Exploring The Spatial Regulation Of Fceri Signaling With Super-Resolution Fluorescence Localization Microscopy

Abstract

Allergic signaling is initiated on the plasma membrane of mast cells through cross-linking of immunoglobulin E (IgE) bound to its receptor, Fc[epsilon]RI, by multivalent antigen. Signaling proceeds through controlled spatial assembly of Fc[epsilon]RI receptors with interaction partners in a process that gives rise to specific signaling outcomes. Antigenstimulated redistribution of signaling molecules has proven difficult to characterize by fluorescence imaging due to the small length scales over which this redistribution occurs. We employ super-resolution fluorescence localization microscopy to measure antigen-stimulated changes in the nanoscale organization and mobility of Fc[epsilon]RI, as well as its spatial association with its signaling partner Lyn kinase, with the goal of understanding the physical mechanisms by which spatial redistribution of signaling molecules on the membrane causes the initiation of the signaling response. Using super-resolution imaging, we record receptor organization and dynamics on live mast cells undergoing antigen-mediated signaling, allowing us to measure nanoscale clustering and diffusion of Fc[epsilon]RI 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. We correlate the time-dependence of the distinct phases with a functional signaling iii response, Ca2+ mobilization, to assess the relevance of each phase to the onset of signaling. We also use super-resolution imaging to measure interactions of Fc[epsilon]RI with Lyn. Because of the improved resolution of our imaging technique, we detect coupling of Lyn to cross-linked IgE-Fc[epsilon]RI that occurs at early stages after antigen stimulation and is associated with the initiation of the stimulated response. Lyn association with Fc[epsilon]RI is assessed through direct imaging, and we can measure the average physical properties of Lyn/Fc[epsilon]RI co-clusters. Through this quantitative approach, we examine mechanisms of regulation of Lyn co-redistribution with IgE-Fc[epsilon]RI. In particular we show that the actin cytoskeleton negatively regulates Fc[epsilon]RI signaling by reducing the local accumulation of Lyn with Fc[epsilon]RI clusters during the onset of the response. The spatial resolution afforded by this technique makes it an effective tool for investigating interactions that give rise to the changes in the organization or mobility of signaling molecules during the initiation of signaling, and how these changes translate into cellular functions. iv