A combination of fluorescence microscopy and chemical inhibitors reveals the timing of early T cell receptor-dependent signal transduction

Karsten Köhler#, Annemarie Lellouch§, Susanne Vollmer#, Bernard Malissen§, Roland Brock#
#Group of Cellular Signal Transduction, Interfaculty Institute for Cell Biology, University of Tübingen, §Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranée- Case 906, 13288 Marseille cedex, France

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 specific cellular responses. Here 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 to define the timing of signalling events. Inhibitors of Src-family tyrosine kinases and actin reorganization were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent T cell contacts. Anti-CD3-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 to T cell receptor complexes was detected by high resolution confocal microscopy. The analyses revealed that at 5 min after receptor engagement Lck activity was required for maintenance of contacts and ZAP-70 clusters. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. Disruption of the dynamics of the actin cytoskeleton inhibited cell spreading, however, was without effect on cell attachment. In summary, the results indicate that in the early phases of signalling kinases act on the CD3 complex in a way that modulates the avidity of these complexes for a polyvalent ligand similar to the one described for integrin-mediated cell adhesion. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.

References: Köhler et al., ChemBioChem, 6 (2005) 152-161