan by an antifungal drug occurs through nanoscale reorganization of glucan exposure geometry. Nanostructuring of glucan might play a role in innate immune activation and provides insights into the physical regulation of glucan exposure.

Antifungal Properties of Cationic Phenylene Ethynylenes and Their Impact on β-Glucan Exposure

ABSTRACT Candida species are the cause of many bloodstream infections through contamination of indwelling medical devices. These infections account for a 40% mortality rate, posing a significant risk to immunocompromised patients. Traditional treatments against Candida infections include amphotericin B and various azole treatments. Unfortunately, these treatments are associated with high toxicity, and resistant strains have become more prevalent. As a new frontier, light-activated phenylene ethynylenes have shown promising biocidal activity against Gram-positive and -negative bacterial pathogens, as well as the environmental yeast Saccharomyces cerevisiae. In this study, we monitored the viability of Candida species after treatment with a cationic conjugated polymer [poly(p-phenylene ethynylene); PPE] or oligomer [“end-only” oligo(p-phenylene ethynylene); EO-OPE] by flow cytometry in order to explore the antifungal properties of these compounds. The oligomer was found to disrupt Candida albicans yeast membrane integrity independent of light activation, while PPE is able to do so only in the presence of light, allowing for some control as to the manner in which cytotoxic effects are induced. The contrast in killing efficacy between the two compounds is likely related to their size difference and their intrinsic abilities to penetrate the fungal cell wall. Unlike EO-OPE-DABCO (where DABCO is quaternized diazabicyclo[2,2,2]octane), PPE-DABCO displayed a strong propensity to associate with soluble β-glucan, which is expected to inhibit its ability to access and perturb the inner cell membrane of Candida yeast. Furthermore, treatment with PPE-DABCO unmasked Candida albicans β-glucan and increased phagocytosis by Dectin-1-expressing HEK-293 cells. In summary, cationic phenylene ethynylenes show promising biocidal activity against pathogenic Candida yeast cells while also exhibiting immunostimulatory effects.