Small, mobile FcepsilonRI receptor aggregates are signaling competent.

Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

[1]  Howland D. T. Jones,et al.  Hyperspectral confocal microscope. , 2006, Applied optics.

[2]  W. Webb,et al.  Lateral diffusion of membrane lipid-anchored probes before and after aggregation of cell surface IgE-receptors , 2003 .

[3]  T. Mazel,et al.  Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells. , 2004, Journal of immunology.

[4]  S. Johansson,et al.  The importance of IgE antibody levels in anti‐IgE treatment , 2006, Allergy.

[5]  Rajat Varma,et al.  Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells , 2007, Proceedings of the National Academy of Sciences.

[6]  T. Jovin,et al.  Rotational dynamics of the Fc epsilon receptor on mast cells monitored by specific monoclonal antibodies and IgE. , 1991, Biochemistry.

[7]  B. Baird,et al.  Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI. , 2002, Journal of immunology.

[8]  Takashi Saito,et al.  Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76 , 2005, Nature Immunology.

[9]  J. Ravetch,et al.  FcR γ chain deletion results in pleiotrophic effector cell defects , 1994, Cell.

[10]  J. Kinet,et al.  The Fc(epsilon)RIbeta subunit functions as an amplifier of Fc(epsilon)RIgamma-mediated cell activation signals. , 1996, Cell.

[11]  J. Rivera,et al.  Molecular regulation of mast cell activation. , 2006, The Journal of allergy and clinical immunology.

[12]  Michael L. Dustin,et al.  The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling , 2000, Nature Immunology.

[13]  W. Webb,et al.  Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization , 1986, The Journal of cell biology.

[14]  J. Kinet,et al.  A Second Amplifier Function for the Allergy-Associated FcεRI-β Subunit , 2000 .

[15]  S. Pierce,et al.  Viewing the antigen‐induced initiation of B‐cell activation in living cells , 2008, Immunological reviews.

[16]  S. Steinberg,et al.  Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy. , 2006, Micron.

[17]  J. Coligan,et al.  The High‐Affinity Immunoglobulin‐E Receptor (FceRI) is Endocytosed by an AP‐2/Clathrin‐Independent, Dynamin‐Dependent Mechanism , 2006, Traffic.

[18]  T. Kawakami,et al.  Pivotal Advance: IgE accelerates in vitro development of mast cells and modifies their phenotype , 2008, Journal of leukocyte biology.

[19]  D. Katz,et al.  Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. , 1980, Journal of immunology.

[20]  T. Jovin,et al.  Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells. , 1986, Biochemistry.

[21]  J. Seagrave,et al.  Relationship of IgE receptor topography to secretion in RBL‐2H3 mast cells , 1991, Journal of cellular physiology.

[22]  J. Keen,et al.  Endocytosis of activated receptors and clathrin-coated pit formation: deciphering the chicken or egg relationship , 1996, The Journal of cell biology.

[23]  G. G. Deanin,et al.  Membrane and cytoskeletal changes associated with IgE-mediated serotonin release from rat basophilic leukemia cells , 1985, The Journal of cell biology.

[24]  Seung Yong Park,et al.  Fc epsilon RI gamma-ITAM is differentially required for mast cell function in vivo. , 2004, Journal of immunology.

[25]  Bridget S Wilson,et al.  Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. , 2004, Molecular biology of the cell.

[26]  J. Kinet,et al.  A second amplifier function for the allergy-associated Fc(epsilon)RI-beta subunit. , 2000, Immunity.

[27]  E. Ortega,et al.  Possible orientational constraints determine secretory signals induced by aggregation of IgE receptors on mast cells. , 1988, The EMBO journal.

[28]  Mark M. Davis,et al.  Direct observation of ligand recognition by T cells , 2002, Nature.

[29]  Rajat Varma,et al.  Actin and agonist MHC–peptide complex–dependent T cell receptor microclusters as scaffolds for signaling , 2005, The Journal of experimental medicine.

[30]  K. Lidke,et al.  Actin restricts FcɛRI diffusion and facilitates antigen-induced receptor immobilization , 2008, Nature Cell Biology.

[31]  T. Mazel,et al.  Dysregulated FcεRI Signaling and Altered Fyn and SHIP Activities in Lyn-Deficient Mast Cells1 , 2004, The Journal of Immunology.

[32]  W. Webb,et al.  Lateral motion and valence of Fc receptors on rat peritoneal mast cells , 1976, Nature.

[33]  I. Pecht,et al.  Immobilization of the type I receptor for IgE initiates signal transduction in mast cells. , 1996, Biochemistry.

[34]  B. Baird,et al.  Rotational motion of monomeric and dimeric immunoglobulin E-receptor complexes. , 1992, Biochemistry.

[35]  R. Weis,et al.  Supported planar membranes in studies of cell-cell recognition in the immune system. , 1986, Biochimica et biophysica acta.

[36]  Byron Goldstein,et al.  Kinetics of Multivalent Antigen DNP-BSA Binding to IgE-FcεRI in Relationship to the Stimulated Tyrosine Phosphorylation of FcεRI , 1998, The Journal of Immunology.

[37]  B. Goldstein,et al.  Kinetics of multivalent antigen DNP-BSA binding to IgE-Fc epsilon RI in relationship to the stimulated tyrosine phosphorylation of Fc epsilon RI. , 1998, Journal of immunology.

[38]  N. Varin‐Blank,et al.  Immobilization and internalization of mutated IgE receptors in transfected cells. , 1991, Journal of immunology.

[39]  J. Kinet,et al.  Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. , 1994, The Journal of biological chemistry.

[40]  J. Oliver,et al.  Roles for Ca2+ stores release and two Ca2+ influx pathways in the Fc epsilon R1-activated Ca2+ responses of RBL-2H3 mast cells. , 1995, Molecular biology of the cell.

[41]  Bridget S. Wilson,et al.  Observing Fcεri Signaling from the Inside of the Mast Cell Membrane , 2000, The Journal of cell biology.

[42]  J. Kinet,et al.  The FcεRIβ Subunit Functions as an Amplifier of FcεRIγ-Mediated Cell Activation Signals , 1996, Cell.

[43]  Keith A. Lidke,et al.  Exploring membrane protein dynamics by multicolor single quantum dot imaging using wide field, TIRF, and hyperspectral microscopy , 2007, SPIE BiOS.

[44]  Diane S Lidke,et al.  Hyperspectral Confocal Fluorescence Imaging: Exploring Alternative Multivariate Curve Resolution Approaches , 2009, Applied spectroscopy.

[45]  Wenxia Song,et al.  Polyvalent Antigens Stabilize B Cell Antigen Receptor Surface Signaling Microdomains 1 , 2003, The Journal of Immunology.

[46]  J. Altman,et al.  Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected]. , 1998, Immunity.

[47]  Howland D. T. Jones,et al.  Weighting hyperspectral image data for improved multivariate curve resolution results , 2008 .

[48]  R. Cherry,et al.  Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers: experimental test of the Saffman-Delbrück equations. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[49]  A. Sigalov.  Multichain immune recognition receptor signaling: different players, same game? , 2004, Trends in immunology.

[50]  W. Webb,et al.  Clustering, mobility, and triggering activity of small oligomers of immunoglobulin E on rat basophilic leukemia cells , 1986, The Journal of cell biology.

[51]  D. Aivazian,et al.  Phosphorylation of T cell receptor ζ is regulated by a lipid dependent folding transition , 2000, Nature Structural Biology.

[52]  J. Oliver,et al.  Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking. , 1995, Molecular biology of the cell.

[53]  K. Mossman,et al.  T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation. , 2008, Biophysical journal.

[54]  Balbino Alarcón,et al.  Recruitment of Nck by CD3ϵ Reveals a Ligand-Induced Conformational Change Essential for T Cell Receptor Signaling and Synapse Formation , 2002, Cell.

[55]  B. Baird,et al.  Bivalent Ligands with Rigid Double-Stranded DNA Spacers Reveal Structural Constraints on Signaling by FcεRI1 , 2002, The Journal of Immunology.

[56]  Akihiro Kusumi,et al.  Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. , 2005, Annual review of biophysics and biomolecular structure.

[57]  J. Kinet,et al.  New developments in FcεRI regulation, function and inhibition , 2007, Nature Reviews Immunology.

[58]  J. Oliver,et al.  Distinct Functions of the FcεR1 γ and β Subunits in the Control of FcεR1-mediated Tyrosine Kinase Activation and Signaling Responses in RBL-2H3 Mast Cells (*) , 1995, The Journal of Biological Chemistry.

[59]  Seung Yong Park,et al.  FcεRIγ-ITAM Is Differentially Required for Mast Cell Function In Vivo1 , 2004, The Journal of Immunology.

[60]  T. Yamamoto,et al.  Impaired tyrosine phosphorylation and Ca2+ mobilization, but not degranulation, in lyn-deficient bone marrow-derived mast cells. , 1997, Journal of immunology.

[61]  J. Ravetch,et al.  FcR gamma chain deletion results in pleiotrophic effector cell defects. , 1994, Cell.