title: Reconstitution of TCR Signaling Using Supported Lipid Bilayers.
abstract: Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro. Our reconstitution demonstrates the sufficiency of these proteins in producing many of the complex behaviors observed during T cell activation. The ability to manipulate all of the components, measure reaction rates, and observe molecular behaviors, including at single molecule resolution, has enabled us to gain insight into some of the important biochemical features of this signaling pathway such as microcluster formation. The same system could be adapted to study other membrane-proximal signaling pathways, including growth factor receptors, death receptors, and Eph receptors.
authors.affiliation: Marine Biological Laboratory, The HHMI Summer Institute, Woods Hole, MA, 02543, USA.|Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California, San Francisco, CA, 94158, USA.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Su
authors.affiliation: Marine Biological Laboratory, The HHMI Summer Institute, Woods Hole, MA, 02543, USA.|Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Jonathon A
authors.initials: JA
authors.lastname: Ditlev
authors.affiliation: Marine Biological Laboratory, The HHMI Summer Institute, Woods Hole, MA, 02543, USA.|Department of Biophysics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Rosen
authors.affiliation: Marine Biological Laboratory, The HHMI Summer Institute, Woods Hole, MA, 02543, USA. ron.vale@ucsf.edu.|Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California, San Francisco, CA, 94158, USA. ron.vale@ucsf.edu.
authors.forename: Ronald D
authors.initials: RD
authors.lastname: Vale
mesh_terms: D000818:Animals; D002474:Cell-Free System / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000276:immunology; D008213:Lymphocyte Activation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000276:immunology; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000737:chemistry* / Q000276:immunology
chemical_list: D008051:Lipid Bilayers; D011948:Receptors, Antigen, T-Cell
keywords: Actin; LAT; Microcluster; Multivalency; Phase separation; Reconstitution; Supported lipid bilayer; TCR