Membrane-Permeant, Bioactivatable Coumarin Derivatives for In-Cell Labelling.

The delivery of small molecule fluorophores with minimal compartmentalization is currently one of the most critical technical problems in intracellular labelling. Here we introduce sulfonated and phosphonated coumarin dyes, demonstrate rapid cell entry via a prodrug approach, and show a lack of interaction with membranes, organelles, or other compartments. The dyes show no specific localization and are evenly distributed in the cells. Our fluorogenic, clickable phosphonate derivatives successfully tagged model targets in intact cells and the increase in brightness upon click reaction was around 60-fold.

In vivo dynamics of acidosis and oxidative stress in the acute phase of an ischemic stroke in a rodent model.

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and H2O2 during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant H2O2 generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo.

Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function.

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.

Two- and three-photon absorption cross-section characterization for high-brightness, cell-specific multiphoton fluorescence brain imaging.

We demonstrate an accurate quantitative characterization of absolute two- and three-photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high-brightness, cell-specific two- and three-photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two-photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep-tissue experiments.

Nitrobenzyl-based fluorescent photocages for spatial and temporal control of signalling lipids in cells.

Here we present a set of fluorescent cages prepared by tethering fluorescent dyes to a photolabile group. The developed molecules enable caging of signalling lipids, their delivery to specific cellular membranes, with further imaging, quantification, and controlled photorelease of active lipids in living cells.

Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide.

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H2O2 dynamics in live cells within a limited field of view. The visualization of H2O2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H2O2 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 H2O2, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. 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 (pKa of 5.9 vs. ≥7.6) and oxidized states (pKa 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 H2O2 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 H2O2 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 H2O2 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 H2O2 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 H2O2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H2O2 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.

Visualization of Intracellular Hydrogen Peroxide with the Genetically Encoded Fluorescent Probe HyPer in NIH-3T3 Cells.

Reactive oxygen species (ROS) are involved in regulating normal physiological cell functions as second messengers as well as nonspecific damage of biomolecules in a pathological process known as oxidative stress. The HyPer family of genetically encoded probes are a useful noninvasive tool for monitoring the real-time dynamics of ROS in individual cells or model organisms. HyPer, the first genetically encoded probe for detection of hydrogen peroxide (H2O2), is oxidized with high specificity and sensitivity by H2O2, leading to ratiometric changes in the fluorescence excitation spectrum of the probe. These changes can be detected with a wide range of commercial confocal and wide-field microscope systems. Here we describe a detailed protocol for ratiometric monitoring of H2O2 produced by D-amino acid oxidase (DAAO) or by NADPH oxidase (NOX) in NIH-3T3 cells using the HyPer probe.

Which Antioxidant System Shapes Intracellular H2O2 Gradients?

Cellular antioxidant systems control the levels of hydrogen peroxide (H2O2) within cells. Multiple theoretical models exist that predict the diffusion properties of H2O2 depending on the rate of H2O2 generation and amount and reaction rates of antioxidant machinery components. Despite these theoretical predictions, it has remained unknown how antioxidant systems shape intracellular H2O2 gradients. The relative role of thioredoxin (Trx) and glutathione systems in H2O2 pattern formation and maintenance is another disputed question. Here, we visualized cellular antioxidant activity and H2O2 gradients formation by exploiting chemogenetic approaches to generate compartmentalized intracellular H2O2 and using the H2O2 biosensor HyPer to analyze the resulting H2O2 distribution in specific subcellular compartments. Using human HeLa cells as a model system, we propose that the Trx system, but not the glutathione system, regulates intracellular H2O2 gradients. Antioxid. Redox Signal. 31, 664-670.

Red fluorescent redox-sensitive biosensor Grx1-roCherry.

Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of - 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local H2O2 production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging.

Thermogenetic stimulation of single neocortical pyramidal neurons transfected with TRPV1-L channels.

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of single neurons using thermosensitive cation channels and IR stimulation. The main advantage of IR stimulation compared to conventional visible light optogenetics is the depth of penetration (up to millimeters). Due to physiological limitations, thermogenetic molecular tools for mammalian brain stimulation remain poorly developed. Here, we tested the possibility of employment of this new technique for stimulation of neocortical neurons. The method is based on activation gating of TRPV1-L channels selectively expressed in specific cells. Pyramidal neurons of layer 2/3 of neocortex were transfected at an embryonic stage using a pCAG expression vector and electroporation in utero. Depolarization and spiking responses of TRPV1L+ pyramidal neurons to IR radiation were recorded electrophysiologically in acute brain slices of adult animals with help of confocal visualization. As TRPV1L-expressing neurons are not sensitive to visible light, there were no limitations of the use of this technique with conventional fluorescence imaging. Our experiments demonstrated that the TRPV1-L+ pyramidal neurons preserve their electrical excitability in acute brain slices, while IR radiation can be successfully used to induce single neuronal depolarization and spiking at near physiological temperatures. Obtained results provide important information for adaptation of thermogenetic technology to mammalian brain studies in vivo.

Pyridinium Analogues of Green Fluorescent Protein Chromophore: Fluorogenic Dyes with Large Solvent-Dependent Stokes Shift.

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.

Thermogenetic neurostimulation with single-cell resolution.

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 Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.

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.

Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide.

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.

HyPer-3: a genetically encoded H(2)O(2) probe with improved performance for ratiometric and fluorescence lifetime imaging.

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.