Esser, J. | bioRxiv Science

bioRxiv2026

ATPLyzer An advanced ratiometric multi-colour biosensor for long-term monitoring of ATP dynamics

Adenosine triphosphate (ATP) is a central molecule in cellular metabolism, serving as the primary energy currency that links catabolic and anabolic pathways. Monitoring intracellular ATP in vivo is essential for understanding the dynamics of metabolic states, as well as intracellular functions and intercellular interactions in health and disease. We report the design and application of ATPLyzer, a series of genetically encoded, ratiometric biosensors for the monitoring of ATP levels in living cells. The matryoshka design consists of an ATP-binding cassette linked to a circularly permutated GFP coupled with an internal large stokes shift reference fluorophore, allowing for single-wavelength excitation and ratiometric output. This design overcomes limitations of conventional biosensors, reliance on dual excitation wavelengths, and susceptibility to photobleaching. Multi-colour ATPLyzer variants with different dissociation constants were characterized in vitro, exhibiting high specificity for ATP over ADP. Monitoring ATP in Escherichia coli confirmed in vivo utility and revealed growth-phase and carbon-supply-dependent ATP dynamics. The ATPLyzer biosensor offers a robust and tuneable tool for minimally invasive, time-resolved monitoring of intracellular ATP dynamics.

bioengineering↗

bioRxiv2025

DNA-binding-independent mechanisms of metabolic regulation by the Drosophila FOXO transcription factor

Forkhead box-O (FOXO) transcription factors are evolutionarily conserved regulators of several biological processes, including development, stress responses, metabolism and ageing. As downstream effectors of nutrient-dependent cell signalling pathways, including insulin/IGF signalling, they integrate signals from multiple stimuli to orchestrate appropriate transcriptional responses to changes in the nutritional environment. Traditionally, FOXO-dependent responses have been attributed to target gene regulation through direct interactions with regulatory regions by DNA-binding via the conserved Forkhead (FH) domain. However, emerging evidence suggests that FOXO proteins may also influence gene expression through DNA-binding-independent mechanisms. However, differences in transcriptional outputs between DNA-binding dependent and independent FOXO functions have yet to be explored. Here, we have used genomic engineering of the endogenous Drosophila foxo locus to disrupt the DNA-binding activity of the single fly FOXO orthologue, allowing us to dissect the in vivo contributions of canonical and non-canonical dFOXO functions. We show that while DNA-binding is essential for several dFOXO-mediated phenotypes including female fecundity, lifespan, and resistance to oxidative and xenobiotic stress, other traits such as adult body size and survival during starvation remain intact. Notably, DNA-binding-deficient dFOXO flies exhibit defective lipid mobilisation under starvation, implicating a DNA-binding-independent role for dFOXO in metabolic regulation. Differential gene expression analysis during starvation in these mutants revealed key transcriptional changes in genes encoding metabolic regulators as well as regulators of transcription and chromatin structure. Together, these findings reveal distinct modes of dFOXO transcriptional regulation that depend on its direct association with DNA.

genetics↗