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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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Diffusion of A488-IgE-FcɛRI, Lyn-EGFP, and PM-EGFP before and after stimulation with antigen. (A, C, and E) FCS autocorrelation curves from single cells: (A) Lyn-EGFP, (C) PM-EGFP, and (E) A488-IgE-FcɛRI. The y-axis is the autocorrelation amplitude as a function of delay time (G(τ)), and each curve is the average of five consecutive 10-s measurements, with error bars as measured SD. The after curves are recorded 30 min after stimulation. (B, D, and F) Histograms of diffusion coefficients compiled from multiple cells: (B) Lyn-EGFP (n = 19 cells), (D) PM-EGFP (n = 12 cells), and (F) A488-IgE-FcɛRI (n = 15 cells). Bin width = 0.3 μm2/ms. (G) Average membrane-bound diffusion coefficients from the designated peaks in B, D, and F, and for cells in the presence of 20 μM PP1 (n = 11 cells). Error bars are the SEM. Multiphoton excitation wavelength = 905 nm and power = 1.4 mW for all measurements.
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fig3: Diffusion of A488-IgE-FcɛRI, Lyn-EGFP, and PM-EGFP before and after stimulation with antigen. (A, C, and E) FCS autocorrelation curves from single cells: (A) Lyn-EGFP, (C) PM-EGFP, and (E) A488-IgE-FcɛRI. The y-axis is the autocorrelation amplitude as a function of delay time (G(τ)), and each curve is the average of five consecutive 10-s measurements, with error bars as measured SD. The after curves are recorded 30 min after stimulation. (B, D, and F) Histograms of diffusion coefficients compiled from multiple cells: (B) Lyn-EGFP (n = 19 cells), (D) PM-EGFP (n = 12 cells), and (F) A488-IgE-FcɛRI (n = 15 cells). Bin width = 0.3 μm2/ms. (G) Average membrane-bound diffusion coefficients from the designated peaks in B, D, and F, and for cells in the presence of 20 μM PP1 (n = 11 cells). Error bars are the SEM. Multiphoton excitation wavelength = 905 nm and power = 1.4 mW for all measurements.

Mentions: Data from individual cells are shown for each of these proteins in Fig. 3 (A, C, and E). The amplitude curves are normalized to facilitate direct comparison of the diffusion times. For Lyn-EGFP (Fig. 3 A) and A488-IgE–labeled FcɛRI (Fig. 3 E) there is an increase in the decay time of the FCS curves from before stimulation (pink) to longer times after 30 min of stimulation by antigen (blue), indicating that both proteins diffuse more slowly through the focal volume and are thus shown to have lower mobility after stimulation. For PM-EGFP (Fig. 3 C), however, there is no detectable shift in the diffusion time as a result of IgE receptor cross-linking with antigen. Fitting each curve requires one or two diffusion coefficients, together with the relative contribution of each. For the inner leaflet–anchored proteins Lyn-EGFP and PM-EGFP it is necessary to use a two-component diffusion fit (Larson et al., 2003; Pyenta et al., 2003), whereas for transmembrane FcɛRI a one component fit is sufficient both before and after stimulation. It has previously been shown that the diffusibility of individual noncross-linked FcɛRI on RBL cells shows diverse diffusive behavior called anomalous subdiffusion, a phenomenon common to cell membrane proteins that is difficult to distinguish from a spread of conventional diffusion coefficients by FCS (Feder et al., 1996).


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)

Diffusion of A488-IgE-FcɛRI, Lyn-EGFP, and PM-EGFP before and after stimulation with antigen. (A, C, and E) FCS autocorrelation curves from single cells: (A) Lyn-EGFP, (C) PM-EGFP, and (E) A488-IgE-FcɛRI. The y-axis is the autocorrelation amplitude as a function of delay time (G(τ)), and each curve is the average of five consecutive 10-s measurements, with error bars as measured SD. The after curves are recorded 30 min after stimulation. (B, D, and F) Histograms of diffusion coefficients compiled from multiple cells: (B) Lyn-EGFP (n = 19 cells), (D) PM-EGFP (n = 12 cells), and (F) A488-IgE-FcɛRI (n = 15 cells). Bin width = 0.3 μm2/ms. (G) Average membrane-bound diffusion coefficients from the designated peaks in B, D, and F, and for cells in the presence of 20 μM PP1 (n = 11 cells). Error bars are the SEM. Multiphoton excitation wavelength = 905 nm and power = 1.4 mW for all measurements.
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fig3: Diffusion of A488-IgE-FcɛRI, Lyn-EGFP, and PM-EGFP before and after stimulation with antigen. (A, C, and E) FCS autocorrelation curves from single cells: (A) Lyn-EGFP, (C) PM-EGFP, and (E) A488-IgE-FcɛRI. The y-axis is the autocorrelation amplitude as a function of delay time (G(τ)), and each curve is the average of five consecutive 10-s measurements, with error bars as measured SD. The after curves are recorded 30 min after stimulation. (B, D, and F) Histograms of diffusion coefficients compiled from multiple cells: (B) Lyn-EGFP (n = 19 cells), (D) PM-EGFP (n = 12 cells), and (F) A488-IgE-FcɛRI (n = 15 cells). Bin width = 0.3 μm2/ms. (G) Average membrane-bound diffusion coefficients from the designated peaks in B, D, and F, and for cells in the presence of 20 μM PP1 (n = 11 cells). Error bars are the SEM. Multiphoton excitation wavelength = 905 nm and power = 1.4 mW for all measurements.
Mentions: Data from individual cells are shown for each of these proteins in Fig. 3 (A, C, and E). The amplitude curves are normalized to facilitate direct comparison of the diffusion times. For Lyn-EGFP (Fig. 3 A) and A488-IgE–labeled FcɛRI (Fig. 3 E) there is an increase in the decay time of the FCS curves from before stimulation (pink) to longer times after 30 min of stimulation by antigen (blue), indicating that both proteins diffuse more slowly through the focal volume and are thus shown to have lower mobility after stimulation. For PM-EGFP (Fig. 3 C), however, there is no detectable shift in the diffusion time as a result of IgE receptor cross-linking with antigen. Fitting each curve requires one or two diffusion coefficients, together with the relative contribution of each. For the inner leaflet–anchored proteins Lyn-EGFP and PM-EGFP it is necessary to use a two-component diffusion fit (Larson et al., 2003; Pyenta et al., 2003), whereas for transmembrane FcɛRI a one component fit is sufficient both before and after stimulation. It has previously been shown that the diffusibility of individual noncross-linked FcɛRI on RBL cells shows diverse diffusive behavior called anomalous subdiffusion, a phenomenon common to cell membrane proteins that is difficult to distinguish from a spread of conventional diffusion coefficients by FCS (Feder et al., 1996).

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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