Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcεRI, 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-FcεRI 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 FcεRI 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 FcεRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcεRI 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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7 November 2005
Article|
November 07 2005
Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells
Daniel R. Larson,
Daniel R. Larson
1School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853
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Julie A. Gosse,
Julie A. Gosse
2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
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David A. Holowka,
David A. Holowka
2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
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Barbara A. Baird,
Barbara A. Baird
2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
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Watt W. Webb
Watt W. Webb
1School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853
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Daniel R. Larson
1School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853
Julie A. Gosse
2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
David A. Holowka
2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
Barbara A. Baird
2Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853
Watt W. Webb
1School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853
Correspondence to Barbara A. Baird: [email protected]; or Watt W. Webb: [email protected]
D.R. Larson and J.A. Gosse contributed equally to this paper.
D.R. Larson's present address is Dept. of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
Abbreviations used in this paper: FCS, fluorescence correlation spectroscopy; huIgE, human IgE; ITAM, immunoreceptor tyrosine-based activation motif; moIgE, mouse monoclonal DNP-specific IgE; RBL, rat basophilic leukemia.
Received:
March 22 2005
Accepted:
September 14 2005
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2005
J Cell Biol (2005) 171 (3): 527–536.
Article history
Received:
March 22 2005
Accepted:
September 14 2005
Citation
Daniel R. Larson, Julie A. Gosse, David A. Holowka, Barbara A. Baird, Watt W. Webb; Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells . J Cell Biol 7 November 2005; 171 (3): 527–536. doi: https://doi.org/10.1083/jcb.200503110
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