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

Abstract

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.

Dectin-1 Molecular Aggregation and Signaling is Sensitive to β-Glucan Structure and Glucan Exposure on Candida albicans Cell Walls

“…In addition, our FLIM-FRET results suggested possible intermolecular interactions between Dectin-1 proteins even before stimulation with β-glucan. We considered this possibility carefully because receptor oligomeric states exist in the basal state for some other C-type lectin receptors, such as DCSIGN, DNGR-1, and NKp80 [68–71]. However, Dectin-1A does not contain cysteine residues in its stalk region which have been shown to be important in the dimerization of other C-type lectin receptors.…”

Dances with Membranes: Breakthroughs from Super-resolution Imaging

“…There are other tests to measure spatial heterogeneity such as the Getis G statistic ( Getis & Ord, 1992 ), which includes different normalizations and weighing parameters dependent on molecular spatial position. These differences result in increased sensitivities to certain cluster parameters ( Itano et al., 2014 ).…”

Toward a new picture of the living plasma membrane

“…In this study, the authors identified that removal of extra‐cellular interactions mediated by Galectin‐9 disrupts the meshwork‐like mesoscale pattern of DC‐SIGN receptors, preventing their enrichment in clathrin‐coated patches on the plasma membrane, consequently affecting its endocytosis. The DC‐SIGN receptor is a classic example of tetramers 70 organizing in nano‐clusters (2–3 tetramers) and further incorporated into micron‐scale localization hotspots, driven at least in two different length scales by extra‐cellular domain interactions 66 . Artificially engineering different ABDs to transmembrane proteins with such defined extra‐cellular interactions, in the appropriate cell system, will help understand the interplay or which interactions (extra‐cellular or indirect cytoskeletal barriers) will emerge as a stronger determinant of the nanoscale and emerging mesoscale distribution of the protein.…”