Automated screening of AURKA activity based on a genetically encoded FRET biosensor using fluorescence lifetime imaging microscopy

Florian Sizaire (1) , Gilles Le Marchand (1) , Jacques Pécréaux (1) , Otmane Bouchareb (2) , Marc Tramier (1, 3)

1 IGDR - Institut de Génétique et Développement de Rennes
2 Inscoper
3 SFR UMS CNRS 3480 - INSERM 018 - Biosit : biologie, santé, innovation technologique

Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust tool to measure Förster Resonance Energy Transfer (FRET) between two fluorescent proteins, mainly when using genetically-encoded FRET biosensors. It is then possible to monitor biological processes such as kinase activity with a good spatiotemporal resolution and accuracy. Therefore, it is of interest to improve this methodology for future high content screening purposes. We here implement a time-gated FLIM microscope that can image and quantify fluorescence lifetime with a higher speed than conventional techniques such as Time-Correlated Single Photon Counting (TCSPC). We then improve our system to perform automatic screen analysis in a 96-well plate format. Moreover, we use a FRET biosensor of AURKA activity, a mitotic kinase involved in several epithelial cancers. Our results show that our system is suitable to measure FRET within our biosensor paving the way to the screening of novel compounds, potentially allowing to find new inhibitors of AURKA activity.

Keywords: Fluorescence lifetime; FRET biosensor; Screening; AURKA 25

Domains: Life Sciences [q-bio]

Florian Sizaire, Gilles Le Marchand, Jacques Pécréaux, Otmane Bouchareb, Marc Tramier. Automated screening of AURKA activity based on a genetically encoded FRET biosensor using fluorescence lifetime imaging microscopy. Methods and Applications in Fluorescence, 2020, 8 (2), pp.024006. ⟨10.1088/2050-6120/ab73f5⟩. ⟨hal-02500623⟩