title: Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells.
abstract: The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRalphabeta heterodimers that bind antigen and cluster of differentiation (CD) 3epsilondelta, epsilongamma, and zetazeta dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, alphabeta heterodimer per complex. We used a form of single-molecule spectroscopy to investigate this question on live T cell hybridomas. The method relies on detecting coincident fluorescence from single molecules labeled with two different fluorophores, as the molecules diffuse through a confocal volume. The fraction of events that are coincident above the statistical background is defined as the "association quotient," Q. In control experiments, Q was significantly higher for cells incubated with wheat germ agglutinin dual-labeled with Alexa488 and Alexa647 than for cells incubated with singly labeled wheat germ agglutinin. Similarly, cells expressing the homodimer, CD28, gave larger values of Q than cells expressing the monomer, CD86, when incubated with mixtures of Alexa488- and Alexa647-labeled antibody Fab fragments. T cell hybridomas incubated with mixtures of anti-TCRbeta Fab fragments labeled with each fluorophore gave a Q value indistinguishable from the Q value for CD86, indicating that the dominant form of the TCR comprises single alphabeta heterodimers. The values of Q obtained for CD86 and the TCR were low but nonzero, suggesting that there is transient or nonrandom confinement, or diffuse clustering of molecules at the T cell surface. This general method for analyzing the subunit composition of protein complexes could be extended to other cell surface or intracellular complexes, and other living cells.
authors.affiliation: Nuffield Department of Clinical Medicine and Medical Research Council Human Immunology Unit, The Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom.
authors.forename: John R
authors.initials: JR
authors.lastname: James
authors.forename: Samuel S
authors.initials: SS
authors.lastname: White
authors.forename: Richard W
authors.initials: RW
authors.lastname: Clarke
authors.forename: Adam M
authors.initials: AM
authors.lastname: Johansen
authors.forename: Paul D
authors.initials: PD
authors.lastname: Dunne
authors.forename: David L
authors.initials: DL
authors.lastname: Sleep
authors.forename: William J
authors.initials: WJ
authors.lastname: Fitzgerald
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
mesh_terms: D000818:Animals; D051940:B7-2 Antigen / Q000737:chemistry; D018106:CD28 Antigens / Q000737:chemistry; D019281:Dimerization; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000737:chemistry; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D051940:B7-2 Antigen; D018106:CD28 Antigens; D010446:Peptide Fragments; D021122:Protein Subunits; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta