Fluorogen-Activating Proteins Provide Tunable Labeling Densities for Tracking FcεRI Independent of IgE

Crosslinking of IgE bound Fc epsilon RI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators that stimulate the allergic response. Here, we present and characterize the use of fluorogen-activating proteins (FAPs) for single particle tracking of Fc epsilon RI to investigate how receptor mobility is influenced after IgE-induced changes in mast cell behavior. FAPs are genetically encoded tags that bind a fluorogen dye and increase its brightness upon binding up to 20,000-fold. We demonstrate that, by titrating fluorogen concentration, labeling densities from ensemble to single particle can be achieved, independent of expression level and without the need for wash steps or photobleaching. The Fc epsilon RI gamma-subunit fused to a FAP (FAP-gamma) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of FceRI at the single molecule level. We show that the FceRI gamma-subunit dynamics are controlled by the IgE-binding alpha-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering Fc epsilon RI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track FceRI relative to dynamin-GFP and find that immobilized receptors readily correlate with locations of dynamin recruitment only under conditions that promote rapid endocytosis. These studies demonstrate the usefulness of the FAP system for single molecule studies and have provided new insights into the relationship among FceRI structure, activity, and mobility.