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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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Observation of fluorescently labeled IgE receptors and Lyn-EGFP kinase on RBL mast cells. (A) Schematic showing the transmembrane receptor, FcɛRI (purple), IgE antibody (yellow) labeled with Alexa Fluor 546 (red stars), Lyn kinase (blue) with EGFP (green stars), a membrane-anchored form of EGFP containing only the myristoylation and palmitoylation sites of Lyn (blue; PM-EGFP), cross-linking antigen (black), and phosphotyrosine sites on Lyn and the receptor (dark red). (B) Side profile of an adherent RBL cell labeled with A546-IgE. The multiphoton excitation volume (arrow) focused on the upper plasma membrane (0.5 × 1.5 μm) is drawn to scale in red. Bar, 5 μm. (C) Dual color cross-correlation FCS observes protein–protein interactions: schematics at left show ellipsoidal focal volume within which fluorescence is excited. Correlation functions (G(τ)) at right are shown for green and red fluorophores and black for the cross-correlation of green with red. The rise of the black cross-correlation curve in the lower example where green and red species are bound together and diffuse together shows that the duration of the interaction exceeds the diffusive dwell time in the focal volume.
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fig1: Observation of fluorescently labeled IgE receptors and Lyn-EGFP kinase on RBL mast cells. (A) Schematic showing the transmembrane receptor, FcɛRI (purple), IgE antibody (yellow) labeled with Alexa Fluor 546 (red stars), Lyn kinase (blue) with EGFP (green stars), a membrane-anchored form of EGFP containing only the myristoylation and palmitoylation sites of Lyn (blue; PM-EGFP), cross-linking antigen (black), and phosphotyrosine sites on Lyn and the receptor (dark red). (B) Side profile of an adherent RBL cell labeled with A546-IgE. The multiphoton excitation volume (arrow) focused on the upper plasma membrane (0.5 × 1.5 μm) is drawn to scale in red. Bar, 5 μm. (C) Dual color cross-correlation FCS observes protein–protein interactions: schematics at left show ellipsoidal focal volume within which fluorescence is excited. Correlation functions (G(τ)) at right are shown for green and red fluorophores and black for the cross-correlation of green with red. The rise of the black cross-correlation curve in the lower example where green and red species are bound together and diffuse together shows that the duration of the interaction exceeds the diffusive dwell time in the focal volume.

Mentions: We approached this problem by directly monitoring the interactions between Lyn, FcɛRI, and other plasma membrane–associated components (Fig. 1 A) using two-photon excitation fluorescence correlation spectroscopy (FCS; Webb, 2001). In this approach, a small diffraction-limited focal volume (∼0.1 μm3 with a cross-sectional area ∼0.1 μm2 on the membrane; Fig. 1 B, red oval intersecting cell membrane) is created by focusing a laser through an objective lens. Because of the nonlinear absorption of photons (two-photon excitation is proportional to I2, where I = intensity of the laser), light is absorbed and fluorescence is emitted only in the vicinity of the focus of the lens, yielding a well defined optical focal volume (Denk et al., 1990). Diffusion of molecules into and out of this focal volume leads to characteristic fluctuations in fluorescence, which in turn can be used to determine dynamic quantities such as diffusion coefficients and reaction kinetics (Magde et al., 1972). If two distinguishable fluorescent species are present, one can analyze correlations between these molecular species indicative of codiffusion and association (Heinze et al., 2000). This phenomenon is illustrated in Fig. 1 C. Cross-correlation is independent of nanoscopic distance and relative orientation between fluorophores, in contrast to an alternative method, Förster resonance energy transfer. Thus, cross-correlation is more effective for studying interactions between membrane-associated proteins in which the labels are on opposite sides of the membrane or are otherwise too spatially separated or misoriented for energy transfer. However, FCS measurements do require a stable target and low-fluorescence background. Thus, assisted by multiphoton microscopy, FCS has begun to become useful in applications to living cell membranes (Schwille et al., 1999).


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)

Observation of fluorescently labeled IgE receptors and Lyn-EGFP kinase on RBL mast cells. (A) Schematic showing the transmembrane receptor, FcɛRI (purple), IgE antibody (yellow) labeled with Alexa Fluor 546 (red stars), Lyn kinase (blue) with EGFP (green stars), a membrane-anchored form of EGFP containing only the myristoylation and palmitoylation sites of Lyn (blue; PM-EGFP), cross-linking antigen (black), and phosphotyrosine sites on Lyn and the receptor (dark red). (B) Side profile of an adherent RBL cell labeled with A546-IgE. The multiphoton excitation volume (arrow) focused on the upper plasma membrane (0.5 × 1.5 μm) is drawn to scale in red. Bar, 5 μm. (C) Dual color cross-correlation FCS observes protein–protein interactions: schematics at left show ellipsoidal focal volume within which fluorescence is excited. Correlation functions (G(τ)) at right are shown for green and red fluorophores and black for the cross-correlation of green with red. The rise of the black cross-correlation curve in the lower example where green and red species are bound together and diffuse together shows that the duration of the interaction exceeds the diffusive dwell time in the focal volume.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Observation of fluorescently labeled IgE receptors and Lyn-EGFP kinase on RBL mast cells. (A) Schematic showing the transmembrane receptor, FcɛRI (purple), IgE antibody (yellow) labeled with Alexa Fluor 546 (red stars), Lyn kinase (blue) with EGFP (green stars), a membrane-anchored form of EGFP containing only the myristoylation and palmitoylation sites of Lyn (blue; PM-EGFP), cross-linking antigen (black), and phosphotyrosine sites on Lyn and the receptor (dark red). (B) Side profile of an adherent RBL cell labeled with A546-IgE. The multiphoton excitation volume (arrow) focused on the upper plasma membrane (0.5 × 1.5 μm) is drawn to scale in red. Bar, 5 μm. (C) Dual color cross-correlation FCS observes protein–protein interactions: schematics at left show ellipsoidal focal volume within which fluorescence is excited. Correlation functions (G(τ)) at right are shown for green and red fluorophores and black for the cross-correlation of green with red. The rise of the black cross-correlation curve in the lower example where green and red species are bound together and diffuse together shows that the duration of the interaction exceeds the diffusive dwell time in the focal volume.
Mentions: We approached this problem by directly monitoring the interactions between Lyn, FcɛRI, and other plasma membrane–associated components (Fig. 1 A) using two-photon excitation fluorescence correlation spectroscopy (FCS; Webb, 2001). In this approach, a small diffraction-limited focal volume (∼0.1 μm3 with a cross-sectional area ∼0.1 μm2 on the membrane; Fig. 1 B, red oval intersecting cell membrane) is created by focusing a laser through an objective lens. Because of the nonlinear absorption of photons (two-photon excitation is proportional to I2, where I = intensity of the laser), light is absorbed and fluorescence is emitted only in the vicinity of the focus of the lens, yielding a well defined optical focal volume (Denk et al., 1990). Diffusion of molecules into and out of this focal volume leads to characteristic fluctuations in fluorescence, which in turn can be used to determine dynamic quantities such as diffusion coefficients and reaction kinetics (Magde et al., 1972). If two distinguishable fluorescent species are present, one can analyze correlations between these molecular species indicative of codiffusion and association (Heinze et al., 2000). This phenomenon is illustrated in Fig. 1 C. Cross-correlation is independent of nanoscopic distance and relative orientation between fluorophores, in contrast to an alternative method, Förster resonance energy transfer. Thus, cross-correlation is more effective for studying interactions between membrane-associated proteins in which the labels are on opposite sides of the membrane or are otherwise too spatially separated or misoriented for energy transfer. However, FCS measurements do require a stable target and low-fluorescence background. Thus, assisted by multiphoton microscopy, FCS has begun to become useful in applications to living cell membranes (Schwille et al., 1999).

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