Maximising FLIM-FRET Biosensor Capabilities for Cancer Studies


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Kirsty Martin1
1CRUK Beatson Institute

Abstract

Background

FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells, including that of the Ras family of GTPases, many of which are known for their critical role in cancer progression. Most biosensors designed to date have been predicated on fluorescent protein pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET.  

Method

Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last two decades, with lifetime imaging systems becoming increasingly accessible and user-friendly, and an entire field of biology dedicated to refining and adapting the different characteristics of existing and novel fluorescent proteins. 

Results

We have exploited this growing pool of fluorescents proteins, identifying long-lifetime mTurquoise2 and the dark acceptor sReACh as components that provide more reliable, reproducible and quantifiable FLIM-FRET data to improve the dynamic range and breadth of application of biosensor technologies. 

Conclusion

Application of these fluorophores to GTPase biosensors of different designs provides a more sensitive set of tools for the analysis of the Ras family in cancer cells, allowing identification of small changes in signal that have the potential to cause large changes in cellular behaviour.