RESUMO
Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.
Assuntos
Trifosfato de Adenosina/química , Membrana Celular/química , Citosol/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/metabolismo , Trifosfato de Adenosina/genética , Bacillus/citologia , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Cinética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteína Vermelha Fluorescente , Proteína Inibidora de ATPaseRESUMO
Engineered fluorescent indicators for visualizing mercury ion (Hg2+) are powerful tools to illustrate the intracellular distribution and serious toxicity of the ion. However, the sensitive and specific detection of Hg2+ in living cells and in vivo is challenging. This paper reported the development of fluorescent indicators for Hg2+ in green or red color by inserting a circularly permuted fluorescent protein into a highly mercury-specific repressor. These sensors provided a rapid, sensitive, specific, and real-time read-out of Hg2+ dynamics in solutions, bacteria, subcellular organelles of mammalian cells, and zebrafish, thereby providing a useful new method for Hg2+ detection and bioimaging. In conjunction with the hydrogen peroxide sensor HyPer, we found mercury uptake would trigger subcellular oxidative events at the single-cell level, and provided visual evidence of the causality of mercury and oxidative damage. These sensors would paint the landscape of mercury toxicity to cell functions.
Assuntos
Técnicas Biossensoriais , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/métodos , Mercúrio/análise , Mercúrio/toxicidade , Mitocôndrias/patologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Consumo de Oxigênio , Peixe-Zebra/metabolismoRESUMO
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Luminescentes/metabolismo , NADP/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sobrevivência Celular , Glucose , Homeostase , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Modelos Moleculares , Estresse Oxidativo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Engenharia de ProteínasRESUMO
High-resolution spatiotemporal imaging of histidine in single living mammalian cells faces technical challenges. Here, we developed a series of ratiometric, highly responsive, and single fluorescent protein-based histidine sensors of wide dynamic range. We used these sensors to quantify subcellular free-histidine concentrations in glucose-deprived cells and glucose-fed cells. Results showed that cytosolic free-histidine concentration was higher and more sensitive to the environment than free histidine in the mitochondria. Moreover, histidine was readily transported across the plasma membrane and mitochondrial inner membrane, which had almost similar transport rates and transport constants, and histidine transport was not influenced by cellular metabolic state. These sensors are potential tools for tracking histidine dynamics inside subcellular organelles, and they will open an avenue to explore complex histidine signaling.
