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Quantitative nanoscale analysis of IgE-Fc?RI clustering and coupling to early signaling proteins.


ABSTRACT: Antigen-mediated cross-linking of IgE bound to its receptor, Fc?RI, initiates a transmembrane signaling cascade that results in mast cell activation in the allergic response. Using immunogold labeling of intact RBL mast cells and scanning electron microscopy (SEM), we visualize molecular reorganization of IgE-Fc?RI and early signaling proteins on both leaflets of the plasma membrane, without the need for ripped off membrane sheets. As quantified by pair correlation analysis, we observe dramatic changes in the nanoscale distribution of IgE-Fc?RI after binding of multivalent antigen to stimulate transmembrane signaling, and this is accompanied by similar clustering of Lyn and Syk tyrosine kinases, and adaptor protein LAT. We find that Lyn co-redistributes with IgE-Fc?RI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-Fc?RI and Lyn in stimulated clusters. Coupling of these proteins is also decreased when membrane cholesterol is reduced either before or after antigen addition. These results provide evidence for involvement of Fc?RI phosphorylation and cholesterol-dependent membrane structure in the interactions that accompany IgE-mediated activation of RBL mast cells. More generally, this SEM view of intact cell surfaces provides new insights into the nanoscale organization of receptor-mediated signaling complexes in the plasma membrane.

SUBMITTER: Veatch SL 

PROVIDER: S-EPMC3376227 | BioStudies | 2012-01-01

REPOSITORIES: biostudies

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