Visualisation of signalling in immune cells.

Currently, a great number of approaches are employed in investigation of the immune system. These range from experiments in live animals and biochemical techniques to investigate whole organs or cell populations down to single cell and molecular techniques to look at dynamics in specific cell-cell interactions. It is the latter approach that this chapter focusses on. The use of Förster resonance energy transfer (FRET) techniques to probe protein-protein interactions that are involved in receptor signalling to the cytoskeleton in intact cells is now well established. Various FRET biosensors are available to visualise several critical cell processes, giving information about activity and the location of key signalling molecules. As a specific set of examples in this chapter, we have generated variants of the original Rho, Rac and Cdc42 "Raichu" probes and improved their fluorophore combination to make them suitable for FLIM. These were employed in a number of assays to determine signal dynamics in T and NK cells. Specific protocols of how to use these probes and technical notes are described.

Integrating receptor signal inputs that influence small Rho GTPase activation dynamics at the immunological synapse.

The Rho GTPase Cdc42 regulates cytoskeletal changes at the immunological synapse (IS) that are critical to T-cell activation. By imaging fluorescent activity biosensors (Raichu) using fluorescence lifetime imaging microscopy, Cdc42 activation was shown to display kinetics that are conditional on the specific receptor input (through two IS-associated receptors, CD3 and beta1 integrin). CD3-triggered Cdc42 activity is dependent on the cyto-2 (NPIY) motif of the beta1 integrin cytoplasmic domain. Perturbations of the ezrin-radixin-moesin (ERM) function blocked CD3- and beta1-dependent increases in Cdc42 activity. Both IS-associated receptors probably lie on a serial molecular pathway and transduce signals through the ERM-dependent machinery that is responsible for the remodeling and stabilization of the synapse. Cdc42 activity is impaired in beta1 integrin-deficient T cells that form conjugates with antigen-presenting cells but is partially restored in the context of an antigen-specific synapse. This restoration of Cdc42 activity is due, at least in part, to the recruitment and activation of beta2 integrin.

satFRET: estimation of Förster resonance energy transfer by acceptor saturation.

We demonstrate theoretically and experimentally the quantification of Förster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as "satFRET" is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546- Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.