RT Journal Article
SR Electronic
T1 Optimised FRET pairs and quantification approaches to detect the activation of Aurora kinase A at mitosis
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 562603
DO 10.1101/562603
A1 Giulia Bertolin
A1 Florian Sizaire
A1 Claire Déméautis
A1 Catherine Chapuis
A1 Fabienne Mérola
A1 Marie Erard
A1 Marc Tramier
YR 2019
UL http://biorxiv.org/content/early/2019/05/13/562603.abstract
AB Genetically-encoded Förster’s Resonance Energy Transfer (FRET) biosensors are indispensable tools to sense the spatiotemporal dynamics of signal transduction pathways. Investigating the crosstalk between different signalling pathways is becoming increasingly important to follow cell development and fate programs. To this end, FRET biosensors must be optimised to monitor multiple biochemical activities simultaneously and in single cells. In addition, their sensitivity must be increased to follow their activation even when the abundance of the biosensor is low.We describe here the development of a second generation of Aurora kinase A/AURKA biosensors. First, we adapt the original AURKA biosensor –GFP-AURKA-mCherry– to multiplex FRET by using dark acceptors as ShadowG or ShadowY. Then, we use the novel superYFP acceptor protein to measure FRET by 2-colour Fluorescence Cross-Correlation Spectroscopy, in cytosolic regions where the abundance of AURKA is extremely low and undetectable with the original AURKA biosensor.These results pave the way to the use of FRET biosensors to follow AURKA activation in conjunction with substrate-based activity biosensors. In addition, they open up the possibility of tracking the activation of small pools of AURKA and its interaction with novel substrates, which would otherwise remain undetectable with classical biochemical approaches.