Super-Resolution Imaging of IgE-FcεRI Stimulated with Structurally Defined Ligands

Abstract

IgE-mediated signaling via its receptor FceRI in mast cells is of interest due to its role in allergic responses, and as a model system for immune receptor signaling. To investigate IgE-FceRI redistribution and dynamics in response to antigen stimulation, we utilize stochastic optical reconstruction microscopy (STORM), a super-resolution imaging technique. STORM permits visualization of IgE at the membrane of live or chemically fixed cells at a resolution of about 30 nm, which allows us to examine the initial events of IgE receptor signaling at a new level of molecular detail.Physiologically, cross-linking of IgE-FceRI by multivalent antigen initiates the transmembrane signal, and we ask how this signaling capacity relates to the cross-linked structure of the clustered IgE-FceRI. To investigate this question, we utilize STORM imaging in conjunction with structurally defined ligands, which correspondingly cross-link IgE with a high level of definition. We use trivalent ligands based on rigid Y-shaped scaffolds of double-stranded DNA, which have one 2,4-dinitrophenyl (DNP) hapten conjugated to each arm. The distance between DNP groups can be tuned by changing the lengths of the arms of the scaffolds. We show through STORM that changes in hapten spacing effectively change the structure of the cross-linked clusters, in terms of their average area and receptor density. To investigate more limited IgE aggregation, we also use other structurally defined ligands, including bivalent DNA-based ligands and monoclonal antibodies specific for IgE or FceRI.Our ongoing work focuses on how these structurally defined IgE-FceRI clusters mediate subsequent molecular events at the plasma membrane. Using two-color super-resolution microscopy, we will characterize the recruitment of early signaling partners, including Lyn tyrosine kinase. We are also investigating whether changes to membrane lipid composition alter clustering of cross-linked IgE-FceRI or consequent interactions with signaling partners.