Subcellular glycan-mannose receptor binding kinetics correlate with myeloid cell function.

Extracting single-molecule lectin binding kinetics from primary cells has not been possible to date. Here, we present Glyco-PAINT-APP (Automated Processing Pipeline), an automated method that enables the extraction of subcellular glycan interaction kinetics using a Points Accumulation for Imaging in Nano-Topography (PAINT)-based approach. This approach leverages an algorithm for precise, high-throughput, subcellular analysis of glycan binding dynamics and facilitates functional correlation studies between glycoform binding patterns and immune cell polarization. Using synthetic glycans and glycosylated antigens, we demonstrate the ability of the technique to automatically correlate glycan binding parameters in subregions of dendritic cell membranes with increased uptake and cross-presentation efficiency of these antigens. Additionally, we show how the method can uncover subtle differences in MR-mediated glycan binding across various MR-expressing primary cells and cell lines. Taken together, Glyco-PAINT-APP enables insights into the cell-intrinsic heterogeneity of glycan-structure-activity relationships in myeloid immune cells.