Plasma Membrane Diffusion Modes of FcεRI Receptor for Immunoglobin E Measured with Imaging Fluorescence Correlation Spectroscopy

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Spatio-temporal organization of the plasma membrane plays a significant role in cell signaling. A prevailing view is that the plasma membrane spatially segregates into ordered lipid (Lo-like) nanodomains co-existing with disordered lipid (Ld-like) regions. Our laboratory, using super-resolution fluorescence imaging, previously showed that immunoglobin E bound to its receptor, FceRI, (IgE-FceRI complex) undergoes time-dependent redistribution on the membrane surface after multivalent ligand stimulation, upon which the receptor is phosphorylated by membrane-anchored Lyn kinase. A large body of evidence shows that this interaction of IgE-FceRI and Lyn occurs in the ordered regions of the plasma membrane enriched in cholesterol. We are examining the nature and dynamics of plasma membrane organization as experienced by IgE-FceRI in resting state and after antigen stimulation. We employ imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS), which maps lateral diffusion with pixel resolution, to investigate the diffusion distribution of IgE-FceRI complexes on the ventral surface of live RBL cells in native and experimentally modulated (e.g., cholesterol depleted) conditions. We also conduct spot variation FCS (svFCS), which analyzes space-dependence of diffusion coefficients from the same set of ITIR-FCS data, to reveal the existence of nanoscopic obstacles (such as nanodomains) that affect the diffusion of IgE-FceRI complexes. These observations are compared with the diffusion behavior of protein markers that are known to prefer ordered or disordered regions of plasma membrane. These results, in conjunction with super-resolution imaging, which determines the size and density of these complexes, will provide a new level of insight into plasma membrane organization and its dynamic remodeling upon ligand-receptor stimulation.