Spatial and Temporal Dynamics of T Cell Receptor Signaling with a Photoactivatable Agonist

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Immunity (Impact Factor: 21.56). 08/2007; 27(1):76-88. DOI: 10.1016/j.immuni.2007.05.017
Source: PubMed


The precise timing of signals downstream of the T cell receptor (TCR) is poorly understood. To address this problem, we prepared major histocompatibility complexes containing an antigenic peptide that is biologically inert until exposed to ultraviolet (UV) light. UV irradiation of these complexes in contact with cognate T cells enabled the high-resolution temporal analysis of signaling. Phosphorylation of the LAT adaptor molecule was observed in 4 s, and diacylglycerol production and calcium flux was observed in 6-7 s. TCR activation also induced cytoskeletal polarization within 2 min. Antibody blockade of CD4 reduced the intensity of LAT phosphorylation and the speed of calcium flux. Furthermore, strong desensitization of diacylglycerol production, but not LAT phosphorylation, occurred shortly after TCR activation, suggesting that different molecular events play distinct signal-processing roles. These results establish the speed and localization of early signaling steps, and have important implications regarding the overall structure of the network.

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Available from: Joycelyn M. Faraj, Jan 29, 2015
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    • "This study of pTyr site dynamics has revealed processes that have been systematically overlooked in the past because of the speed with which they occur. We have monitored the phosphosite dynamics of early TCR signaling with finer temporal resolution than in previous proteomic studies of TCR signaling (see Table S4 in File S2 and references cited therein) and with greater breadth than earlier studies of early TCR signaling events employing relatively low-throughput assays [37], [38], and we developed a mechanistic model for TCR signaling that reproduces measured time courses of phosphorylation for a greater number of specific sites than previously developed models for immunoreceptor signaling (see Table S5 in File S2 and references cited therein). "
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    • "We next examined whether the V3 linker might also influence the kinetics of nPKC recruitment. For this purpose, we utilized a TCR photoactivation and imaging assay based on a caged form of the MCC-I-Ek complex that is nonstimulatory until irradiated with UV light [19]. 5C.C7 T cells are attached to coverslips coated with this caged pMHC. "
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