Research ResourceCell Biology

Studying the Dynamics of SLP-76, Nck, and Vav1 Multimolecular Complex Formation in Live Human Cells with Triple-Color FRET

Sci. Signal.  24 Apr 2012:
Vol. 5, Issue 221, pp. rs3
DOI: 10.1126/scisignal.2002423

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Watching Protein Complex Assembly

In response to the activation of the T cell receptor (TCR) on T cells, various kinases, adaptors, and signaling proteins assemble to form various multimolecular complexes to mediate T cell activation. The complex containing the adaptor proteins SLP-76 and Nck and the guanine nucleotide exchange factor Vav1 is required for the cytoskeletal rearrangements that occur in response to TCR stimulation. Pauker et al. developed an imaging technique (involving triple-color FRET analysis) to monitor the dynamics of the various interactions between SLP-76, Nck, and Vav1 in stimulated live T cells and observed formation of the tripartite complex. They found that Nck and Vav1 were preassembled as heterodimers before TCR stimulation and that mutation of a critical residue in Vav1 disrupted actin rearrangement. This study establishes the use of this technique in the investigation of other multiprotein complexes in live cells.