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

James JR, White SS, Clarke RW, Johansen AM, Dunne PD, Sleep DL, Fitzgerald WJ, Davis SJ, Klenerman D. (2007), Proc Natl Acad Sci U S A. 104, 17662-7

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.

Key figure: Control experiments confirming single-molecule level fluorescence detection

(A) V_β8-KMAC92.6 cells incubated with Alexa488-labeled anti-TCRβ Fab fragments (nonbinding, blue trace) and anti-CD3ε Fab fragments (binding, red trace). Fluorescence bursts are only observed for the anti-CD3ε Fab, indicating that the bursts result from the binding of the fluorescent Fabs to their target antigens at the cell surface. (B) A DO11.10 cell incubated with Alexa488-labeled anti-TCRβ Fab fragments was scanned across the diameter of the cell by using a scan rate of 1 μm/25 s. Fluorescence bursts are only observed at the perimeter of the cell. (C) A DO11.10 cell stained with the DiO membrane probe; single-molecule fluorescence bursts are observed at the same focus height (15 μm) as the bursts detected in Fig. 2 B. (D) Fluorescence from Yae5B3K cells incubated with Alexa647-labeled anti-CD3ε Fab fragments and excited at different laser powers. No differences in the bursts was observed, beyond a 50% reduction in overall fluorescence, for cells illuminated with the laser power routinely used (1 μW; dark-blue trace) versus half that typically used (0.5 μW; blue trace), suggesting that optical trapping effects are absent.