Abstract
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.
Highlights
Antigen-stimulated phosphorylation of Fc⑀RI by Lyn has been shown to occur in a cholesterol-dependent manner during the initial biochemical step of the allergic response in RBL-2H3 mast cells [6], and these cholesterol-rich membrane domains have been shown to protect Lyn from tyrosine phosphatases, thereby enhancing its activity [44]
We used a fluorescence dynamics assay to measure changes in membrane structure that occur as RBL mast cells are stimulated with multivalent antigen
Our experiments are performed at room temperature, which reduces the rate of signaling as compared with that occurring at 37 °C, and previous in vivo tyrosine phosphorylation assays performed on suspended RBL cells at 37 °C revealed maximal Fc⑀RI phosphorylation between 2 and 5 min after stimulation with the same concentration of DNP-BSA used in these studies [6, 26]
Summary
Cell Preparation—RBL-2H3 mast cells were maintained and harvested as described previously [6, 31]. Complementary 1P (480 nm, 4.2 MHz) timeresolved fluorescence measurements were carried out to enhance the time resolution and signal-to-noise ratio as described previously [25, 27] In these studies, the laser beam was strategically positioned at areas of interest on the cell membrane. For time-resolved fluorescence anisotropy, 1P laser pulses were positioned on regions of interest on the cells, or a single cell was averaged via scanning 2P-FLIM, and the epi-fluorescence polarizations were resolved and detected simultaneously as described earlier Both polarization images and time-resolved fluorescence polarization decays were constructed using the SPC830 module. We used time-resolved fluorescence polarization anisotropy to readily apparent in conventional imaging To overcome this examine the validity of the dipole moment randomization of limitation, we performed excited state dynamics experiments both the donor and acceptor [24]. The rate of energy transfer (in nsϪ1) is defined as kFRET ϭ ((D)((1/ET) Ϫ 1))Ϫ1 [33]
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