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From T-Cell Antigen Receptor Engagement to Cytoskeleton Reorganization
In the present study, we focused on the dynamic interactions that occur between signaling molecules crucial for translating TCR engagement into localized actin polymerization. This goal was accomplished by using advanced molecular imaging techniques to observe T-cell spreading at the level of single activated T cells. During these events, we characterized the dynamic localization of the proteins regulating actin polymerization and demonstrated the molecular interactions involved in this process (Figure 1). We defined protein-protein interactions by performing quantitative characterization at the Angstrom level using fluorescence resonance energy transfer (FRET) techniques. Figure 1. Molecular interactions leading to T-cell receptor (TCR)–induced actin polymerization. T-cell activation is initiated by antigen presenting cells (APCs) containing stimulatory major histocompatibility complex (MHC)–peptide complexes. Phosphorylation of the TCR (black circles) is mediated by Src family protein tyrosine kinases. ZAP-70 is recruited to the phosphorylated TCR subunits through its SH2 domain. It is phosphorylated and activated by the Src family protein tyrosine kinases. The linker for the activation of T cells (LAT) is tyrosine phosphorylated by ZAP-70. LAT contains nine tyrosine residues that, when phosphorylated, act as docking sites for adapter proteins such as Grb2 and Gads. SLP-76 is recruited by the LAT-nucleated complex through its interaction with the SH3 domains of Gads. SLP-76 associates with the SH2 domain of Nck. Nck binds WASp, which in turn binds the Arp2/3 complex that mediates actin polymerization. Our studies demonstrated that actin polymerization began at the site of TCR engagement and then migrated to the cellular periphery. Actin polymerization was driven by the Wiskott-Aldrich Syndrome protein (WASp) and was dependent on its dynamic localization. We demonstrated the critical role of TCR-induced tyrosine phosphorylation of the adaptor proteins LAT and SLP-76 in recruiting other adaptors, Nck and WASp, that are required for actin polymerization. The use of mutant T-cell lines lacking LAT or SLP-76 revealed a complexity in the mechanism of actin polymerization. In LAT-deficient cells, Nck and WASp did not cluster at the TCR. Nck also failed to cluster at the TCR in SLP-76–deficient cells. Reconstitution of these cells with the appropriate cDNA (LAT or SLP-76) restored normal recruitment of the tagged molecules at the TCR. However, reconstitution of LAT-deficient cells with a mutant form of LAT lacking four critical tyrosine residues, the sites of important phosphorylation, failed to reconstitute Nck recruitment. In the deficient cells, analysis of actin status revealed significant, albeit incomplete, inhibition of polymerization. The role of Nck in T-cell antigen receptor action has recently received much attention. Gil et al. (Cell 109: 901–12, 2002) reported experiments indicating that Nck directly binds TCR/CD3ε chains upon TCR engagement due to induced exposure of proline-rich sequences within the CD3ε. Recruitment of Nck to CD3ε was found to precede phosphorylation of it and other TCR subunits. Also, this binding was found to be independent of SLP-76. The cellular reagents used in our current study enabled us to test these observations. Two of our results contradicted the study. First, the Src kinase inhibitor PP2 was used to block proximal tyrosine phosphorylation. Despite TCR-induced clustering, no activated ZAP-70, SLP-76, or Nck was detected in clusters at the cell membrane. We attribute this failure of recruitment to the lack of TCR phosphorylation induced by Lck or Fyn, the consequent lack of ZAP-70 recruitment and activation, followed by the failure of any ZAP-70 SH2–mediated interactions. Since our means of activation is via CD3ε-mediated clustering of TCRs, the failure of Nck recruitment argues against recruitment being dependent on CD3ε conformational changes exposing Nck binding sites. Also, the expression of YFP-Nck in the SLP-76–deficient cells was used to test whether SLP-76 is necessary for Nck recruitment. In the absence of SLP-76, Nck was not recruited to the TCR, whose activation was confirmed by the presence of pZAP-70 at the TCR. Restoration of Nck clustering in these cells was demonstrated following reexpression of SLP-76. Thus, Nck recruitment in our studies requires tyrosine phosphorylation and SLP-76. To confirm that the same protein recruitment and interactions occur in normal, non-transformed cells, we adapted the assay to human peripheral blood lymphocytes. Though the kinetics were slower, the pattern of protein localization in these cells was similar to that observed with Jurkat cells. Nck and WASp were initially recruited to the TCR, where they co-localized with activated ZAP-70 and then migrated to the periphery where they accumulated at an actin-rich circumferential ring. In conclusion, advances in confocal microscopy and imaging techniques, such as FRET and time-resolved imaging of fluorescent chimeras, enabled our study of actin polymerization and reorganization at the TCR. This process plays a major role in immunological synapse formation and amplification of the immune response. |
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the T-cell receptor (TCR) binds foreign antigens on antigen presenting cells
(APC), multiple proteins redistribute to form the immunological synapse. Organization
of the synapse and the creation of a tight seal between the T cell and APC depend
on actin polymerization. Studies using biochemical and imaging techniques have
shown the formation of signaling assemblies at the TCR; however, studies of
the molecular interactions linking the TCR to the cytoskeleton proteins in live
cells have been rare.