The lymphocyte-specific Src family protein tyrosine kinase p56(Lck) (Lck) is essential for T cell development and activation and, hence, for adaptive immune responses. The mechanism by which Lck activity is directed toward specific substrates in response to T cell receptor (TCR) activation remains elusive. We used fluorescence lifetime imaging microscopy to assess the activation-dependent spatiotemporal changes in the conformation of Lck in live human T cells. Kinetic analysis of the fluorescence lifetime of Lck biosensors enabled the direct visualization of the dynamic local opening of 20% of the total amount of Lck proteins after activation of T cells with antibody against CD3 or by superantigen-loaded antigen-presenting cells. Parallel biochemical analysis of TCR complexes revealed that the conformational changes in Lck correlated with the induction of Lck enzymatic activity. These data show the dynamic, local activation through conformational change of Lck at sites of TCR engagement.

The enzymatic activity of the Src family tyrosine kinase p56 (Lck) is tightly controlled by differential phosphorylation of two tyrosine residues, Tyr and Tyr Phosphorylation of Tyr and the conformational opening of Lck are believed to activate the kinase, whereas Tyr 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 Tyr to induce T cell activation. Consistent with the FLIM measurements, an optimized immunofluorescence microscopy protocol revealed that the TCR-stimulated phosphorylation of Lck at Tyr 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 Tyr plays a role in Lck function that goes beyond inducing an open conformation of the kinase.

Glycolytic oscillations of intact yeast cells of the strain Saccharomyces carlsbergensis were investigated at both the levels of cell populations and of individual cells. Individual cells showed glycolytic oscillations even at very low cell densities (e.g. 1.0105 cells/ml). By contrast, the collective behaviour on the population level was cell density-dependent: at high cell densities it is oscillatory, but below the threshold density of 1.0106 cells/ml the collective dynamics becomes quiescent. We demonstrate that the transition in the collective dynamics is caused by the desynchronisation of the oscillations of individual cells. This is characteristic for a Kuramoto transition. Spatially resolved measurements at low cell densities revealed that even cells that adhere to their neighbours oscillated with their own, independent frequencies and phases.

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.

We present a detector system with a microchannel plate based photomultiplier tube (MCP-PMT) and its application for fluorescence lifetime imaging (FLIM) in visible light. A capacity coupled imaging technique (charge image) combined with a charge division anode is employed for the positional readout. Using an artificial neural network's (ANN) computation model we are able to reconstruct the position of the incident photon as precise as 20 microns over the detector active area of 25 mm diameter. Thus, the resulting image quality corresponds roughly to a megapixel conventional CCD camera. Importantly, it is feasible to reach such resolution using only 9 charge acquisition channels supporting the anode structure of 14 interconnected readout electrodes. Additionally, the system features better than 50 ps temporal resolution allowing single photon counting FLIM acquisition with a regular fluorescence wide-field microscope.