Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Oct. 27, 2023

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- intra-cellular dynamics L-lactate are currently hampered by limited selection performance L-lactate-specific genetically encoded biosensors. Here we now report spectrally functionally orthogonal pair high-performance biosensors: green fluorescent extracellular biosensor, designated eLACCO2.1, red intracellular R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization robust fluorescence response. To best our knowledge, R-iLACCO1 its affinity variants exhibit larger responses than any previously reported biosensor. We demonstrate spatially multiplexed imaging coexpression cultured cells, vivo mice.

Language: Английский

---

Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals

Chemical Reviews, Journal Year: 2024, Volume and Issue: 124(22), P. 12573 - 12660

Published: Nov. 13, 2024

Cellular function is controlled through intricate networks of signals, which lead to the myriad pathways governing cell fate. Fluorescent biosensors have enabled study these signaling in living systems across temporal and spatial scales. Over years there has been an explosion number fluorescent biosensors, as they become available for numerous targets, utilized spectral space, suited various imaging techniques. To guide users this extensive biosensor landscape, we discuss critical aspects proteins consideration development, smart tagging strategies, historical recent types, grouped by target, with a focus on design applications sensors systems.

Language: Английский

---

Engineering a cpGFP-based biosensor for enhanced quantification of glycolate production in Escherichia coli

Talanta, Journal Year: 2025, Volume and Issue: 286, P. 127529 - 127529

Published: Jan. 13, 2025

Language: Английский

---

Synthesis and application of a photocaged-l-lactate for studying the biological roles of l-lactate

Communications Chemistry, Journal Year: 2025, Volume and Issue: 8(1)

Published: April 5, 2025

L-Lactate, once considered a metabolic waste product of glycolysis, is now recognized as vitally important metabolite and signaling molecule in multiple biological pathways. However, exploring L-lactate's emerging intra- extra-cellular roles hindered by lack tools to perturb L-lactate concentration intracellularly extracellularly. Photocaged compounds are powerful way introduce bioactive molecules with spatiotemporal precision using illumination. Here, we report the development photocaged derivative L-lactate, 4-methoxy-7-nitroindolinyl-L-lactate (MNI-L-lac), that releases upon We validated MNI-L-lac cell culture demonstrating photorelease elicits response from genetically encoded extra- intracellular biosensors (eLACCO1, eLACCO2.1, R-iLACCO1.2). To demonstrate utility MNI-L-lac, employed activate G protein-coupled receptor 81 (GPR81), revealed inhibition adenylyl cyclase activity concomitant decrease cAMP. These results indicate may be useful for perturbing endogenous order investigate

Language: Английский

---

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

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: April 17, 2025

Abstract Pyruvate is the end-product of glycolysis and a central metabolite involved in many biochemical pathways. However, lack high-performance (i.e., Δ F / 0 > 10) single fluorescent protein (FP)-based biosensors has hindered efforts to investigate physiological role pyruvate. Here, we present GreenPy1 ApplePy1 series, which are green FP (GFP)-based red (RFP)-based pyruvate biosensors, respectively. Both series exhibit large fluorescence intensity change (Δ ∼ 20 >40) range affinities (10s μM several mM). We demonstrate utility these for multicolor imaging concentration changes mammalian cells.

Language: Английский

---

A novel genetically encoded fluorescent probe for rapid Hg2 + analysis in environmental samples and elucidation of cellular uptake pathways

Sensors and Actuators B Chemical, Journal Year: 2025, Volume and Issue: 439, P. 137842 - 137842

Published: April 23, 2025

Language: Английский

---

Simultaneous Quantification of Intracellular pH and Lactate at the Single-Cell Level Using a Genetically Encoded Fluorescence Lifetime Biosensor

Sensors and Actuators B Chemical, Journal Year: 2025, Volume and Issue: unknown, P. 137979 - 137979

Published: May 1, 2025

Language: Английский

---

Transmembrane Protease Serine 11B Modulates Lactate Transport Through SLC16A1 in Pancreatic Ductal Adenocarcinoma—A Functional Link to Phenotype Heterogeneity

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(11), P. 5398 - 5398

Published: June 4, 2025

Tumor cell heterogeneity, e.g., in stroma-rich pancreatic ductal adenocarcinoma (PDAC), includes a differential metabolism of lactate. While being secreted as waste product by most cancer cells characterized the glycolytic Warburg metabolism, it is utilized subset highly malignant running reverse metabolism. Key drivers lactate transport are carrier proteins SLC16A1 (import/export) and SLC16A3 (export). Expression function both carriers controlled chaperone Basigin (BSG), which itself functionally transmembrane protease serine 11B (TMPRSS11B). In this study we explored impact TMPRSS11B on phenotype PDAC under conditions. Amongst panel lines, Panc1 BxPc3 were identified to express at high level, whilst other lines such T3M4 did not. ShRNA-mediated knock-down enhanced import through SLC16A1, shown GFP/iLACCO1 uptake assay, whereas TMPRSS1B overexpression dampened SLC16A1-driven uptake. Moreover, differentially impacted proliferation chemoresistance conditions or cells, respectively, well their stemness properties indicated altered colony formation rates expression stem markers Nanog, Sox2, KLF4 Oct4. These effects depended BSG gene silencing. Immunohistochemical analysis revealed reciprocal together with some areas tumor tissues from patients. Those regions exhibiting low no but concomitant greater amounts KLF4. contrast, largely negative for expression. Thus, adds metabolic heterogeneity its absence supports enhancement BSG-supported subsequent alterations towards stemness.

Language: Английский

---

Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons

ACS Sensors, Journal Year: 2024, Volume and Issue: 9(6), P. 3394 - 3402

Published: June 1, 2024

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement biological imaging applications. In an effort to accelerate SFPBs, we report modified transposons transposase-based creation libraries FPs randomly inserted into analyte binding domains, vice versa. These feature ends that are optimized minimize length linkers connect FP domain. We rationalized shorter between domains should result in more effective allosteric coupling binding-dependent conformational change domain fluorescence modulation chromophore As a proof concept, employed end-modified Mu discovery SFPB prototypes based on insertion two circularly permuted red (mApple FusionRed) proteins l-lactate spermidine. Using analogous approach, discovered calcium ion (Ca

Language: Английский

---

High-Performance Chemigenetic Potassium Ion Indicator

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 27, 2024

Potassium ion (K

Language: Английский

---