Molecular perspective of antigen-mediated mast cell signaling.
Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that Förster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.
Davey
Angel M
AM
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Krise
Keith M
KM
Sheets
Erin D
ED
Heikal
Ahmed A
AA
D000818:Animals; D000941:Antigens / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006834:Hydrazines / Q000494:pharmacology; D007073:Immunoglobulin E / Q000737:chemistry; D007700:Kinetics; D008407:Mast Cells / Q000166:cytology*; D021962:Membrane Microdomains / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D051381:Rats; D017455:Receptors, IgE / Q000737:chemistry* / Q000502:physiology; D015398:Signal Transduction
C417561:Alexa 488 hydrazide; D000941:Antigens; D003432:Cross-Linking Reagents; D006834:Hydrazines; D017455:Receptors, IgE; D007073:Immunoglobulin E