Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcɛRI, 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-FcɛRI 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 FcɛRI 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 FcɛRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcɛRI 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.

• Publication year

  2005

• Venue

  Journal of Cell Biology

• Publication date

  2005-11-07

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1083/jcb.200503110PMID 16275755PMCID 2171255
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• Transmembrane Sequences Are Determinants of Immunoreceptor Signaling 1

  2005cited by this paper
• Reconstitution of Regulated Phosphorylation of FcϵRI by a Lipid Raft-excluded Protein-tyrosine Phosphatase*

  2005influential reference
• Transmembrane Sequences Are Determinants of Immunoreceptor Signaling

  2005influential reference
• Lipid segregation and IgE receptor signaling: a decade of progress.

  2005cited by this paper
• Membrane elasticity in giant vesicles with fluid phase coexistence.

  2005cited by this paper
• Lyn tyrosine kinase: accentuating the positive and the negative.

  2005cited by this paper
• Visualization of plasma membrane compartmentalization with patterned lipid bilayers.

  2004influential reference
• Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells.

  2004cited by this paper
• Dysregulated FcεRI Signaling and Altered Fyn and SHIP Activities in Lyn-Deficient Mast Cells1

  2004cited by this paper
• The role of the FcepsilonRI beta-chain in allergic diseases.

  2004cited by this paper
• Negative Regulation of Immunoglobulin E–dependent Allergic Responses by Lyn Kinase

  2004cited by this paper
• The Role of the FcεRI β-Chain in Allergic Diseases

  2004cited by this paper
• In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells.

  2004cited by this paper
• Lateral diffusion of membrane lipid-anchored probes before and after aggregation of cell surface IgE-receptors

  2003cited by this paper
• Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension

  2003cited by this paper
• Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations

  2003cited by this paper
• Mechanism of Lck recruitment to the T-cell receptor cluster as studied by single-molecule-fluorescence video imaging.

  2003cited by this paper
• Mast cell signal transduction from the high-affinity IgE receptor.

  2003influential reference
• Direct measurement of Gag–Gag interaction during retrovirus assembly with FRET and fluorescence correlation spectroscopy

  2003influential reference
• Asymmetric localization of flotillins/reggies in preassembled platforms confers inherent polarity to hematopoietic cells

  2003cited by this paper
• Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells.

  2003influential reference
• Cutting Edge: Transmembrane Phosphoprotein Csk-Binding Protein/Phosphoprotein Associated With Glycosphingolipid-Enriched Microdomains as a Negative Feedback Regulator of Mast Cell Signaling Through the FcεRI

  2002cited by this paper
• Macromolecular protein signaling complexes and mast cell responses: a view of the organization of IgE-dependent mast cell signaling.

  2002cited by this paper
• Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

  2002influential reference
• Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

  2001cited by this paper
• Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components.

  2001cited by this paper
• Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcepsilon receptor I aggregation.

  2001cited by this paper
• Fluorescence correlation spectroscopy: inception, biophysical experimentations, and prospectus.

  2001cited by this paper
• Interactions between Fc(epsilon)RI and lipid raft components are regulated by the actin cytoskeleton.

  2000cited by this paper
• Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis.

  2000cited by this paper
• Sequential Requirements of the N-Terminal Palmitoylation Site and SH2 Domain of Src Family Kinases in the Initiation and Progression of FcɛRI Signaling

  2000cited by this paper
• Observing Fcεri Signaling from the Inside of the Mast Cell Membrane

  2000cited by this paper
• The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology.

  1999cited by this paper
• The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology.

  1999cited by this paper
• Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

  1999cited by this paper
• The Lck SH3 Domain Is Required for Activation of the Mitogen-activated Protein Kinase Pathway but Not the Initiation of T-cell Antigen Receptor Signaling*

  1999influential reference
• Compartmentalized Activation of the High Affinity Immunoglobulin E Receptor within Membrane Domains*

  1997cited by this paper
• Inhibition of Lyn function in mast cell activation by SH3 domain binding peptides.

  1997cited by this paper
• Src family‐selective tyrosine kinase inhibitor, PP1, inhibits both FcεRI‐ and Thy‐1‐mediated activation of rat basophilic leukemia cells

  1997cited by this paper
• Antigen-mediated IGE receptor aggregation and signaling: a window on cell surface structure and dynamics.

  1996cited by this paper
• Constrained diffusion or immobile fraction on cell surfaces: a new interpretation.

  1996cited by this paper
• Chemical cross-linking of IgE-receptor complexes in RBL-2H3 cells.

  1995cited by this paper
• Aggregation of the high-affinity IgE receptor and enhanced activity of p53/56lyn protein-tyrosine kinase.

  1994cited by this paper
• Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor.

  1994cited by this paper
• Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation.

  1994influential reference
• Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins

  1994cited by this paper
• Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction.

  1992cited by this paper
• Calcium spiking.

  1991cited by this paper
• Two-photon laser scanning fluorescence microscopy.

  1990cited by this paper
• Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells.

  1989influential reference
• Complete structure and expression in transfected cells of high affinity IgE receptor

  1989cited by this paper
• Molecular crowding on the cell surface.

  1988cited by this paper
• Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization

  1986influential reference
• IgE‐induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing clones

  1981cited by this paper
• THE INTERACTION OF IgE WITH RAT BASOPHILIC LEUKEMIA CELLS

  1974cited by this paper
• THE INTERACTION OF IgE WITH RAT BASOPHILIC LEUKEMIA CELLS

  1974cited by this paper
• Thermodynamic Fluctuations in a Reacting System-Measurement by Fluorescence Correlation Spectroscopy

  1972cited by this paper

• The Utility of Fluorescence Recovery after Photobleaching (FRAP) to Study the Plasma Membrane

  2023cites this paper
• Recent advances in nanotechnology approaches for non-viral gene therapy.

  2022cites this paper
• Lipid-based and protein-based interactions synergize transmembrane signaling stimulated by antigen clustering of IgE receptors

  2021cites this paper
• Delivery Systems for Nucleic Acids and Proteins: Barriers, Cell Capture Pathways and Nanocarriers

  2021cites this paper
• Alpha-linolenic acid inhibits IgE-mediated anaphylaxis by inhibiting Lyn kinase and suppressing mast cell activation.

  2021cites this paper
• Lipid-based, protein-based, and steric interactions synergize to facilitate transmembrane signaling stimulated by antigen-clustering of IgE receptors

  2020cites this paper
• Self-assembly of poly(allylamine)/siRNA nanoparticles, their intracellular fate and siRNA delivery.

  2019cites this paper
• Biological Membrane Organization and Cellular Signaling.

  2019cites this paper
• Imaging FCS delineates subtle heterogeneity in plasma membranes of resting mast cells

  2019cites this paper
• Membrane Biophysics: New Insights and Methods

  2018influential citation
• Analysis and Applications of Single-Molecule Fluorescence in Live Cell Membranes

  2018cites this paper
• Impact of ConcanavalinA affinity in the intracellular fate of Protein Corona on Glucosamine Au nanoparticles

  2018cites this paper
• The FcεRI signaling cascade and integrin trafficking converge at patterned ligand surfaces

  2017cites this paper
• The Biophysics of Cell Membranes

  2017cites this paper
• Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

  2017cites this paper
• Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy

  2017cites this paper
• Contribution of plasma membrane lipid domains to red blood cell (re)shaping

  2017cites this paper
• Lipid domains and membrane (re)shaping : from biophysics to biology

  2017cites this paper
• Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy.

  2017cites this paper
• Graphene Oxide Nanosheets Stimulate Ruffling and Shedding of Mammalian Cell Plasma Membranes.

  2016cites this paper
• Open Peer Review

  2016cites this paper
• Lipid Rafts, Pseudotyping, and Virus-Like Particles: Relevance of a Novel, Configurable, and Modular Antigen-Presenting Platform

  2016cites this paper
• Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling

  2016influential citation
• Investigation of Membrane Domains around the B Cell Receptor using Super-Resolution Microscopy.

  2016cites this paper
• Erratum: Steady-state cross-correlations for live two-colour super-resolution localization data sets

  2015cites this paper
• Current approaches to studying membrane organization

  2015cites this paper
• Analysis of Membrane‐Protein Complexes by Single‐Molecule Methods

  2015cites this paper
• Polyene sphingolipids with latent fluorescence: new tools to study the biophysical properties of cellular membranes

  2015cites this paper
• Steady-state cross-correlations for live two-colour super-resolution localization data sets

  2015cites this paper
• Dances with Membranes: Breakthroughs from Super-resolution Imaging

  2015cites this paper
• High Spatiotemporal Bioimaging Techniques to Study the Plasma Membrane Nanoscale Organization

  2014cites this paper
• Coupling Across Plasma Membrane in Ac vation of the EGF receptor ' ' ' *

  2014cites this paper
• Time-Resolved Fluorescence Spectroscopy Measures Clustering and Mobility of a G Protein-Coupled Receptor Opsin in Live Cell Membranes

  2014cites this paper
• Near-Field Fluorescence Cross-Correlation Spectroscopy on Planar Membranes

  2014cites this paper
• Ultrasensitive MAPK/Erk activation in absence of protein synthesis in Xenopus oocytes

  2013cites this paper
• Conformational coupling across the plasma membrane in activation of the EGF receptor.

  2013cites this paper
• Dual-color fluorescence cross-correlation spectroscopy with continuous laser excitation in a confocal setup.

  2013cites this paper
• Distinct stages of stimulated FcεRI receptor clustering and immobilization are identified through superresolution imaging.

  2013influential citation
• Advanced microscopy reveals membrane and cytoskeletal dynamics in immune cells

  2013cites this paper
• Lipid rafts generate digital‐like signal transduction in cell plasma membranes

  2012cites this paper
• A Mechanistic Model of Early FcεRI Signaling: Lipid Rafts and the Question of Protection from Dephosphorylation

  2012cites this paper
• Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic model.

  2012cites this paper
• Correcting for Spectral Cross-Talk in Dual-Color Fluorescence Cross-Correlation Spectroscopy

  2012cites this paper
• Membrane mechanisms for signal transduction: the coupling of the meso-scale raft domains to membrane-skeleton-induced compartments and dynamic protein complexes.

  2012cites this paper
• Quantitative nanoscale analysis of IgE-FcεRI clustering and coupling to early signaling proteins.

  2012cites this paper
• Fluorescence Correlation Spectroscopy to Study Membrane Organization and Interactions

  2012cites this paper
• 2.11 Fluorescence Correlation Spectroscopy

  2012cites this paper
• Deciphering Cell Membrane Organization Based on Lateral Diffusion Measurements by Fluorescence Correlation Spectroscopy at Different Length Scales

  2012cites this paper
• Dynamics and distribution of immunoglobolin E receptors : a dialog between experiment and theory

  2012cites this paper
• STUDYING MEMBRANE ANCHOR ORGANIZATION IN LIVING CELL MEMBRANES

  2011cites this paper
• Lateral dynamics of proteins with polybasic domain on anionic membranes: a dynamic Monte-Carlo study.

  2011cites this paper
• Spatio-temporal signaling in mast cells.

  2011cites this paper
• Cellular responses to patterned poly(acrylic acid) brushes.

  2011cites this paper
• Detecting nanodomains in living cell membrane by fluorescence correlation spectroscopy.

  2011cites this paper
• Impaired FcεRI stability, signaling, and effector functions in murine mast cells lacking glycosylphosphatidylinositol-anchored proteins.

  2011cites this paper
• An array of planar apertures for near-field fluorescence correlation spectroscopy.

  2011cites this paper
• Correlation Functions Quantify Super-Resolution Images and Estimate Apparent Clustering Due to Over-Counting

  2011cites this paper
• Computational study of electrostatic contribution to membrane dynamics

  2011cites this paper
• Modeling Membrane Domains

  2011cites this paper
• Regulating lipid organization and investigating membrane protein properties in physisorbed polymer-tethered membranes

  2011cites this paper
• THE ROLE OF DIFFUSION AND MEMBRANE TOPOGRAPHY IN THE INITIATION OF HIGH AFFINITY IGE RECEPTOR SIGNALING

  2011cites this paper
• Hemifusion and lateral lipid domain partition in lipid membranes of different complexity

  2011cites this paper
• Spatial organization of transmembrane receptor signalling

  2010cites this paper
• Relationships between plasma membrane microdomains and HIV‐1 assembly

  2010cites this paper
• Endocytosis of nanomedicines.

  2010cites this paper
• What precedes the initial tyrosine phosphorylation of the high affinity IgE receptor in antigen‐activated mast cell?

  2010cites this paper
• Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles.

  2010cites this paper
• Investigating Cytoskeletal Regulation Of Ige Receptor Signaling By Micropatterned Ligand Arrays

  2010cites this paper
• Distribution and dynamics of rat basophilic leukemia immunoglobulin E receptors (FcepsilonRI) on planar ligand-presenting surfaces.

  2010cites this paper
• Fluorescence Correlation Spectroscopy Studies of DNA Binding to Catanionic Surfactant Vesicles

  2010cites this paper
• Single Molecule Spectroscopy Illuminating the Molecular Dynamics of Life

  2010cites this paper
• Lipid Rafts, Pseudotyping, and Virus-Like Particles: Relevance of a Novel, Configurable, and Modular Antigen-Presenting Platform

  2010cites this paper
• Estrogen receptor interactions and dynamics monitored in live cells by fluorescence cross-correlation spectroscopy.

  2010cites this paper
• HIV-1 Nef Binds a Subpopulation of MHC-I throughout Its Trafficking Itinerary and Down-regulates MHC-I by Perturbing Both Anterograde and Retrograde Trafficking*

  2010cites this paper
• Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

  2009cites this paper
• Chapter 9 Endothelial Adhesive Platforms Organize Receptors to Promote Leukocyte Extravasation

  2009cites this paper
• Physics puzzles on membrane domains posed by cell biology

  2009cites this paper
• Single Molecule Fluorescence in Membrane Biology

  2009cites this paper
• The fine structure of the influenza virus envelope and the concept of transmembrane asymmetry of lateral domains in biomembranes

  2009cites this paper
• Tracking microdomain dynamics in cell membranes.

  2009cites this paper
• Going beyond 2D: following membrane diffusion and topography in the IgE-Fc[epsilon]RI system using 3-dimensional tracking microscopy

  2009cites this paper
• Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

  2009cites this paper
• Fluorescence correlation spectroscopy in membrane structure elucidation.

  2009cites this paper
• Caught in the act: quantifying protein behaviour in living cells.

  2009cites this paper
• Measurement of Molecular Mobility with Fluorescence Correlation Spectroscopy

  2009cites this paper
• A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

  2009cites this paper
• Adapters in the organization of mast cell signaling

  2009cites this paper
• Optimized protocol of a frequency domain fluorescence lifetime imaging microscope for FRET measurements

  2009cites this paper
• Fluorescence cross-correlation spectroscopy using single wavelength laser

  2009cites this paper
• Membrane heterogeneities in the formation of B cell receptor–Lyn kinase microclusters and the immune synapse

  2008cites this paper
• Probing the dynamics of protein-protein interactions at neuronal contacts by optical imaging.

  2008cites this paper
• Protein-protein interactions determined by fluorescence correlation spectroscopy.

  2008cites this paper
• Real-time cross-correlation image analysis of early events in IgE receptor signaling.

  2008cites this paper
• Chapter 1: In vivo applications of fluorescence correlation spectroscopy.

  2008cites this paper
• Fc receptors.

  2008cites this paper
• New concepts for fluorescence correlation spectroscopy on membranes.

  2008cites this paper
• Lipid rafts, cholesterol, and the brain.

  2008cites this paper
• Enhanced FRET contrast in lifetime imaging

  2008cites this paper
• Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches.

  2008cites this paper
• Cell Membrane Dynamics

  2008cites this paper