FcεRI Signal Propagation is Regulated through Transient Binding of Syk

Abstract

The high affinity IgE receptor, FceRI, serves as the primary immunoreceptor on mast cells and basophils.Crosslinking of IgE-bound FceRI via multivalent antigen initiates Src family kinase mediated phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs), recruitment of the kinase Syk, and propagation of signaling through the scaffolding protein Linker for Activation of T-cells (LAT). While the sequence of these signaling events has been well studied using biochemical techniques, the biophysical mechanisms that regulate FceRI signaling are unclear because protein activation and dynamic protein-protein interactions are difficult to quantify on living cells. Using two-color single molecule imaging in live cells, we quantify how FceRI aggregation influences SYK recruitment and LAT mobility.We imaged the basal surface of RBL-2H3 cells expressing GFP-Syk using TIRF microscopy and upon crosslinking of FceRI observed an increase in the membrane localization of GFP-Syk. The formation of GFP-Syk clusters directly co-localized with crosslinked FceRI and the intensity of these clusters increased linearly with FceRI aggregate size. Interestingly, while ensemble measurements showed that FceRI and GFP-Syk were co-localized over minutes, single molecule imaging revealed that GFP-Syk clusters are in fact maintained through a continuous exchange of transiently bound GFP-Syk. We quantify the residency time of individual GFP-Syk molecules at FceRI aggregates and explore the relationship between FceRI aggregate size and Syk residency time. The observed transient GFP-Syk binding supports the hypothesis that phosphorylated SYK moves from FceRI clusters to bind and activate LAT. To explore this mechanism further, we quantify the influence of FceRI crosslinking on the spatiotemporal distribution of LAT. We have generated a Fluorogen Activating Protein (FAP) -tagged LAT construct and take advantage of the far-red emission of the FAP probe to simultaneously track FAP-LAT mobility with respect to FceRI/SYK patches.