Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcεRI Aggregation and Structural Changes.

Immunoreceptor tyrosine-based activation motif (ITAM)-containing Fc receptors are critical components of the innate and adaptive immune systems. FcεRI mediates the allergic response via crosslinking of IgE-bound receptors by multivalent antigens. Yet, the underlying molecular mechanisms that govern the response of FcεRI to specific antigens remain poorly understood. We compared responses induced by two antigens with distinct geometries, high valency DNP-BSA and trivalent DF3, and found unique secretion and receptor phosphorylation profiles that are due to differential recruitment of Lyn and SHIP1. To understand how these two antigens can cause such markedly different outcomes, we used direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging combined with Bayesian Grouping of Localizations (BaGoL) analysis to compare the nanoscale characteristics of FcεRI aggregates. DF3 aggregates were found to be smaller and more densely packed than DNP-BSA aggregates. Using lifetime-based Förster resonance energy transfer (FRET) measurements, we discovered that FcεRI subunits undergo structural rearrangements upon crosslinking with either antigen, and in response to interaction with monovalent antigen presented on a supported lipid bilayer. The extent of conformational change is positively correlated with signaling efficiency. Finally, we provide evidence for forces in optimizing FcεRI signaling, such that immobilizing DF3 on a rigid surface promoted degranulation while increasing DNP-BSA flexibility lowered degranulation. These results provide a link between the physical attributes of allergens, including size, shape, valency, and flexibility, and FcεRI signaling strength. Thus, the antigen modulates mast cell outcomes by creating unique aggregate geometries that tune FcεRI conformation, phosphorylation and signaling partner recruitment.

Docking of Syk to FcεRI is enhanced by Lyn but limited in duration by SHIP1.

The high-affinity immunoglobulin E (IgE) receptor, FcεRI, is the primary immune receptor found on mast cells and basophils. Signal initiation is classically attributed to phosphorylation of FcεRI ß- and γ-subunits by the Src family kinase (SFK) Lyn, followed by the recruitment and activation of the tyrosine kinase Syk. FcεRI signaling is tuned by the balance between Syk-driven positive signaling and the engagement of inhibitory molecules, including SHIP1. Here, we investigate the mechanistic contributions of Lyn, Syk, and SHIP1 to the formation of the FcεRI signalosome. Using Lyn-deficient RBL-2H3 mast cells, we found that another SFK can weakly monophosphorylate the γ-subunit, yet Syk still binds the incompletely phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs). Once recruited, Syk further enhances γ-phosphorylation to propagate signaling. In contrast, the loss of SHIP1 recruitment indicates that Lyn is required for phosphorylation of the ß-subunit. We demonstrate two noncanonical Syk binding modes, trans γ-bridging and direct ß-binding, that can support signaling when SHIP1 is absent. Using single particle tracking, we reveal a novel role of SHIP1 in regulating Syk activity, where the presence of SHIP1 in the signaling complex acts to increase the Syk:receptor off-rate. These data suggest that the composition and dynamics of the signalosome modulate immunoreceptor signaling activities.

High-speed single molecule imaging datasets of membrane proteins in rat basophilic leukemia cells.

A high-speed fluorescence microscope operating at a 490 Hz frame rate was used to image two different membrane proteins- the high-affinity IgE receptor FcɛRI, a transmembrane protein, and an outer-leaflet GPI-anchored protein. The IgE receptor was imaged via IgE labeled with Janelia Fluor 646 and the GPI-anchored protein was imaged using a GPI-GFP fusion protein and an ATTO 647 N labeled anti-GFP nanobody. Data was collected for both proteins in untreated cells and cells that had actin stabilized by phalloidin. This dataset can be used for development and testing of single-particle tracking methods on experimental data and to explore the hypothesis that the actin cytoskeleton may affect the movement of membrane proteins.

Combinatorial diversity of Syk recruitment driven by its multivalent engagement with FcεRIγ.

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αßγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk-FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk's bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.