| Autor: |
James, John R., White, Samuel S., Clarke, Richard W., Johansen, Adam M., Dunne, Paul D., David L. Sleep, Fitzgerald, William J., Davis, Simon J., Klenerman, David |
| Předmět: |
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| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; 11/6/2007, Vol. 104 Issue 45, p17662-17667, 6p, 1 Chart, 2 Graphs |
| Abstrakt: |
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) 3ϵϵδ, 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-Iabeled 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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