De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling.

The enzymatic activity of the Src family tyrosine kinase p56Lck (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr394 and Tyr505 Phosphorylation of Tyr394 and the conformational opening of Lck are believed to activate the kinase, whereas Tyr505 phosphorylation is thought to generate a closed, inactive conformation of Lck. We investigated whether the conformation of Lck and its phosphorylation state act in concert to regulate the initiation of T cell receptor (TCR) signaling. With a sensitive biosensor, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the conformations of wild-type Lck and its phosphorylation-deficient mutants Y394F and Y505F and the double mutant Y394F/Y505F in unstimulated T cells and after TCR stimulation. With this approach, we separated the conformational changes of Lck from the phosphorylation state of its regulatory tyrosines. We showed that the conformational opening of Lck alone was insufficient to initiate signaling events in T cells. Rather, Lck additionally required phosphorylation of Tyr394 to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr394 occurred preferentially at the plasma membrane of Jurkat cells and primary human T cells. Our study supports the hypothesis that T cell activation through the TCR complex is accompanied by the de novo activation of Lck and that phosphorylation of Tyr394 plays a role in Lck function that goes beyond inducing an open conformation of the kinase.

Wide-field fluorescence lifetime imaging with multi-anode detectors.

Fluorescence lifetime imaging microscopy (FLIM) has become a powerful and widely used tool to monitor inter- and intramolecular dynamics of fluorophore-labeled proteins inside living cells.Here, we present recent achievements in the construction of a positional sensitive wide-field single-photon counting detector system to measure fluorescence lifetimes in the time domain and demonstrate its usage in FRET applications.The setup is based on a conventional fluorescence microscope equipped with synchronized short-pulse lasers that illuminate the entire field of view at minimal invasive intensities, thereby enabling long-term experiments of living cells. The system is capable to acquire single-photon counting images and measures directly the transfer rate of fast photophysical processes as, for instance, FRET, in which it can resolve complex fluorescence decay kinetics.

Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.