Jhawar, A. | Search bioRxiv

bioRxiv2026

Revealing the spatiotemporal dynamics of methionine metabolism with a genetically encoded single-fluorophore biosensor

Methionine is an essential amino acid, used for protein synthesis, redox homeostasis, and methylation reactions throughout the cell. However, the compartmentalized dynamics of methionine have remained elusive, due to a lack of available tools to measure methionine with high spatial and temporal resolution. To address this limitation, we have developed a single fluorescent protein-based methionine optical reporter (Meteor) which reports subcellular changes in methionine with high dynamic range. Using Meteor, we demonstrate the subcellular uptake of methionine in multiple cell lines into several locations, including the mitochondrial matrix. Furthermore, we use Meteor to illuminate the dynamics of the methionine cycle in the cytoplasm and nucleus, finding cancer cells can rapidly increase methionine from metabolic precursors in both locations. Finally, demonstrated that Meteor can be used to visualize methionine dynamics in vivo using Caenorhabditis elegans. Thus, we have developed a new tool to measure methionine dynamics across scales with high dynamic range and spatiotemporal resolution.

biochemistry↗

bioRxiv2025

Spatial regulation of AMPK activity under oxidative stress requires LKB1

AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, with over 100 identified downstream targets throughout the cell. In response to cellular stress, including energetic stress, AMPK is activated via binding of AMP and phosphorylation by upstream kinases, including liver kinase B1 (LKB1). We and others have found that the activation of AMPK in response to cellular stress has distinct subcellular mechanisms, indicating compartmentalized regulation of AMPK signaling. Oxidative stress is known to stimulate AMPK activity, but how AMPK is spatially regulated by oxidative stress is underexplored. Using a single-fluorophore excitation-ratiometric AMPK activity reporter (ExRai AMPKAR), we find that oxidative stress induced by hydrogen peroxide (H2O2) results in AMPK activity with distinct spatiotemporal dynamics. We found that in the cytoplasm, nucleus, outer mitochondrial membrane, and cytosolic lysosomal surface, phosphorylation of AMPK by LKB1 is required for AMPK activity. Using a biosensor for ATP, we found at the cytoplasm and lysosome local ATP depletion dictates kinetics of AMPK activity. Using a multi- omics approach, we discover that in response to oxidative stress, AMPK mediates significant metabolic and gene expression changes, including upregulation of oxidative stress response through nuclear factor erythroid 2-related factor 2 (NRF2). Expanding on this identified mechanism, we find that non-small cell lung cancers harboring Kelch-like ECH-associated protein 1 (KEAP1) mutations have a functionally deficient LKB1-AMPK signaling network in response to oxidative stress. Altogether, this work provides new insights into how the subcellular environment influences localized AMPK activity, and identifies how AMPK regulates the cellular response to oxidative stress.

biochemistry↗