James, John R., White, S. S., Clarke, R. W., Johansen, Adam M., Dunne, P. D., Sleep, D. L., Fitzgerald, W. J., Davis, S. J. and Klenerman, D. (2007) Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells. Proceedings of the National Academy of Sciences, Vol.104 (No.45). pp. 17662-17667. doi:10.1073/pnas.0700411104

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Abstract

The T cell receptor (TCR) expressed on most T cells is a protein complex consisting of TCRαβ heterodimers that bind antigen and cluster of differentiation (CD) 3εδ, εγ, and ζζ dimers that initiate signaling. A long-standing controversy concerns whether there is one, or more than one, αβ 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-TCRβ 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 αβ 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.

Item Type:	Journal Article
Divisions:	Faculty of Science > Statistics
Journal or Publication Title:	Proceedings of the National Academy of Sciences
ISSN:	0027-8424
Official Date:	2007
Dates:	Date Event 2007 Published
Volume:	Vol.104
Number:	No.45
Page Range:	pp. 17662-17667
DOI:	10.1073/pnas.0700411104
Status:	Peer Reviewed
Access rights to Published version:	Restricted or Subscription Access

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