Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells

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.