Journal
JOURNAL OF CELL BIOLOGY
Volume 171, Issue 3, Pages 527-536Publisher
ROCKEFELLER UNIV PRESS
DOI: 10.1083/jcb.200503110
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Funding
- NIAID NIH HHS [R01 AI018306, AI18306] Funding Source: Medline
- NIBIB NIH HHS [9 P41 EB001976, P41 EB001976] Funding Source: Medline
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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.
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