Biology subjects

Hario, S.

Publications and source records attributed to Hario, S..

3 recordsLinked to original sources

bioRxiv2026

Ratiometric Fluorescent Protein Biosensors Reveal Citrate Dynamics and Cellular Heterogeneity

Citrate is a central intermediate metabolite linking the tricarboxylic acid cycle and lipid biosynthesis. Tools for monitoring of spatiotemporal citrate dynamics are critical for getting a better understanding of cellular metabolism. Here, we develope genetically encoded excitation ratiometric biosensors for citrate, based on our previous intensiometric green fluorescence protein-based citrate biosensor, Citron1. We find that a single mutation in the Citron1 chromophore-forming tripeptide provided an excitation ratiometric response. Further rounds of directed evolution yield highly responsive variants, exhibiting citrate-dependent fluorescence changes between two excitation peaks. When expressed in mammalian cells, these biosensors enable citrate dynamics to be monitored in both the cytosol and mitochondria. Comparative analysis across multiple human breast cancer cell lines uncovers cell line-specific differences in citrate levels and their heterogeneity, which could be linked to their malignancy. Furthermore, flow cytometry-based measurements in mouse embryonic stem cells demonstrate the proteomics signatures underlying the population-level variability in citrate concentrations and citrate rewiring during stem cell differentiation. Together, these results show that these excitation ratiometric citrate biosensors enable quantitative, compartment-resolved, and population-scale analysis of cellular metabolism.

molecular biology↗

bioRxiv2025

High-performance genetically-encoded green and red fluorescent biosensors for pyruvate

Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, a lack of high-performance (i.e., {Delta}F/F0 > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate the physiological role of pyruvate. Here, we present the GreenPy1 and ApplePy1 series, which are green FP (GFP)-based and red FP (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change ({Delta}F/F0 [~] 20 to >40) and a range of affinities (10s of M to several mM). We demonstrate the utility of these pyruvate biosensors for multicolor imaging of metabolite concentration changes in mammalian cells.

molecular biology↗

bioRxiv2022

High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate

L-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long been generally considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of L-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of L-lactate, we have developed a series of high-performance ({Delta}F/F = 15 to 30 in vitro), intensiometric, genetically-encoded green fluorescent protein (GFP)-based intracellular L-lactate biosensors with a range of affinities. We evaluated the performance of these biosensors by in vitro and live-cell characterization and demonstrated the utility with imaging applications in several cell lines.

bioengineering↗