Chemical inhibitors when timing is critical: a pharmacological concept for the maturation of T cell contacts.
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.
Köhler
Karsten
K
Group of Cellular Signalling, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
Lellouch
Annemarie C
AC
Vollmer
Susanne
S
Stoevesandt
Oda
O
Hoff
Antje
A
Peters
Lasse
L
Rogl
Hans
H
Malissen
Bernard
B
Brock
Roland
R
D000818:Animals; D017252:CD3 Complex / Q000502:physiology; D006801:Humans; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000037:antagonists & inhibitors / Q000502:physiology; D018613:Microscopy, Confocal / Q000379:methods; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000201:enzymology* / Q000276:immunology; D013997:Time Factors; D051746:ZAP-70 Protein-Tyrosine Kinase
D017252:CD3 Complex; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human