Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

YTnC2, an improved genetically encoded green calcium indicator based on toadfish troponin C.

Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best-suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7-fold higher molecular brightness and 6.4-fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant.

Combined Structural and Computational Study of the mRubyFT Fluorescent Timer Locked in Its Blue Form.

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFTS148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFTS148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C-O- fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.

FNCaMP, ratiometric green calcium indicator based on mNeonGreen protein.

Neurobiologists widely use green genetically encoded calcium indicators (GECIs) for visualization of neuronal activity. Among them, ratiometric GECIs allow imaging of both active and non-active neuronal populations. However, they are not popular, since their properties are inferior to intensiometric GCaMP series of GECIs. The most characterized and developed ratiometric green GECI is FGCaMP7. However, the dynamic range and sensitivity of its large Stock's shift green (LSS-Green) form is significantly lower than its Green form and its molecular design is not optimal. To address these drawbacks, we engineered a ratiometric green calcium indicator, called FNCaMP, which is based on bright mNeonGreen protein and calmodulin from A. niger and has optimal NTnC-like design. We compared the properties of the FNCaMP and FGCaMP7 indicators in vitro, in mammalian cells, and in neuronal cultures. Finally, we obtained and analyzed X-ray structure of the FNCaMP indicator.

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers.

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis.

cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals.

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca2+ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively.

LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes' Shift.

Red fluorescent proteins with a large Stokes' shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes' shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein.

The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

Rapid directed molecular evolution of fluorescent proteins in mammalian cells.

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of fluorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2 × 107 independent random genes of fluorescent proteins expressed in HEK cells, completing one iteration of directed evolution in a course of 8 days. We employed this approach to develop a set of green and near-infrared fluorescent proteins with enhanced intracellular brightness. The developed near-infrared fluorescent proteins demonstrated high performance for fluorescent labeling of neurons in culture and in vivo in model organisms such as Caenorhabditis elegans, Drosophila, zebrafish, and mice. Spectral properties of the optimized near-infrared fluorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red fluorescent proteins, as well as dual-color near-infrared fluorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced fluorescent proteins will find wide application for in vivo multicolor imaging of small model organisms.

LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift.

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs' performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus.

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4-6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3-3.5, respectively.

GAF-CaMP3-sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity.

The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2-superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2-sfGFP indicator, named GAF-CaMP3-sfGFP. The GAF-CaMP3-sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2-sfGFP, in cultured HeLa cells, GAF-CaMP3-sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3-sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3-sfGFP showed a linear DF/F response in the range of 0-20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome.

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (Kd of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy.

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.

NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites.

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.

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.