An Ultrasensitive Fluorescent Biosensor for Multi‐modal Kinase Activity Detection and High‐resolution Imaging in Awake Mice

Abstract

Fluorescent protein (FP)‐based kinase activity biosensors are powerful tools for probing the spatiotemporal dynamics of signaling pathways in living cells. Kinase activity imaging is frequently used to unravel the molecular regulation of Protein Kinase A (PKA), a prototypical kinase that integrates multiple pathways to orchestrate diverse physiological processes. Yet the limited sensitivity of most kinase biosensors restricts their reliable application in physiologically relevant in vivo systems or clinically useful high‐content detection modalities. We previously developed an excitation‐ratiometric PKA activity reporter (ExRai‐AKAR) which utilizes a PKA substrate peptide and phosphoamino acid‐binding forkhead‐associated 1 (FHA1) domain to modulate the peak excitation wavelength of cpEGFP in response to phosphorylation by PKA. Here, we report the further development of a new, ultrasensitive excitation‐ratiometric PKA activity reporter (ExRai‐AKAR2) obtained via high‐throughput linker library screening. This powerful and versatile molecular tool enables sensitive and rapid monitoring of live‐cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting, and one‐ or two‐photon imaging. In vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion. The performance of ExRai‐AKAR2 promises to successfully expand kinase activity imaging beyond the petri dish.Support or Funding InformationThis work was supported by Brain Initiative Grant R01 MH111516 and by R35 CA197622, R01 DK073368, and R01 GM111665 (to J.Z.).