Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these ...reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.
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•HyPer7 is an ultrasensitive, ultrafast, and pH-stable indicator for H2O2•Being controlled by the Trx system, H2O2 does not diffuse out from mitochondria•Intensity of the H2O2 gradient associates with the stability of cellular protrusions•H2O2 gradients can be visualized in wounded tissues with HyPer7
Pak et al. have developed HyPer7, a next-generation genetically encoded fluorescent probe for H2O2 detection. Importantly, HyPer7 is resistant to pH changes. Applying HyPer7 to study H2O2 diffusion from the mitochondrial matrix, the authors decipher the topology of H2O2 production by Complex I and visualize oxidant gradients in cell motility and wounded tissue.
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.
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.
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.
SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.
The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies.
•A new ratiometric genetically encoded pH-sensor SypHer-2 is developed.•SypHer-2 has brighter fluorescence signal in cells compared to SypHer.•Using SypHer-2 we registered activity-associated pH oscillations in neurons.•Intracellular pH drop precedes Xenopus laevis tadpole tail regeneration event.
Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader ...application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen-vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca
imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.
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.
Novel fluorogenic dyes based on the GFP chromophore are developed. The compounds contain a pyridinium ring instead of phenolate and feature large Stokes shifts and solvent-dependent variations in the ...fluorescence quantum yield. Electronic structure calculations explain the trends in solvatochromic behavior in terms of the increase of the dipole moment upon excited-state relaxation in polar solvents associated with the changes in bonding pattern in the excited state. A unique combination of such optical characteristics and lipophilic properties enables using one of the new dyes for imaging the membrane structure of endoplasmic reticulum. An extremely high photostability (due to a dynamic exchange between the free and absorbed states) and selectivity make this compound a promising label for this type of cellular organelles.
High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for ...intracellular H2O2 exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H2O2 gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H2O2 levels both in vitro and in vivo.
Hydrogen peroxide (H
O
) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H
O
...dynamics in live cells within a limited field of view. The visualization of H
O
within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H
O
concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H
O
, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from
.
When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (p
of 5.9 vs. ≥7.6) and oxidized states (p
of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H
O
and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H
O
when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H
O
and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H
O
upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H
O
with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H
O
indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.
A versatile synthetic approach to accessing unsymmetrically substituted (trans‐A2)BC‐type porphyrins bearing two heteroatoms at the macrocycle periphery is developed. For this purpose, experimental ...conditions for the substitution of the bromine atom in zinc 5‐bromo‐15‐(diethoxyphosphoryl)‐10,20‐diphenylporphyin (2) by S‐, O‐, and N‐nucleophiles were explored. SNAr reactions afford AlkO‐, ArO‐, and AlkS‐substituted porphyrins in good to high yields. In contrast, SNAr reactions of 2 with N‐nucleophiles lead to meso‐amino‐substituted porphyrins in preparative yields only with cyclic secondary amines. Primary amines, anilines and azacrowns may also react with bromide 2 but the palladium catalyst is needed to obtain the products in acceptable yields. The interest of the compounds under investigation for biomimetic assembly of tetrapyrroles was demonstrated by the studies of self‐assembly of ditopic morpholinyl‐substituted porphyrin 5a in the solid state. Moreover, we have prepared emissive porphyrin monolayers at the air/water interface and revealed that these porphyrin films were suitable for detection of zinc(II) ions in aqueous solutions.
Transition‐metal‐catalyzed and catalyst‐free conditions were explored for the reactions of meso‐bromo(diethoxyphosphoryl)‐diphenylporphyrin with of S‐, O‐, and N‐nucleophiles to develop concise and versatile procedures for the accessing (trans‐A2)BC‐type porphyrins bearing the electron withdrawing diethoxyphosphoryl group and the electron donating RO, RS, or R2N substituents at the macrocycle.
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