title: Visualization of TCR nanoclusters via immunogold labeling, freeze-etching, and surface replication.
abstract: T cells show high sensitivity for antigen, even though their T-cell antigen receptor (TCR) has a low affinity for its ligand, a major histocompatibility complex molecule presenting a short pathogen-derived peptide. Over the past few years, it has become clear that these paradoxical properties rely at least in part on the organization of cell surface-expressed TCRs in TCR nanoclusters. We describe a protocol, comprising immunogold labeling, cell surface replica generation, and electron microscopy (EM) analysis that allows nanoscale resolution of the distribution of TCRs and other cell surface molecules of cells grown in suspension. Unlike most of the light microscopy-based single-molecule resolution techniques, this technique permits visualization of these molecules on cell surfaces that do not adhere to an experimental support. Given the potential of adhesion-induced receptor redistributions, our technique is a relevant complement to the substrate adherence-dependent techniques. Furthermore, it does not rely on introduction of fluorescently labeled recombinant molecules and therefore allows direct analysis of nonmanipulated primary cells.
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies, Albert Ludwigs University Freiburg, Freiburg, Germany; Centre for Chronic Immunodeficiency (CCI), University Clinics Freiburg and Medical Faculty, Albert Ludwigs University Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg, Freiburg, Germany.
authors.forename: Gina J
authors.initials: GJ
authors.lastname: Fiala
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Rejas
authors.forename: Wolfgang W
authors.initials: WW
authors.lastname: Schamel
authors.forename: Hisse M
authors.initials: HM
authors.lastname: van Santen
mesh_terms: D000538:Aluminum Silicates / Q000737:chemistry; D001402:B-Lymphocytes / Q000378:metabolism / Q000648:ultrastructure*; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D005613:Freeze Etching; D015870:Gene Expression; D017979:Gold Colloid / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D046529:Microscopy, Electron, Transmission; D055503:Protein Multimerization; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods*; D013499:Surface Properties; D013601:T-Lymphocytes / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D000538:Aluminum Silicates; D017979:Gold Colloid; D011948:Receptors, Antigen, T-Cell; C011934:mica
keywords: Cell surface proteins; Cell surface replicas; Immunogold labeling; Receptor clustering; T cell receptor; Transmission electron microscopy