Biology subjects

Kapelczak, E. D.

Publications and source records attributed to Kapelczak, E. D..

4 recordsLinked to original sources

bioRxiv2026

Flexibility of systemic one-carbon metabolism partially buffers dietary methyl donor deficiency

Choline is a methyl-rich nutrient used in lipid synthesis and catabolized to support one-carbon metabolism. Choline consumption in most humans remains less than the suggested adequate intake, yet how systemic metabolism compensates for choline deficiency is not fully described. Here, we use in vivo stable isotope tracing to explore the fate of choline in mammalian tissues. We find that choline is catabolized in the liver to support both the methionine cycle and the mitochondrial folate cycle in addition to its role in lipid synthesis. When dietary methyl donors are deficient, surprisingly, we find maintained systemic choline and methionine fluxes, but diminished contribution of choline to the folate cycle. To compensate for dietary methyl deficiency, systemic flux of serine is doubled by increased kidney synthesis which supplies one-carbon units for increased methionine synthesis in the liver. Our study suggests that systemic one-carbon flexibility can compensate for nutritional methyl deficiency by inter-organ nutrient exchange.

physiology↗

bioRxiv2026

Optical metabolic imaging of the tricarboxylic acid cycle

The tricarboxylic acid (TCA) cycle lies at the core of cellular metabolism, integrating energy production, biosynthesis and redox homeostasis, yet direct quantitative imaging of its activity in living systems remains challenging. Here we introduce MATRIX-SRS (Metabolic Activity TRacing of the trIcarboXylic acid cycle by Stimulated Raman Scattering microscopy), a platform enabling spatially resolved quantification of TCA-linked metabolism in live cells. Using emerging deuterium-labeled probes, MATRIX-SRS visualizes subcellular TCA-associated carbon-deuterium bonds in live cancer cells and neurons. We then integrate density functional theory, reaction network mapping, and hyperspectral MATRIX-SRS to construct a robust in situ metabolic quantification pipeline. Integrating MATRIX-SRS with isotope-tracing mass spectrometry, we reveal a global attenuation of TCA activity during epithelial-to-mesenchymal transition, providing deep molecular insights. Applying this framework, we further quantify changes in deuterium-labeled biomass in absolute concentrations for the first time, under native and drug-treated conditions, establishing a generalizable foundation for live quantitative spatial metabolomics.

biochemistry↗

bioRxiv2026

Illuminating spatial dynamics of glutamine metabolism with a sensitive genetically encoded biosensor

Glutamine is the most abundant amino acid in serum, used as a key nutrient by cells for protein synthesis, energy production, carbon and nitrogen metabolism, and cellular redox balance. The use of glutamine in the cell is highly compartmentalized, but the dynamics of glutamine metabolism across organelles and individual cells are not fully understood. To illuminate subcellular glutamine dynamics, we developed a green fluorescent protein-based intracellular glutamine optical reporter, iGlo. We find iGlo is sensitive and specific for glutamine and can be used to measure glutamine uptake, production, and consumption with high spatiotemporal resolution in multiple cell types. Furthermore, multiplexed imaging of iGlo with a lactate biosensor in single cells reveals the temporal crosstalk between glucose and glutamine metabolism to maintain energy homeostasis. Thus, iGlo enables the sensitive and precise study of compartmentalized glutamine dynamics and represents a new and enhanced tool for studying the spatiotemporal dynamics and regulation of metabolism.

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↗