Quantitative Nanoscale Analysis of IgE-Fc蔚RI Clustering and Coupling to Early Signaling Proteins

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• 作者：Sarah L. Veatch ; Ethan N. Chiang ; Prabuddha Sengupta ; David A. Holowka ; Barbara A. Baird
• 刊名：The Journal of Physical Chemistry B
• 出版年：2012
• 出版时间：June 14, 2012
• 年：2012
• 卷：116
• 期：23
• 页码：6923-6935
• 全文大小：646K
• 年卷期：v.116,no.23(June 14, 2012)
• ISSN：1520-5207

文摘

Antigen-mediated cross-linking of IgE bound to its receptor, Fc蔚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蔚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蔚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蔚RI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-Fc蔚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蔚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.