Determining physicochemical parameters in living cells requires methods adapted to the complexity and constraints of biological environments. We are working on developing robust, quantitative, and multimodal imaging techniques that rely on a thorough understanding of the underlying photophysical processes and the development of dedicated instruments, such as the FLIM BI-FLUOR microscope available at the SpICy platform.

In particular, FRET between two fluorescent proteins fused to proteins of interest allows us to visualize their interaction and gain insight into the topology or stoichiometry of this interaction within the cellular environment. We detect FRET by measuring the fluorescence lifetime of the donor partner in imaging mode (FLIM). This strategy, which enables quantitative FRET analysis, is undergoing a revival thanks to new generation of detectors and image analysis methods.

We have developed a strategy that combines FRET-FLIM and FCCS to study the NADPH oxidase of phagocytes. This enzyme produces superoxide anions, a precursor to reactive oxygen species (ROS) that are essential for host responses to microbial infections. It consists of two membrane subunits (NOX2 and p22) and three cytosolic subunits (p40, p47, and p67). We have characterized the intermolecular and intramolecular interactions of the cytosolic subunits and elucidated their conformation, stoichiometry, interaction fraction, and affinities in living cells. Additionally, by combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we constructed a 3D model of the entire cytosolic complex [Ziegler2019, Valenta2022].

We are developing new projects to detect membrane contact sites (postdoc M. Abdesselem, thesis D. Zamiati, ANR ApicolipidTraffic 2023-2027).