Chemical inhibitors when timing is critical: a pharmacological concept for the maturation of T cell contacts |
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Authors: | Köhler Karsten Lellouch Annemarie C Vollmer Susanne Stoevesandt Oda Hoff Antje Peters Lasse Rogl Hans Malissen Bernard Brock Roland |
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Affiliation: | Group of Cellular Signalling, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany. |
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Abstract: | Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts. |
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