ABSTRACT
Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells. This is referred to as a "reversed" pH gradient, and is essential in supporting their accelerated growth rate, invasion and migration, and in suppressing anti-tumor immunity, the promotion of metabolic coupling with fibroblasts and in preventing apoptosis. Moreover, abnormal pH, both pHi and pHe, contribute to drug resistance in cancers. Therefore, the development of methods for measuring pH in living tumor cells is likely to lead to better understanding of tumor biology and to open new ways for cancer treatment. Genetically encoded, fluorescent, pH-sensitive probes represent promising instruments enabling the subcellular measurement of pHi with unrivaled specificity and high accuracy. Here, we describe a protocol for pHi imaging at a microscopic level in HeLa tumor spheroids, using the genetically encoded ratiometric (dual-excitation) pHi indicator, SypHer2.
Subject(s)
Bacterial Proteins/genetics , Biosensing Techniques , Cytoplasm/chemistry , Luminescent Proteins/genetics , Optical Imaging/methods , Spheroids, Cellular/metabolism , Bacterial Proteins/metabolism , Gene Expression , Genes, Reporter , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , HeLa Cells , Humans , Hydrogen-Ion Concentration , Lentivirus/genetics , Lentivirus/metabolism , Luminescent Proteins/metabolism , Optical Imaging/instrumentation , Protein Isoforms/genetics , Protein Isoforms/metabolism , Spheroids, Cellular/ultrastructure , Transfection , Tumor Cells, CulturedABSTRACT
Stimulated emission depletion (STED) microscopy is a popular super resolution imaging technique. Not only synthetic dyes and fluorescent proteins can be utilized as STED fluorophores, but also genetically encoded biosensors. Fusing the biosensor with proteins of interest allows subdiffraction imaging of intracellular macromolecular architecture with simultaneous extraction of functional information about cellular activities. Here, we describe a protocol for live-cell STED microscopy of the HyPer2 biosensor fused to cytoskeletal filaments.
Subject(s)
Biosensing Techniques/methods , Cytoskeletal Proteins/metabolism , Fluorescent Dyes/metabolism , Microscopy, Fluorescence/methods , Animals , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Hydrogen Peroxide/metabolism , Mice , NIH 3T3 Cells , Single-Cell AnalysisABSTRACT
BACKGROUND: SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. METHODS: Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. RESULTS: Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. CONCLUSIONS: SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. GENERAL SIGNIFICANCE: The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.
Subject(s)
Hydrogen-Ion Concentration , Neurosciences , Regeneration/physiology , Animals , Calcium/metabolism , Fluorescence , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Radiometry , Xenopus laevis/physiologyABSTRACT
Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.
Subject(s)
Biosensing Techniques/methods , Cytoskeleton/ultrastructure , Cytoskeleton/chemistry , Fluorescent Dyes/chemistry , Hydrogen Peroxide/chemistry , Microscopy, FluorescenceABSTRACT
Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca(2+) uptake.
Subject(s)
Hydrogen Peroxide/chemistry , Luminescent Proteins/chemistry , Microscopy, Fluorescence , Reactive Oxygen Species/chemistry , Calcium/chemistry , Cytoplasm/chemistry , Electron Transport , Fluorescent Dyes/chemistry , HEK293 Cells , HeLa Cells , Humans , Kinetics , Mitochondria/metabolism , Oxidation-Reduction , Recombinant Proteins/chemistry , Signal Transduction , Spectrophotometry , Time Factors , Red Fluorescent ProteinABSTRACT
Understanding of redox signaling requires data on the spatiotemporal distribution of hydrogen peroxide (H(2)O(2)) within the cell. The fluorescent reporter HyPer is a powerful instrument for H(2)O(2) imaging. However, rapid diffusion of HyPer throughout the nucleocytoplasmic compartment does not allow visualization of H(2)O(2) gradients on the micrometer scale. Here we dramatically improved the spatial resolution of H(2)O(2) imaging by applying subcytoplasmic targeting of HyPer. The membrane-attached reporters identified "microdomains" of elevated H(2)O(2) levels within the cytoplasm of the cells exposed to growth factors. We demonstrate that diffusion of H(2)O(2) across the cytoplasm was strongly limited, providing evidence that H(2)O(2) acts locally inside cells.
Subject(s)
Cells/metabolism , Hydrogen Peroxide/metabolism , Animals , Diffusion , HeLa Cells , Humans , Mice , NIH 3T3 Cells , Oxidation-Reduction , Signal TransductionABSTRACT
Hydrogen peroxide is an important second messenger controlling intracellular signaling cascades by selective oxidation of redox active thiolates in proteins. Changes in intracellular [H(2)O(2)] can be tracked in real time using HyPer, a ratiometric genetically encoded fluorescent probe. Although HyPer is sensitive and selective for H(2)O(2) due to the properties of its sensing domain derived from the Escherichia coli OxyR protein, many applications may benefit from an improvement of the indicator's dynamic range. We here report HyPer-2, a probe that fills this demand. Upon saturating [H(2)O(2)] exposure, HyPer-2 undergoes an up to sixfold increase of the ratio F500/F420 versus a threefold change in HyPer. HyPer-2 was generated by a single point mutation A406V from HyPer corresponding to A233V in wtOxyR. This mutation was previously shown to destabilize interface between monomers in OxyR dimers. However, in HyPer-2, the A233V mutation stabilizes the dimer and expands the dynamic range of the probe.
