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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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Time variation of diffusion and cross-correlation for Lyn-EGFP and A546-IgE-FcɛRI during stimulation with antigen. (A and B) Two separate single cell measurements of the time variation of diffusion of Lyn-EGFP (green) and the cross-correlation of Lyn-EGFP with IgE-FcɛRI (black). Cross-correlation of Lyn-EGFP with IgE-FcɛRI in the absence of stimulation (gray) comes from a separate measurement on a different cell. The y-axis scale on the left side is the ratio of the amplitude of the cross-correlation G cross (0) and the amplitude of the receptor auto-correlation G FcɛRI(0), and is proportional to the fraction of Lyn-EGFP associated with the receptor (Eq. 1). The black and gray curves are plotted on this axis. The y-axis on the right side represents diffusion coefficients for Lyn-EGFP. The green curve is plotted on this axis. The gray bar indicates the time of antigen addition. (C) Time-resolved cross-correlation averaged over multiple cells. Black, Lyn-EGFP–IgE-FcɛRI in stimulated responding cells (n = 7 cells); green, PM-EGFP–IgE-FcɛRI in stimulated cells (n = 6 cells); gray, Lyn-EGFP–IgE-FcɛRI in unstimulated cells (n = 3 cells). The blue circles show relative FcɛRI β tyrosine phosphorylation, measured by antiphosphotyrosine Western blotting under the same experimental conditions. (D) Time-resolved diffusion averaged over multiple stimulated cells. Green, Lyn-EGFP (n = 7 cells); blue, PM-EGFP (n = 6 cells); red, IgE-FcɛRI (n = 13 cells). Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.
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fig5: Time variation of diffusion and cross-correlation for Lyn-EGFP and A546-IgE-FcɛRI during stimulation with antigen. (A and B) Two separate single cell measurements of the time variation of diffusion of Lyn-EGFP (green) and the cross-correlation of Lyn-EGFP with IgE-FcɛRI (black). Cross-correlation of Lyn-EGFP with IgE-FcɛRI in the absence of stimulation (gray) comes from a separate measurement on a different cell. The y-axis scale on the left side is the ratio of the amplitude of the cross-correlation G cross (0) and the amplitude of the receptor auto-correlation G FcɛRI(0), and is proportional to the fraction of Lyn-EGFP associated with the receptor (Eq. 1). The black and gray curves are plotted on this axis. The y-axis on the right side represents diffusion coefficients for Lyn-EGFP. The green curve is plotted on this axis. The gray bar indicates the time of antigen addition. (C) Time-resolved cross-correlation averaged over multiple cells. Black, Lyn-EGFP–IgE-FcɛRI in stimulated responding cells (n = 7 cells); green, PM-EGFP–IgE-FcɛRI in stimulated cells (n = 6 cells); gray, Lyn-EGFP–IgE-FcɛRI in unstimulated cells (n = 3 cells). The blue circles show relative FcɛRI β tyrosine phosphorylation, measured by antiphosphotyrosine Western blotting under the same experimental conditions. (D) Time-resolved diffusion averaged over multiple stimulated cells. Green, Lyn-EGFP (n = 7 cells); blue, PM-EGFP (n = 6 cells); red, IgE-FcɛRI (n = 13 cells). Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.

Mentions: Two such time courses from single cells are shown in Fig. 5 (A and B). Consistent with the steady-state FCS measurements made before and after stimulation (Fig. 3), there is a time-dependent decrease in Lyn-EGFP diffusion after stimulation (Fig. 5, A and B, green curves). The time courses for these diffusion changes are somewhat different for each of the cells shown. As measured by cross-correlation analysis, the amount of Lyn-EGFP associated with IgE-FcɛRI increases after stimulation, and these interactions show time-dependent changes that also exhibit somewhat different patterns for each cell (Fig. 5, A and B, black curves). As apparent from these and other data, interactions between Lyn-EGFP and FcɛRI are detectable after a variable lag time and appear to undergo rapid, transient variations after an initial increase in association that occurs as the diffusion of Lyn-EGFP is decreasing. These transient interactions occur with varying magnitudes over the ∼30 min of observation, whereas Lyn-EGFP diffusion remains persistently reduced after the onset of its stimulation-dependent decrease. By comparison, Lyn-EGFP–FcɛRI association in unstimulated cells is consistently low and invariant over the entire measurement period (Fig. 5, A and B, gray curves). Cells that have been monitored in this manner for 30 min at 21°C remain functionally competent, as indicated by their ruffling response to antigen when warmed to 37°C (unpublished data).


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)

Time variation of diffusion and cross-correlation for Lyn-EGFP and A546-IgE-FcɛRI during stimulation with antigen. (A and B) Two separate single cell measurements of the time variation of diffusion of Lyn-EGFP (green) and the cross-correlation of Lyn-EGFP with IgE-FcɛRI (black). Cross-correlation of Lyn-EGFP with IgE-FcɛRI in the absence of stimulation (gray) comes from a separate measurement on a different cell. The y-axis scale on the left side is the ratio of the amplitude of the cross-correlation G cross (0) and the amplitude of the receptor auto-correlation G FcɛRI(0), and is proportional to the fraction of Lyn-EGFP associated with the receptor (Eq. 1). The black and gray curves are plotted on this axis. The y-axis on the right side represents diffusion coefficients for Lyn-EGFP. The green curve is plotted on this axis. The gray bar indicates the time of antigen addition. (C) Time-resolved cross-correlation averaged over multiple cells. Black, Lyn-EGFP–IgE-FcɛRI in stimulated responding cells (n = 7 cells); green, PM-EGFP–IgE-FcɛRI in stimulated cells (n = 6 cells); gray, Lyn-EGFP–IgE-FcɛRI in unstimulated cells (n = 3 cells). The blue circles show relative FcɛRI β tyrosine phosphorylation, measured by antiphosphotyrosine Western blotting under the same experimental conditions. (D) Time-resolved diffusion averaged over multiple stimulated cells. Green, Lyn-EGFP (n = 7 cells); blue, PM-EGFP (n = 6 cells); red, IgE-FcɛRI (n = 13 cells). Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.
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fig5: Time variation of diffusion and cross-correlation for Lyn-EGFP and A546-IgE-FcɛRI during stimulation with antigen. (A and B) Two separate single cell measurements of the time variation of diffusion of Lyn-EGFP (green) and the cross-correlation of Lyn-EGFP with IgE-FcɛRI (black). Cross-correlation of Lyn-EGFP with IgE-FcɛRI in the absence of stimulation (gray) comes from a separate measurement on a different cell. The y-axis scale on the left side is the ratio of the amplitude of the cross-correlation G cross (0) and the amplitude of the receptor auto-correlation G FcɛRI(0), and is proportional to the fraction of Lyn-EGFP associated with the receptor (Eq. 1). The black and gray curves are plotted on this axis. The y-axis on the right side represents diffusion coefficients for Lyn-EGFP. The green curve is plotted on this axis. The gray bar indicates the time of antigen addition. (C) Time-resolved cross-correlation averaged over multiple cells. Black, Lyn-EGFP–IgE-FcɛRI in stimulated responding cells (n = 7 cells); green, PM-EGFP–IgE-FcɛRI in stimulated cells (n = 6 cells); gray, Lyn-EGFP–IgE-FcɛRI in unstimulated cells (n = 3 cells). The blue circles show relative FcɛRI β tyrosine phosphorylation, measured by antiphosphotyrosine Western blotting under the same experimental conditions. (D) Time-resolved diffusion averaged over multiple stimulated cells. Green, Lyn-EGFP (n = 7 cells); blue, PM-EGFP (n = 6 cells); red, IgE-FcɛRI (n = 13 cells). Multiphoton excitation wavelength = 860 nm; power = 1.4 mW.
Mentions: Two such time courses from single cells are shown in Fig. 5 (A and B). Consistent with the steady-state FCS measurements made before and after stimulation (Fig. 3), there is a time-dependent decrease in Lyn-EGFP diffusion after stimulation (Fig. 5, A and B, green curves). The time courses for these diffusion changes are somewhat different for each of the cells shown. As measured by cross-correlation analysis, the amount of Lyn-EGFP associated with IgE-FcɛRI increases after stimulation, and these interactions show time-dependent changes that also exhibit somewhat different patterns for each cell (Fig. 5, A and B, black curves). As apparent from these and other data, interactions between Lyn-EGFP and FcɛRI are detectable after a variable lag time and appear to undergo rapid, transient variations after an initial increase in association that occurs as the diffusion of Lyn-EGFP is decreasing. These transient interactions occur with varying magnitudes over the ∼30 min of observation, whereas Lyn-EGFP diffusion remains persistently reduced after the onset of its stimulation-dependent decrease. By comparison, Lyn-EGFP–FcɛRI association in unstimulated cells is consistently low and invariant over the entire measurement period (Fig. 5, A and B, gray curves). Cells that have been monitored in this manner for 30 min at 21°C remain functionally competent, as indicated by their ruffling response to antigen when warmed to 37°C (unpublished data).

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