Single Particle Tracking using Fluorogen Activating Peptides to Investigate FcεRI Signaling Dynamics

Abstract

The high affinity IgE receptor, FceRI, serves as the primary immunoreceptor on mast cells and basophils. Cross-linking of IgE-bound FceRI via multivalent antigen initiates a signaling cascade that ultimately results in the release of key mediators for an allergic response. Priming with IgE has been traditionally thought of as a passive event, yet certain forms of IgE termed ‘cytokinergic’ IgEs (cIgE) have been shown to induce activation independent of antigen. The mechanism of action of cIgEs remains unclear. To determine whether changes in receptor mobility are associated with cIgE activation, we have developed a fluorogen activating peptide (FAP)-tagged FceRI ɣ-subunit for single particle tracking. FAPs are genetically expressible tags that bind an exogenous fluorogen dye. The titration of the fluorogen dye allows the labeling density to be adjusted at the time of imaging without the need to photo-bleach or photo-activate a population of dye molecules. We find that even in the presence of concentrations of cIgE that induce robust signaling, FceRI remains highly mobile.We take advantage of the FAP system to additionally investigate the mobility of the downstream scaffolding protein Linker for Activation of T-cells (LAT). The mechanism through which FceRI crosslinking propagates signaling to LAT remains a topic of debate as LAT has been previously shown to form clusters segregated from FceRI after activation. However, these studies were carried out using high concentrations of multivalent allergen that resulted in large immobile FceRI aggregates. The far-red emission of the MG2P fluorogen allows for simultaneous tracking of FAP-LAT mobility with respect to FceRI labeled with Alexa488-IgE. Using FAP-tagged LAT, we investigate how LAT mobility and distribution change over a range of activating conditions, in which FceRI goes from mobile to immobile crosslinked aggregates.