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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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Average cross-correlation values before and after stimulation by antigen. Cells were stimulated by antigen in the absence or presence of 1 μM cytochalasin D. Error bars represent the SEM for PM-EGFP–FcɛRI (n = 3), Lyn-EGFP–FcɛRI (n = 7), Lyn-EGFP–FcɛRI with cytochalasin D (n = 4), and Lyn-EGFP–chimeric αTζ with cytochalasin D (n = 7).
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fig6: Average cross-correlation values before and after stimulation by antigen. Cells were stimulated by antigen in the absence or presence of 1 μM cytochalasin D. Error bars represent the SEM for PM-EGFP–FcɛRI (n = 3), Lyn-EGFP–FcɛRI (n = 7), Lyn-EGFP–FcɛRI with cytochalasin D (n = 4), and Lyn-EGFP–chimeric αTζ with cytochalasin D (n = 7).

Mentions: One possible explanation for the association between Lyn-EGFP and FcɛRI detected by cross-correlation is the binding of the SH2 domain of Lyn to the phosphorylated β subunit of FcɛRI (Kihara and Siraganian, 1994). To test this, we used a single chain chimeric IgE receptor, αTζ, which has been shown to mediate antigen-stimulated degranulation when cross-linked at 37°C, but which does not associate with FcɛRI β or mediate its phosphorylation after stimulation (Gosse et al., 2005). Average values for associations of Lyn-EGFP with FcɛRI and with αTζ before and after cell stimulation, as detected by cross-correlation, are summarized in Fig. 6. For these comparisons, inhibition of actin polymerization by cytochalasin D was necessary for FCS measurements with αTζ to prevent αTζ internalization, which occurs more rapidly after antigen cross-linking than does FcɛRI internalization (unpublished data). As shown in Fig. 6, the stimulated interaction between Lyn-EGFP and FcɛRI exhibits partial sensitivity to cytochalasin D, suggesting some role for the actin cytoskeleton in mediating this association. In the presence of cytochalasin D, the stimulated increase in cross-correlation between Lyn-EGFP and αTζ is similar to that for Lyn-EGFP and FcɛRI. Cross-correlation between PM-EGFP and FcɛRI does not increase significantly due to stimulation, as also seen in Fig. 5. Thus, these results indicate that the stimulated association of Lyn-GFP with IgE receptors detected by FCS cross-correlation is sensitive to inhibition of actin polymerization, but is independent of interactions between Lyn-EGFP and FcɛRI β.


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)

Average cross-correlation values before and after stimulation by antigen. Cells were stimulated by antigen in the absence or presence of 1 μM cytochalasin D. Error bars represent the SEM for PM-EGFP–FcɛRI (n = 3), Lyn-EGFP–FcɛRI (n = 7), Lyn-EGFP–FcɛRI with cytochalasin D (n = 4), and Lyn-EGFP–chimeric αTζ with cytochalasin D (n = 7).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171255&req=5

fig6: Average cross-correlation values before and after stimulation by antigen. Cells were stimulated by antigen in the absence or presence of 1 μM cytochalasin D. Error bars represent the SEM for PM-EGFP–FcɛRI (n = 3), Lyn-EGFP–FcɛRI (n = 7), Lyn-EGFP–FcɛRI with cytochalasin D (n = 4), and Lyn-EGFP–chimeric αTζ with cytochalasin D (n = 7).
Mentions: One possible explanation for the association between Lyn-EGFP and FcɛRI detected by cross-correlation is the binding of the SH2 domain of Lyn to the phosphorylated β subunit of FcɛRI (Kihara and Siraganian, 1994). To test this, we used a single chain chimeric IgE receptor, αTζ, which has been shown to mediate antigen-stimulated degranulation when cross-linked at 37°C, but which does not associate with FcɛRI β or mediate its phosphorylation after stimulation (Gosse et al., 2005). Average values for associations of Lyn-EGFP with FcɛRI and with αTζ before and after cell stimulation, as detected by cross-correlation, are summarized in Fig. 6. For these comparisons, inhibition of actin polymerization by cytochalasin D was necessary for FCS measurements with αTζ to prevent αTζ internalization, which occurs more rapidly after antigen cross-linking than does FcɛRI internalization (unpublished data). As shown in Fig. 6, the stimulated interaction between Lyn-EGFP and FcɛRI exhibits partial sensitivity to cytochalasin D, suggesting some role for the actin cytoskeleton in mediating this association. In the presence of cytochalasin D, the stimulated increase in cross-correlation between Lyn-EGFP and αTζ is similar to that for Lyn-EGFP and FcɛRI. Cross-correlation between PM-EGFP and FcɛRI does not increase significantly due to stimulation, as also seen in Fig. 5. Thus, these results indicate that the stimulated association of Lyn-GFP with IgE receptors detected by FCS cross-correlation is sensitive to inhibition of actin polymerization, but is independent of interactions between Lyn-EGFP and FcɛRI β.

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