Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

被引:94
作者
Larson, DR
Gosse, JA
Holowka, DA
Baird, BA [1 ]
Webb, WW
机构
[1] Cornell Univ, Dept Chem & Biol Chem, Ithaca, NY 14853 USA
[2] Cornell Univ, Sch Appl & Engn Phys, Ithaca, NY 14853 USA
关键词
D O I
10.1083/jcb.200503110
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), Fc epsilon RI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-Fc epsilon RI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between Fc epsilon RI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between Fc epsilon RI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked Fc epsilon RI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein-protein interactions in intact, living cells.
引用
收藏
页码:527 / 536
页数:10
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