Super-Resolution Localization Microscopy Identifies Distinct Stages of Antigen-Induced IgE Receptor Cross-Linking and Immobilization in Rbl-2H3 Mast Cells

Abstract

Cross-linking of immunoglobulin E (IgE) bound to its receptor, FceRI, by multivalent antigen initiates a transmembrane signaling cascade essential for mast cell activation and important for inflammatory immune responses and allergic disease. In this study, we apply super-resolution fluorescence localization microscopy to record receptor organization and dynamics on live RBL-2H3 mast cells undergoing antigen-mediated signaling, allowing us to measure nanoscale clustering and diffusion of FceRI simultaneously. Through comparison of cross-linking-induced changes in these properties as a function of time, we are able to resolve two distinct temporal phases of receptor clustering and immobilization. Additionally, we correlate the time-dependence of the distinct phases with a functional signaling response, Ca2+ mobilization. In the first phase of receptor clustering and immobilization, receptors slow dramatically with a relatively small average increase in clustering, and individual receptors appear to transiently associate with small clusters. This first phase occurs before Ca2+ mobilization and concurrently with initial signaling steps. At later times, receptor-rich clusters become increasingly dense while receptors remain predominately immobilized. These latter behaviors are observed at times following the initiation of the Ca2+ response, and we conclude that although cross-linking is necessary for commencement of downstream signaling, receptor assembly into large, densely packed clusters at later times is likely associated with termination of the stimulated response. These findings motivate future study of the physical interactions that give rise to the observed changes in receptor organization and mobility, and how these translate into cellular functions. In ongoing experiments, we are exploring the requirements of signaling for receptor cross-linking through the use of antigens with controlled structure and valency, and we will correlate our observations with functional responses such as Ca2+ mobilization and receptor association with downstream signaling partners.