Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.

Herein, we show that a far-red arylidene-squaraine dye is stable against nucleophiles, in contrast to arene-squaraines. Owing to the fluorescence enhancement in apolar media together with high brightness and photostability, this dye was successfully applied to detect the oxytocin G protein-coupled receptor and monitor its internalization in living cells.

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four "EF hand" type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.