Mapping for Specialized Domains for FCeRI Signaling &Internalization

Summary

Principal Investigator: Bridget S Wilson
Abstract: DESCRIPTION (provided by applicant): By crosslinking IgE bound to the high affinity IgE receptor, FceRI, allergens initiate a complex sequence of events leading to release of mast cell inflammatory mediators. We have used a combination of high resolution microscopy techniques and lipidomics to show that IgE receptors localize to distinct microdomains in the plasma membrane during signaling and receptor internalization. Our central hypothesis is that this membrane organization is critical to the regulation of mast cell responses. Based upon recent evidence, we propose that membrane proteins are concentrated in "protein islands," protein and cholesterol-rich domains separated by expanses of mostly lipid. IgE receptors are transiently confined within these small, heterogenous islands, providing one mechanism for receptor clustering observed by EM. Molecular segregation can occur within islands, facilitating signal transduction and at least two pathways of endocytosis. Cytoskeletal elements, including actin-based corrals and cortical cytoskeleton networks, restrict receptor mobility and may also serve as tethers for protein islands. In this proposal, we plan to test several hypotheses related to our model. In Aim 1, we will develop new monovalent probes for plasma membrane lipids. We will use both transmission and X- ray spectral electron microscopy to map the distributions of tagged lipids on membrane sheets. We will also use fluorescent cholesterol derivatives to determine if cholesterol-rich regions in the outer and the inner leaflet of the membrane bilayers overlap and to study cholesterol diffusion relative to FceRI. Results in the rat basophilic leukemia (RBL-2H3) cell line will be confirmed in BMMCs. In Aim 2, we will test the hypothesis that receptor diffusion and antigen-stimulated signal initiation is modified by part-time residency in protein islands and by elements of the cortical cytoskeleton. We have documented the expression of 2 a spectrin and 3 [unreadable] spectrin isoforms in RBL-2H3 cells. We will use RNA interference and transient transfection methods to disrupt the spectrin cortical network. Single particle tracking (SPT) with multiple colors of monomeric IgE-quantum dot probes will reveal consequences of this disruption for receptor mobility and clustering behavior. Stochastic modeling methods will be used to determine a best fit model for the anomalous diffusion and clustering of IgE receptors, comparing contributions of 0.5-1 micron corrals, smaller cortical cytoskeletal subdomains and protein islands. Assays for calcium flux, secretory response and cytokine production will correlate functional responses with receptor immobilization, clustering and desensitization. In Aim 3, we will test the hypothesis that both clathrin-dependent and clathrin-independent endocytic pathways mediate internalization of receptors from primary signaling domains. We will isolate CLICs (clathrin-independent vesicular carriers) responsible for FceRI endocytosis in the absence of clathrin. We will use a combination of proteomics, live cell and electron microscopy, pharmacologic, and molecular biology approaches to identify and characterize molecular regulators of non-clathrin-mediated endocytosis. A particular focus in this aim is the small GTPase, Arf6. Results of these analyses will contribute important new insight specifically into the molecular events that initiate allergic inflammation and more generally into the nature and assembly of membrane microdomains controlling signal transduction and membrane trafficking in immune cells. Public Health Relevance: Allergic diseases are on the rise in the United States. Mast cells and basophils are the principal mediators of the allergic response, through activation of the high affinity IgE receptor, FceRI. When these receptors are crosslinked by polyvalent allergens, the cells release inflammatory mediators such as histamine and leukotrienes. We use sophisticated microscopy techniques and analytical approaches to study the behavior of IgE receptors in the mast cell membrane. We have discovered that the plasma membrane has a rich topography, that we believe is critical to the regulation of mast cell responses. The landscape of the mast cell membrane undergoes dramatic change during cell activation and receptors come together into large clusters in order to signal to the cell interior. The signaling process is limited in part by uptake of the receptors into the cell, a process referred to as endocytosis. In this proposal, we will study the dynamic behavior of receptors in live cell membranes using brightly fluorescent nanoprobes called quantum dots. We will also use our innovative electron microscopy methods to map the distributions of lipids, receptors and other proteins across the mast cell landscape. We will gain important insight into the mechanisms that drive receptor endocytosis. Our work is applicable to other cell types, particularly other cells of the immune system. Results of these analyses will contribute important new insight into the nature and assembly of membrane microdomains controlling immune cell signaling.
Funding Period: 2002-04-01 - 2015-01-31
more information: NIH RePORT

Top Publications

  1. ncbi Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy
    Jun Zhang
    Department of Computer Science, University of New Mexico, Albuquerque, NM 87110, USA
    Micron 37:14-34. 2006
  2. pmc Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces
    Amanda Carroll-Portillo
    Department of Pathology, University of New Mexico, Albuquerque, NM 87131, USA
    J Immunol 184:1328-38. 2010
  3. pmc Distribution and dynamics of rat basophilic leukemia immunoglobulin E receptors (FcepsilonRI) on planar ligand-presenting surfaces
    Kathrin Spendier
    Consortium of the Americas for Interdisciplinary Science, Department of Pathology, University of New Mexico, Albuquerque, New Mexico, USA
    Biophys J 99:388-97. 2010
  4. pmc SERS nanosensors that report pH of endocytic compartments during FcεRI transit
    K L Nowak-Lovato
    Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
    Anal Bioanal Chem 398:2019-29. 2010
  5. pmc Time-resolved three-dimensional molecular tracking in live cells
    Nathan P Wells
    Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States
    Nano Lett 10:4732-7. 2010
  6. pmc Sensitivity analysis predicts that the ERK-pMEK interaction regulates ERK nuclear translocation
    K Radhakrishnan
    University of New Mexico School of Medicine, Department of Pathology and Cancer Center, Albuquerque, NM, USA
    IET Syst Biol 3:329-41. 2009
  7. pmc Spatio-temporal signaling in mast cells
    Bridget S Wilson
    Department of Pathology, University of New Mexico Albuquerque, New Mexico, USA
    Adv Exp Med Biol 716:91-106. 2011
  8. pmc Using hierarchical clustering and dendrograms to quantify the clustering of membrane proteins
    Flor A Espinoza
    Department of Mathematics and Statistics, University of New Mexico, Albuquerque, NM 87131 1141, USA
    Bull Math Biol 74:190-211. 2012
  9. pmc Novel mechanism for Fc{epsilon}RI-mediated signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and the selective influence of STAT5B over mast cell cytokine production
    Nicholas A Pullen
    Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23284, USA
    J Biol Chem 287:2045-54. 2012
  10. pmc Insights into cell membrane microdomain organization from live cell single particle tracking of the IgE high affinity receptor FcϵRI of mast cells
    Flor A Espinoza
    Department of Mathematics and Statistics, University of New Mexico, Albuquerque, 87131 1141, USA
    Bull Math Biol 74:1857-911. 2012

Research Grants

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
  2. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
  3. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
  4. DEVELOPMENT AND CONTROL OF PULMONARY ALVEOLAR STABILITY
    Samuel Hawgood; Fiscal Year: 2013

Detail Information

Publications22

  1. ncbi Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy
    Jun Zhang
    Department of Computer Science, University of New Mexico, Albuquerque, NM 87110, USA
    Micron 37:14-34. 2006
    ..Our analyses, and the resulting programs for automating data collection and for carrying out statistical and clustering analyses provide toolboxes specialized for the spatiotemporal analysis and modeling of signal transduction pathways...
  2. pmc Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces
    Amanda Carroll-Portillo
    Department of Pathology, University of New Mexico, Albuquerque, NM 87131, USA
    J Immunol 184:1328-38. 2010
    ....
  3. pmc Distribution and dynamics of rat basophilic leukemia immunoglobulin E receptors (FcepsilonRI) on planar ligand-presenting surfaces
    Kathrin Spendier
    Consortium of the Americas for Interdisciplinary Science, Department of Pathology, University of New Mexico, Albuquerque, New Mexico, USA
    Biophys J 99:388-97. 2010
    ..The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes...
  4. pmc SERS nanosensors that report pH of endocytic compartments during FcεRI transit
    K L Nowak-Lovato
    Chemistry Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA
    Anal Bioanal Chem 398:2019-29. 2010
    ..These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors...
  5. pmc Time-resolved three-dimensional molecular tracking in live cells
    Nathan P Wells
    Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States
    Nano Lett 10:4732-7. 2010
    ..During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell...
  6. pmc Sensitivity analysis predicts that the ERK-pMEK interaction regulates ERK nuclear translocation
    K Radhakrishnan
    University of New Mexico School of Medicine, Department of Pathology and Cancer Center, Albuquerque, NM, USA
    IET Syst Biol 3:329-41. 2009
    ..Our study also identifies biological experiments that can verify this explanation...
  7. pmc Spatio-temporal signaling in mast cells
    Bridget S Wilson
    Department of Pathology, University of New Mexico Albuquerque, New Mexico, USA
    Adv Exp Med Biol 716:91-106. 2011
    ..The dynamic relationships between receptor diffusion, aggregation state, clustering, signal initiation and signal strength are discussed in the context of these recent findings...
  8. pmc Using hierarchical clustering and dendrograms to quantify the clustering of membrane proteins
    Flor A Espinoza
    Department of Mathematics and Statistics, University of New Mexico, Albuquerque, NM 87131 1141, USA
    Bull Math Biol 74:190-211. 2012
    ....
  9. pmc Novel mechanism for Fc{epsilon}RI-mediated signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation and the selective influence of STAT5B over mast cell cytokine production
    Nicholas A Pullen
    Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23284, USA
    J Biol Chem 287:2045-54. 2012
    ..Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcεRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function...
  10. pmc Insights into cell membrane microdomain organization from live cell single particle tracking of the IgE high affinity receptor FcϵRI of mast cells
    Flor A Espinoza
    Department of Mathematics and Statistics, University of New Mexico, Albuquerque, 87131 1141, USA
    Bull Math Biol 74:1857-911. 2012
    ..The fit for short jumps suggests that the motion of the quantum dots can be modeled as diffusion in a fractal space of dimension less than two...
  11. pmc Caught in the act: quantifying protein behaviour in living cells
    Diane S Lidke
    Department of Pathology and Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
    Trends Cell Biol 19:566-74. 2009
    ..In this review, we illustrate the power of emerging fluorescence microscopy techniques to capture and quantify protein dynamics...
  12. pmc Small, mobile FcepsilonRI receptor aggregates are signaling competent
    Nicholas L Andrews
    Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131, USA
    Immunity 31:469-79. 2009
    ..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...
  13. pmc Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging
    D J Frankel
    Biomolecular Materials and Interfaces Department, MS1413 Sandia National Laboratories, Albuquerque, New Mexico 87185, USA
    Biophys J 90:2404-13. 2006
    ..Overall the data suggest that signaling and endocytosis occur in mast cells from raised membrane regions that depend on cholesterol for their integrity and may be organized in specific relationship with the cortical cytoskeleton...
  14. pmc Reaction diffusion modeling of calcium dynamics with realistic ER geometry
    Shawn Means
    Sandia National Laboratory, Albuquerque, New Mexico, USA
    Biophys J 91:537-57. 2006
    ..Simulation results also suggest that the buffering capacity of the ER, and not restricted diffusion, is the predominant factor influencing average luminal calcium concentrations...
  15. pmc Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton
    Björn F Lillemeier
    Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA
    Proc Natl Acad Sci U S A 103:18992-7. 2006
    ..Abundant actin staining and inhibitor studies show that these structures are connected to the cytoskeleton and at least partially depend on it for their formation and/or maintenance...
  16. pmc Activated N-formyl peptide receptor and high-affinity IgE receptor occupy common domains for signaling and internalization
    Mei Xue
    Department of Pathology and Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131, USA
    Mol Biol Cell 18:1410-20. 2007
    ..The observation of receptors in lightly coated membrane invaginations suggests that, despite the lack of caveolin, hematopoietic cells harbor caveolae-like structures that are candidates for nonclathrin-mediated endocytosis...
  17. ncbi FcepsilonRI and Thy-1 domains have unique protein and lipid compositions
    Zurab Surviladze
    Department of Pathology, University of New Mexico, Albuquerque, NM, USA
    J Lipid Res 48:1325-35. 2007
    ..5-3 times more abundant in FcRI domains than in the Thy-1 microdomains, whereas most diacyl GPE molecular species were equally abundant in the two domains...
  18. ncbi Markov random field modeling of the spatial distribution of proteins on cell membranes
    Jun Zhang
    Department of Computer Science, University of New Mexico, Albuquerque, NM 87131, USA
    Bull Math Biol 70:297-321. 2008
    ..The work is an important step toward a more complete understanding of membrane spatial organization and dynamics during signaling...
  19. doi Exploring membrane domains using native membrane sheets and transmission electron microscopy
    Bridget S Wilson
    Dept of Pathology and Cancer Center, University of New Mexico School of Medicine, Albuquerque, USA
    Methods Mol Biol 398:245-61. 2007
    ..Probe coordinates are extracted from digitized images and the distributions of the probes are analyzed with respect to each other and to membrane features like clathrin-coated pits, caveolae, and the cortical cytoskeleton...
  20. pmc Stochastic modeling of calcium in 3D geometry
    Tomas Mazel
    Department of Pathology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
    Biophys J 96:1691-706. 2009
    ..The explicit consideration of organelle spatial relationships represents an important step toward building a comprehensive, realistic model of cellular calcium dynamics...
  21. pmc Time series analysis of particle tracking data for molecular motion on the cell membrane
    Wenxia Ying
    Department of Mathematics and Statistics, University of New Mexico, Albuquerque, NM, 87131 1141, USA
    Bull Math Biol 71:1967-2024. 2009
    ..In this case, we introduce the notion of an instantaneous diffusion constant. All of the diffusion constants show a strong consistency for most of the biological data...
  22. pmc The architectural relationship of components controlling mast cell endocytosis
    Cedric Cleyrat
    Department of Pathology University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
    J Cell Sci 126:4913-25. 2013
    ..Intersections between these specialized domains might represent sorting stations that direct cargo to specific endocytic pathways. ..

Research Grants31

  1. RAGE and Mechanisms of Vascular Dysfunction
    Shi Fang Yan; Fiscal Year: 2013
    ..Using novel and state-of-the-art techniques, floxed mice and molecular approaches to gene regulation, we are well-positioned to lead the study of RAGE in the next cycle of this Program. ..
  2. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
  3. TSH RECEPTOR MULTIMERIZATION
    TERRY FRANCIS DAVIES; Fiscal Year: 2013
    ....
  4. DEVELOPMENT AND CONTROL OF PULMONARY ALVEOLAR STABILITY
    Samuel Hawgood; Fiscal Year: 2013
    ..abstract_text> ..