Super-Resolution Imaging of IgE Receptor Clustering Initiated by Structurally-Defined Ligands in Rbl Mast Cells

Abstract

Cross-linking of IgE bound to its receptor, FceRI, by multivalent ligands initiates a transmembrane signaling cascade that activates allergic and inflammatory responses in mast cells. Super-resolution fluorescence localization microscopy is an effective tool for quantitative nanoscale imaging of the distribution and ligand-induced redistribution of FceRI. We use structurally-defined trivalent ligands specific for anti-dinitrophenyl (DNP) IgE, for which the ligand size and valency is precisely controlled, to cross-link IgE-FceRI. These ligands are based on a Y-shaped double-stranded DNA scaffold with DNP groups conjugated to each of the three 5’ ends. The distance between DNP groups is fixed by the length of single-stranded oligonucleotides annealed to form the Y-shaped structure. As described previously for these Yn-DNP3 ligands, (Sil et al., 2007, ACS Chem Biol) some (but not other) cell signaling pathways depend on inter-DNP distances, ranging from 5 – 15 nm, which constrain the proximity of ligand-bound IgE-receptors. We are investigating the organization and dynamics of ligand-induced IgE-receptor clustering to gain new information about interactions of cross-linked IgE-receptors within the membrane that accompany or limit transmembrane signaling. We image fixed and live cells and compare Yn-DNP3 ligands with the heterogeneous ligand, DNP20-BSA. IgE is labeled with a photo-switchable probe and IgE-receptor clustering is quantified in super-resolution images with spatial correlation functions. In living cells, we record single-receptor trajectories, and receptor mobility is monitored in real time through analysis of populations of tracks. Both DNP20-BSA and Y16-DNP3 cause a sharp decrease in mobility upon ligand addition, but produce IgE-receptor clusters that appear distinctive in size and density. Y16-DNP3–stimulated changes occur on a shorter time scale when matched for DNP concentration with DNP20-BSA. Continuing work with Yn-DNP3 ligands will probe clustering-dependent IgE-receptor association with membrane structures and downstream signaling partners.