Genetically Encoded FRET Biosensor for Visualizing EphA4 Activity in Different Compartments of the Plasma Membrane

The EphA4 receptor tyrosine kinaseis well-known for its pivotal role in development, cancer progression, and neurological disorders. However, how EphA4 kinase activity is regulated in time and space still remains unclear. To visualize EphA4 activity in different membrane microdomains, we developed a sensitive EphA4 biosensorbased on Forster resonance energy transfer( FRET), and targeted it in or outside raft-like microdomains in the plasma membrane. We showed that our biosensorcan produce a robust and specific FRETresponse upon EphA4 activation, both in vitro and in live cells. Interestingly, we observed stronger FRETresponses for the non- rafttargeting biosensorthan for the rafttargeting biosensor, suggesting that stronger EphA4 activation may occur in non- raftregions. Further investigations revealed the importance of the actin cytoskeleton in suppressing EphA4 activity in raft-like microdomains. Therefore, our FRET-based EphA4 biosensorcould serve as a powerful tool to visualize and investigate...