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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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Fluorescence autocorrelation and cross-correlation curves for A546-IgE-FcɛRI and Lyn-EGFP measured on a single cell before and after stimulation with antigen. (A) Before stimulation; (B) 6 min after stimulation; and (C) 30 min after stimulation. Each curve is the average of five 10-s measurements on a single focal spot. Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.
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fig4: Fluorescence autocorrelation and cross-correlation curves for A546-IgE-FcɛRI and Lyn-EGFP measured on a single cell before and after stimulation with antigen. (A) Before stimulation; (B) 6 min after stimulation; and (C) 30 min after stimulation. Each curve is the average of five 10-s measurements on a single focal spot. Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.

Mentions: To investigate whether changes in Lyn-EGFP mobility that result from antigen stimulation are due to interaction between Lyn-EGFP and FcɛRI, time-resolved fluorescence cross-correlation was measured between these proteins on live cells. Cross-correlation relies on simultaneous monitoring of the fluorescence from two probes (in this case Alexa Fluor 546 coupled to IgE and EGFP attached to Lyn) as they diffuse through the focal volume. If the two probes diffuse as a single entity, there will be a cross-correlation between their fluorescence fluctuations. The extent of cross-correlation is proportional to the amplitude of the cross-correlation curve, which is the value of the correlation at τ = 0 delay time (e.g., G(0) ≡ G(τ) at τ = 0). This amplitude has a straightforward interpretation for autocorrelation measurements: the autocorrelation amplitude is the inverse of the average number of molecules in the focal volume (i.e., G(0) = 0.1 corresponds to 10 molecules in the focal volume). For cross-correlation measurements, the amplitude of the cross-correlation curve provides a measure of association between the two probes. The fractional association of Lyn-EGFP, F, is proportional to the quotient of the cross-correlation amplitude and the individual autocorrelation amplitude of A546-IgE-FcɛRI such that (1)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}F{\propto}{G_{cross}\left({\mathrm{0}}\right)}/{G_{Fc{\mathrm{{\epsilon}}}RI}\left({\mathrm{0}}\right)}{\mathrm{,}}\end{equation*}\end{document}where Gcross (0) is the amplitude of the cross-correlation and GFcɛRI (0) is the amplitude of the A546-IgE-FcɛRI autocorrelation. In the simple case of a 1:1 binding stoichiometry and absence of background fluorescence interference, the proportionality relationship can be expressed as equality (Heinze et al., 2000). Because the relationship between A546-IgE-FcɛRI fluorescence and Lyn-EGFP fluorescence is likely to be more complicated than a 1:1 ratio, we restrict expression to the proportionality in Eq. 1. This behavior is illustrated in Fig. 4 with autocorrelation curves for Lyn-EGFP (green) and A546-IgE-FcɛRI (red) and cross-correlation curves (black) at time points before and after stimulation of a single cell by antigen. Before stimulation, the amplitudes of the Lyn-EGFP and FcɛRI autocorrelations are each greater than the cross-correlation amplitude (Fig. 4 A); also note that the FcɛRI curve has a slower decay than the Lyn-EGFP curve, as expected for the more slowly diffusing transmembrane receptor. At 6 min after stimulation, the cross-correlation amplitude has increased relative to the amplitudes of the Lyn-EGFP and A546-IgE-FcɛRI curves and overlaps the FcɛRI curve (Fig. 4 B). At 30 min after stimulation, the cross-correlation amplitude has decreased back to a value similar to that before stimulation (Fig. 4 C), indicating that the fractional association between Lyn-EGFP and the receptor has decreased from the value at 6 min after stimulation. For the example shown in Fig. 4, the number of Lyn-EGFP molecules in the focal volume is less than the number of A546-IgE-FcɛRI molecules (the amplitude GLyn(0) is greater than the amplitude GFcɛRI (0)). The overlap between the cross-correlation and the A546-IgE-FcɛRI autocorrelation indicates that all the Lyn-EGFP in the focal volume is associated with the labeled receptor and that a fraction of the total labeled receptor is associated with Lyn.


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)

Fluorescence autocorrelation and cross-correlation curves for A546-IgE-FcɛRI and Lyn-EGFP measured on a single cell before and after stimulation with antigen. (A) Before stimulation; (B) 6 min after stimulation; and (C) 30 min after stimulation. Each curve is the average of five 10-s measurements on a single focal spot. Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.
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fig4: Fluorescence autocorrelation and cross-correlation curves for A546-IgE-FcɛRI and Lyn-EGFP measured on a single cell before and after stimulation with antigen. (A) Before stimulation; (B) 6 min after stimulation; and (C) 30 min after stimulation. Each curve is the average of five 10-s measurements on a single focal spot. Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.
Mentions: To investigate whether changes in Lyn-EGFP mobility that result from antigen stimulation are due to interaction between Lyn-EGFP and FcɛRI, time-resolved fluorescence cross-correlation was measured between these proteins on live cells. Cross-correlation relies on simultaneous monitoring of the fluorescence from two probes (in this case Alexa Fluor 546 coupled to IgE and EGFP attached to Lyn) as they diffuse through the focal volume. If the two probes diffuse as a single entity, there will be a cross-correlation between their fluorescence fluctuations. The extent of cross-correlation is proportional to the amplitude of the cross-correlation curve, which is the value of the correlation at τ = 0 delay time (e.g., G(0) ≡ G(τ) at τ = 0). This amplitude has a straightforward interpretation for autocorrelation measurements: the autocorrelation amplitude is the inverse of the average number of molecules in the focal volume (i.e., G(0) = 0.1 corresponds to 10 molecules in the focal volume). For cross-correlation measurements, the amplitude of the cross-correlation curve provides a measure of association between the two probes. The fractional association of Lyn-EGFP, F, is proportional to the quotient of the cross-correlation amplitude and the individual autocorrelation amplitude of A546-IgE-FcɛRI such that (1)\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}F{\propto}{G_{cross}\left({\mathrm{0}}\right)}/{G_{Fc{\mathrm{{\epsilon}}}RI}\left({\mathrm{0}}\right)}{\mathrm{,}}\end{equation*}\end{document}where Gcross (0) is the amplitude of the cross-correlation and GFcɛRI (0) is the amplitude of the A546-IgE-FcɛRI autocorrelation. In the simple case of a 1:1 binding stoichiometry and absence of background fluorescence interference, the proportionality relationship can be expressed as equality (Heinze et al., 2000). Because the relationship between A546-IgE-FcɛRI fluorescence and Lyn-EGFP fluorescence is likely to be more complicated than a 1:1 ratio, we restrict expression to the proportionality in Eq. 1. This behavior is illustrated in Fig. 4 with autocorrelation curves for Lyn-EGFP (green) and A546-IgE-FcɛRI (red) and cross-correlation curves (black) at time points before and after stimulation of a single cell by antigen. Before stimulation, the amplitudes of the Lyn-EGFP and FcɛRI autocorrelations are each greater than the cross-correlation amplitude (Fig. 4 A); also note that the FcɛRI curve has a slower decay than the Lyn-EGFP curve, as expected for the more slowly diffusing transmembrane receptor. At 6 min after stimulation, the cross-correlation amplitude has increased relative to the amplitudes of the Lyn-EGFP and A546-IgE-FcɛRI curves and overlaps the FcɛRI curve (Fig. 4 B). At 30 min after stimulation, the cross-correlation amplitude has decreased back to a value similar to that before stimulation (Fig. 4 C), indicating that the fractional association between Lyn-EGFP and the receptor has decreased from the value at 6 min after stimulation. For the example shown in Fig. 4, the number of Lyn-EGFP molecules in the focal volume is less than the number of A546-IgE-FcɛRI molecules (the amplitude GLyn(0) is greater than the amplitude GFcɛRI (0)). The overlap between the cross-correlation and the A546-IgE-FcɛRI autocorrelation indicates that all the Lyn-EGFP in the focal volume is associated with the labeled receptor and that a fraction of the total labeled receptor is associated with Lyn.

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
Related in: MedlinePlus