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

Larson DR, Gosse JA, Holowka DA, Baird BA, Webb WW - J. Cell Biol. (2005)

Bottom Line: During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity.Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI 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.

View Article: PubMed Central - PubMed

Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, 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-FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI 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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Spatial distribution of A546-IgE-FcɛRI and Lyn-EGFP before and after stimulation with antigen. (A and B) Multiphoton images showing equatorial distributions of Lyn-EGFP (green; A) and Alexa 546-IgE-FcɛRI (red; B) before stimulation with cross-linking antigen. (C and D) Equatorial distributions of Lyn-EGFP (C) and A546-IgE-FcɛRI (D) after stimulation with cross-linking antigen for 40 min at 21°C. Bar, 5 μm. Multiphoton excitation wavelength = 860 nm; power = 2.0 mW.
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fig2: Spatial distribution of A546-IgE-FcɛRI and Lyn-EGFP before and after stimulation with antigen. (A and B) Multiphoton images showing equatorial distributions of Lyn-EGFP (green; A) and Alexa 546-IgE-FcɛRI (red; B) before stimulation with cross-linking antigen. (C and D) Equatorial distributions of Lyn-EGFP (C) and A546-IgE-FcɛRI (D) after stimulation with cross-linking antigen for 40 min at 21°C. Bar, 5 μm. Multiphoton excitation wavelength = 860 nm; power = 2.0 mW.

Mentions: Interactions between IgE-FcɛRI and Lyn are not readily observable by fluorescence imaging under our conditions of stimulation. Before stimulation with cross-linking antigen (DNP-BSA), both Lyn-EGFP and A546-IgE-FcɛRI appear uniformly distributed in the plasma membrane (Fig. 2, A and B). After cross-linking with DNP-BSA for 40 min at 21°C, A546-IgE-FcɛRI aggregates into discrete puncta at the cell surface (Fig. 2 D), and these persist and grow for longer time periods, eventually forming large domains if cells are held at 4°C to slow internalization (Thomas et al., 1994). Nevertheless, Lyn-EGFP appeared to remain uniformly distributed at optical resolution during the time of our measurements at 21°C (Fig. 2 C). Thus, coredistribution of Lyn-EGFP accompanying microscopically detectable clustering of IgE receptors is not readily detectable under these conditions, possibly because only a small fraction of the available Lyn-EGFP binds in its competition with the excess coexisting unlabeled Lyn. Furthermore, previous biochemical coimmunoprecipitation studies have suggested that this receptor–kinase interaction is weak and/or transient (Pribluda et al., 1994; Siraganian, 2003). Transient colocalization of Lyn with cross-linked FcɛRI has been detected by electron microscopy (Wilson et al., 2000), but real-time kinetics of these interactions have not been observed.


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

Larson DR, Gosse JA, Holowka DA, Baird BA, Webb WW - J. Cell Biol. (2005)

Spatial distribution of A546-IgE-FcɛRI and Lyn-EGFP before and after stimulation with antigen. (A and B) Multiphoton images showing equatorial distributions of Lyn-EGFP (green; A) and Alexa 546-IgE-FcɛRI (red; B) before stimulation with cross-linking antigen. (C and D) Equatorial distributions of Lyn-EGFP (C) and A546-IgE-FcɛRI (D) after stimulation with cross-linking antigen for 40 min at 21°C. Bar, 5 μm. Multiphoton excitation wavelength = 860 nm; power = 2.0 mW.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171255&req=5

fig2: Spatial distribution of A546-IgE-FcɛRI and Lyn-EGFP before and after stimulation with antigen. (A and B) Multiphoton images showing equatorial distributions of Lyn-EGFP (green; A) and Alexa 546-IgE-FcɛRI (red; B) before stimulation with cross-linking antigen. (C and D) Equatorial distributions of Lyn-EGFP (C) and A546-IgE-FcɛRI (D) after stimulation with cross-linking antigen for 40 min at 21°C. Bar, 5 μm. Multiphoton excitation wavelength = 860 nm; power = 2.0 mW.
Mentions: Interactions between IgE-FcɛRI and Lyn are not readily observable by fluorescence imaging under our conditions of stimulation. Before stimulation with cross-linking antigen (DNP-BSA), both Lyn-EGFP and A546-IgE-FcɛRI appear uniformly distributed in the plasma membrane (Fig. 2, A and B). After cross-linking with DNP-BSA for 40 min at 21°C, A546-IgE-FcɛRI aggregates into discrete puncta at the cell surface (Fig. 2 D), and these persist and grow for longer time periods, eventually forming large domains if cells are held at 4°C to slow internalization (Thomas et al., 1994). Nevertheless, Lyn-EGFP appeared to remain uniformly distributed at optical resolution during the time of our measurements at 21°C (Fig. 2 C). Thus, coredistribution of Lyn-EGFP accompanying microscopically detectable clustering of IgE receptors is not readily detectable under these conditions, possibly because only a small fraction of the available Lyn-EGFP binds in its competition with the excess coexisting unlabeled Lyn. Furthermore, previous biochemical coimmunoprecipitation studies have suggested that this receptor–kinase interaction is weak and/or transient (Pribluda et al., 1994; Siraganian, 2003). Transient colocalization of Lyn with cross-linked FcɛRI has been detected by electron microscopy (Wilson et al., 2000), but real-time kinetics of these interactions have not been observed.

Bottom Line: During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity.Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI 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.

View Article: PubMed Central - PubMed

Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcepsilonRI, 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-FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcepsilonRI 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.

Show MeSH