Lateral Diffusion of Membrane Lipid-Anchored Probes before and after Aggregation of Cell Surface IgE-Receptors

Abstract

Cross-linking of the high-affinity IgE receptor (FcεRI) by multivalent antigen on mast cells initiates transmembrane signaling by coupling of FcεRI with the cytoplasmic tyrosine kinase Lyn in coalesced protein−lipid rafts. As part of our ongoing investigation of these membrane-based interactions, we employed fluorescence correlation spectroscopy and fluorescence photobleaching recovery to compare lateral mobilities of representative components: Cy3−IgE−FcεRI, a transmembrane protein; DiI−C16, an outer leaflet lipid probe with two saturated acyl chains; PM−EGFP, a Lyn analogue anchored to the inner leaflet via the saturated acyl chains palmitate and myristate; and EGFP−GG, a control protein anchored to the inner leaflet via an unsaturated geranylgeranyl chain and an adjacent polybasic amino acid sequence. Interpreting the data with both free diffusion and anomalous subdiffusion models, we find that EGFP−GG diffuses faster than PM−EGFP on average. Both inner leaflet probes are sensitive to day-to-day variation in diffusion properties but typically diffuse faster than DiI−C16, which is faster than the transmembrane receptor Cy3−IgE−FcεRI. Large-scale cross-linking of Cy3−IgE−FcεRI markedly decreases the mobility of this receptor, DiI−C16, and PM−EGFP, whereas EGFP−GG mobility changes little. Quantitative parameters derived from the diffusion data characterize the environment of the coalesced protein−lipid rafts in which Lyn interacts with IgE−FcεRI after antigen cross-linking, and which also concentrate other raft markers such as DiI−C16. Furthermore, the differences of the diffusion properties for DiI−C16 compared to PM−EGFP and EGFP−GG are consistent with the view that the nature of rafts differs between the outer and inner leaflets of the plasma membrane.