Super-Resolution Tracking and Imaging of IgE Receptors during Stimulation by Antigen

Abstract

In mast cells, cross-linking of IgE bound to FceR1 receptors with multivalent antigen initiates cell activation and consequent inflammatory responses. Clustered IgE-FceRI interacts with early signaling partners such as Lyn, Syk, and LAT, initiating a signaling cascade. Our previous scanning electron microscopy (SEM) studies quantified the nanoscale co-redistribution of FceRI with Lyn, Syk, and LAT within the plasma membrane upon antigen stimulation. Here we use super-resolution localization microscopy to localize proteins on the cell surface with sub-diffraction resolution using a TIR fluorescence microscope. We present, in snapshots, the time-dependence of IgE receptor reorganization after antigen addition using chemically fixed cells and quantify the extent of clustering using pair autocorrelation functions. We have validated our methods for quantifying and correcting for artifactual clustering due to probe over-counting through two-color experiments where individual IgE proteins are labeled with one of two species of distinguishable fluorescent probe and imaged at sub-diffraction resolution in multiple color channels. Here clustering is quantified using pair cross-correlation functions. Results of one- and two-color super-resolution measurements agree well with conclusions from SEM experiments. We have also extended this technique to imaging living cells at room temperature to measure IgE receptor redistribution in real time. We quantify the organization and diffusion behavior of proteins undergoing stimulated responses with the excellent statistics afforded by the technique. Live cell experiments are conducted under a variety of stimulation conditions, such as in the presence of drugs that perturb membrane physical state or affect actin cytoskeleton assembly. Our results confirm that super-resolution imaging is an effective tool for quantitative imaging of cellular components at the nanoscale. Our ongoing studies are providing new information to clarify the physical basis for spatial assembly of specific proteins in the plasma membrane during early signaling events.