Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells - Université de Rennes Accéder directement au contenu
Article Dans Une Revue Journal of visualized experiments : JoVE Année : 2020

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Résumé

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded Förster's Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.
Fichier principal
Vignette du fichier
Bertolin et al_Revised.pdf (1.25 Mo) Télécharger le fichier
Origine : Fichiers produits par l'(les) auteur(s)

Dates et versions

hal-02929723 , version 1 (25-11-2020)

Identifiants

Citer

Giulia Bertolin, Gilles Le Marchand, Marc Tramier. Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells. Journal of visualized experiments : JoVE, 2020, 161, ⟨10.3791/61611⟩. ⟨hal-02929723⟩
164 Consultations
104 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More