Measuring AMPK Activity in Vivo Using a Genetically‐Encoded Biosensor

Abstract

Adenosine monophosphate‐activated kinase (AMPK) is a highly conserved heterotrimeric protein complex that functions in a broad spectrum of cellular stress response pathways. Most work investigating the cellular dynamics of AMPK activity has been limited to either whole‐organ/tissue or, in the case of smaller genetic models like Caenorhabditis elegans, whole‐organism analyses. Some limitations of these approaches include: 1) An inability to observe cellular heterogeneity of AMPK activation; 2) a lack of the temporal dynamics; 3) an inability to correlate AMPK dynamics with physiological states of a living organism. Thus, we sought to adapt a genetically‐coded AMPK biosensor, called AMPKAR‐EV (Kongaya et al., 2017), for use in C. elegans, which is transparent and genetically tractable. The AMPKAR‐EV biosensor uses fluorescence resonance energy transfer (FRET) to report AMPK phosphorylation of substrates. We expressed codon‐adapted AMPKAR‐EV in C. elegans and made three separate transgenic lines expressing it in intestines, body wall muscles, and neurons. As expected, we find that the biosensor responds to conditions that promote AMPK activation. We crossed the lines expressing AMPKAR‐EV in the intestines into a number of different genetic mutants, which we either generated using CRISPR or obtained from previous studies. For context, work in mammals suggests that protein kinase A (PKA) phosphorylates AMPK at serine 173 (S173 ‐ inhibitory site) and prevents catalytic phosphorylation at threonine 172 (T172 ‐ activation site). These sites are conserved to C. elegans but located at serine 244 (S244) and threonine 243 (T243) respectively; however, the significance of these regulatory sites have not been specifically studied. To test these mechanisms more precisely, we made two C. elegans strains that also express AMPKAR‐EV, one that alters the activation site (aak‐2(T243A)) and another that alters the inhibitory site (aak‐2(S244G)). The third strain we constructed expresses AMPKAR‐EV in the kin‐2(ce179) mutant background, which exhibits constitutively active PKA. Using these strains we are optimizing AMPKAR‐EV in vivo and testing the hypothesis that PKA inhibits AMPK at the S244 inhibitory site, which prevents its phosphorylation at the activation site. We envision that AMPKAR‐EV will be of broad use in C. elegans as an organism well‐known for its study of behavior, aging, and longevity.