Kiryl Piatkevich | Massachusetts Institute of Technology (MIT) (original) (raw)

Papers by Kiryl Piatkevich

International Journal of Molecular Sciences, 2021

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficient... more 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...

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracell... more 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 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 C.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 fl...

Cell, 2020

Highlights d Clustering fluorescent sensors at points in cells enables many to be imaged at once ... more Highlights d Clustering fluorescent sensors at points in cells enables many to be imaged at once d Modular reagent design allows existing sensors to be easily adapted to cluster d Such ''signaling reporter islands'' (SiRIs) are safe and robust in cells and in vivo d SiRIs reveal relationships between components of signal transduction networks

Near-infrared (NIR) genetically-encoded calcium ion (Ca2+) indicators (GECIs) can provide advanta... more Near-infrared (NIR) genetically-encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross-talk with visible-light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report two improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and C. elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

International Journal of Molecular Sciences, 2020

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for vi... more 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 enviro...

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their a... more Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual cor...

International Journal of Molecular Sciences, 2019

A variety of genetically encoded calcium indicators are currently available for visualization of ... more 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 photostab...

International Journal of Molecular Sciences, 2019

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in ... more 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...

Applied Sciences, 2019

Our ability to investigate the brain is limited by available technologies that can record biologi... more Our ability to investigate the brain is limited by available technologies that can record biological processes in vivo with suitable spatiotemporal resolution. Advances in optogenetics now enable optical recording and perturbation of central physiological processes within the intact brains of model organisms. By monitoring key signaling molecules noninvasively, we can better appreciate how information is processed and integrated within intact circuits. In this review, we describe recent efforts engineering genetically-encoded fluorescence indicators to monitor neuronal activity. We summarize recent advances of sensors for calcium, potassium, voltage, and select neurotransmitters, focusing on their molecular design, properties, and current limitations. We also highlight impressive applications of these sensors in neuroscience research. We adopt the view that advances in sensor engineering will yield enduring insights on systems neuroscience. Neuroscientists are eager to adopt suitabl...

Scientific reports, Jan 15, 2018

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller ... more 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 show...

Nature neuroscience, 2017

Optogenetic control of individual neurons with high temporal precision within intact mammalian br... more Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high-efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons. In combination with two-photon holographic stimulation, we found that this somatic CoChR (soCoChR) enabled photostimulation of individual cells in mouse cortical brain slices with single-cell resolution and <1-ms temporal precision. We used soCoChR to perform connectivity mapping on in...

Nature chemical biology, 2018

We developed a new way to engineer complex proteins toward multidimensional specifications using ... more We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neu...

Biophysical journal, Jan 7, 2017

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from ba... more Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in ...

Light: Science & Applications, 2016

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in ... more Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two-and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.

PLOS ONE, 2017

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fung... more Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Barykina, Natalia V. et al. "Green Fluorescent Genetically Encoded Calcium Indicator Based on calmodulin/M13-Peptide from Fungi." Edited by Eugene A.

Scientific Reports, 2016

Genetically encoded calcium indicators (GECIs) are mainly represented by two-or one-fluorophoreba... more Genetically encoded calcium indicators (GECIs) are mainly represented by two-or one-fluorophorebased sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca 2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca 2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca 2+binding sites. The engineered sensor, called NTnC, uses TnC as the Ca 2+-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 Ca 2+. Using NTnC, we have visualized Ca 2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca 2+ 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 Ca 2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope. Optical techniques using genetically encoded calcium indicators (GECIs) based on fluorescent proteins (FPs) are broadly applied for in vivo visualization of neuronal activity. FP-based calcium indicators (or sensors) can be classified into two major designs (Fig. 1a). The first class of GECIs includes the FRET (fluorescence resonance energy transfer)-based family of sensors, which is composed of two fluorescent proteins, one acting as a donor and another as an acceptor, with a Ca 2+-binding domain located between them 1. The latter can be represented by calmodulin (CaM) in combination with the M13 peptide from myosin light chain kinase (CaM/M13) or by a minimal Ca 2+-binding motif from the C-terminal domain of troponin C (TnC). In the first type of FRET sensor, CaM carries four calcium ion-binding

Nature biotechnology, Sep 4, 2016

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on dif... more Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.

Chemistry & Biology, 2014

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by incre... more A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Methods in Cell Biology, 2011

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide... more Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and METHODS IN CELL BIOLOGY, VOL 102

Scientific Reports, 2013

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the ... more Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties. G FP-like fluorescent proteins (FPs) are indispensable imaging tools for all areas of biomedical research 1-3. Three dimensional structures of GFP-like proteins are highly conserved, consisting of a beta-barrel formed by about 220-240 amino acids. A chromophore is buried inside the barrel, shielded by the protein matrix from stochastic interactions with solvent molecules. The rigid environment provided by protein scaffold prevents thermal isomerization and nonfluorescent relaxation of the chromophore. A wide variety of FP spectral phenotypes originate from two major contributing factors: the chemical structure of the chromophore, and interactions occurring between the chromophore, both in the ground and excited states, and its immediate environment. Based on the chemical structure, which to a large extent determines the spectral properties of FPs, the chromophores can be classified into several groups 4. Most of the red and far-red FPs contain so called DsRedlike chromophores 5 , which can exist in either neutral or anionic states. Neutral DsRed-like chromophores absorb blue-cyan light and emit green-yellow, whereas the anionic forms possess excitation and emission maxima at ,560-580 and 570-610 nm, respectively 4,6,7. However, fluorescence spectra can be significantly perturbed by changes in the immediate chromophore environment. Spectroscopic studies combined with high resolution crystal structures revealed the interactions responsible for the bathochromic shift of fluorescence in several red and far-red FPs. Among the most common modifications of the chromophore environment in far-red FPs is the introduction of a hydrogen bond between the chromophore and its immediate environment. An important example is the hydrogen bond between the N-acylimine oxygen of the DsRed-like chromophore and a water molecule or a side chain of an amino acid (Figure 1A). This type of interaction has been observed in mNeptune 8 , eqFP650 (Ref. 9,10), eqFP670 (Ref. 9,10), mRojoA 11 , mRouge 11 , which possess water-mediated hydrogen bonds. In mPlum 12 and its variant mPlum/E16Q 13 , the N-acylimine oxygen of the chromophore forms direct hydrogen bonds with the side chain functionalities of Glu16 and Gln16, respectively 13. A hydrogen bond between the protonated Glu215 carboxyl group and the imidazolinone ring nitrogen was proposed to account for the

International Journal of Molecular Sciences, 2021

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficient... more 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...

In vivo imaging of model organisms is heavily reliant on fluorescent proteins with high intracell... more 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 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 C.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 fl...

Cell, 2020

Highlights d Clustering fluorescent sensors at points in cells enables many to be imaged at once ... more Highlights d Clustering fluorescent sensors at points in cells enables many to be imaged at once d Modular reagent design allows existing sensors to be easily adapted to cluster d Such ''signaling reporter islands'' (SiRIs) are safe and robust in cells and in vivo d SiRIs reveal relationships between components of signal transduction networks

Near-infrared (NIR) genetically-encoded calcium ion (Ca2+) indicators (GECIs) can provide advanta... more Near-infrared (NIR) genetically-encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross-talk with visible-light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report two improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and C. elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

International Journal of Molecular Sciences, 2020

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for vi... more 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 enviro...

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their a... more Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual cor...

International Journal of Molecular Sciences, 2019

A variety of genetically encoded calcium indicators are currently available for visualization of ... more 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 photostab...

International Journal of Molecular Sciences, 2019

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in ... more 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...

Applied Sciences, 2019

Our ability to investigate the brain is limited by available technologies that can record biologi... more Our ability to investigate the brain is limited by available technologies that can record biological processes in vivo with suitable spatiotemporal resolution. Advances in optogenetics now enable optical recording and perturbation of central physiological processes within the intact brains of model organisms. By monitoring key signaling molecules noninvasively, we can better appreciate how information is processed and integrated within intact circuits. In this review, we describe recent efforts engineering genetically-encoded fluorescence indicators to monitor neuronal activity. We summarize recent advances of sensors for calcium, potassium, voltage, and select neurotransmitters, focusing on their molecular design, properties, and current limitations. We also highlight impressive applications of these sensors in neuroscience research. We adopt the view that advances in sensor engineering will yield enduring insights on systems neuroscience. Neuroscientists are eager to adopt suitabl...

Scientific reports, Jan 15, 2018

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller ... more 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 show...

Nature neuroscience, 2017

Optogenetic control of individual neurons with high temporal precision within intact mammalian br... more Optogenetic control of individual neurons with high temporal precision within intact mammalian brain circuitry would enable powerful explorations of how neural circuits operate. Two-photon computer-generated holography enables precise sculpting of light and could in principle enable simultaneous illumination of many neurons in a network, with the requisite temporal precision to simulate accurate neural codes. We designed a high-efficacy soma-targeted opsin, finding that fusing the N-terminal 150 residues of kainate receptor subunit 2 (KA2) to the recently discovered high-photocurrent channelrhodopsin CoChR restricted expression of this opsin primarily to the cell body of mammalian cortical neurons. In combination with two-photon holographic stimulation, we found that this somatic CoChR (soCoChR) enabled photostimulation of individual cells in mouse cortical brain slices with single-cell resolution and <1-ms temporal precision. We used soCoChR to perform connectivity mapping on in...

Nature chemical biology, 2018

We developed a new way to engineer complex proteins toward multidimensional specifications using ... more We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neu...

Biophysical journal, Jan 7, 2017

Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from ba... more Several series of near-infrared (NIR) fluorescent proteins (FPs) were recently engineered from bacterial phytochromes but were not systematically compared in neurons. To fluoresce, NIR FPs utilize an enzymatic derivative of heme, the linear tetrapyrrole biliverdin, as a chromophore whose level in neurons is poorly studied. Here, we evaluated NIR FPs of the iRFP protein family, which were reported to be the brightest in non-neuronal mammalian cells, in primary neuronal culture, in brain slices of mouse and monkey, and in mouse brain in vivo. We applied several fluorescence imaging modes, such as wide-field and confocal one-photon and two-photon microscopy, to compare photochemical and biophysical properties of various iRFPs. The iRFP682 and iRFP670 proteins exhibited the highest brightness and photostability under one-photon and two-photon excitation modes, respectively. All studied iRFPs exhibited efficient binding of the endogenous biliverdin chromophore in cultured neurons and in ...

Light: Science & Applications, 2016

Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in ... more Three-photon wide-field depth-resolved excitation is used to overcome some of the limitations in conventional point-scanning two-and three-photon microscopy. Excitation of chromophores as diverse as channelrhodopsins and quantum dots is shown, and a penetration depth of more than 700 μm into fixed scattering brain tissue is achieved, approximately twice as deep as that achieved using two-photon wide-field excitation. Compatibility with live animal experiments is confirmed by imaging the cerebral vasculature of an anesthetized mouse; a complete focal stack was obtained without any evidence of photodamage. As an additional validation of the utility of wide-field three-photon excitation, functional excitation is demonstrated by performing three-photon optogenetic stimulation of cultured mouse hippocampal neurons expressing a channelrhodopsin; action potentials could reliably be excited without causing photodamage.

PLOS ONE, 2017

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fung... more Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Barykina, Natalia V. et al. "Green Fluorescent Genetically Encoded Calcium Indicator Based on calmodulin/M13-Peptide from Fungi." Edited by Eugene A.

Scientific Reports, 2016

Genetically encoded calcium indicators (GECIs) are mainly represented by two-or one-fluorophoreba... more Genetically encoded calcium indicators (GECIs) are mainly represented by two-or one-fluorophorebased sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca 2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca 2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca 2+binding sites. The engineered sensor, called NTnC, uses TnC as the Ca 2+-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 Ca 2+. Using NTnC, we have visualized Ca 2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca 2+ 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 Ca 2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope. Optical techniques using genetically encoded calcium indicators (GECIs) based on fluorescent proteins (FPs) are broadly applied for in vivo visualization of neuronal activity. FP-based calcium indicators (or sensors) can be classified into two major designs (Fig. 1a). The first class of GECIs includes the FRET (fluorescence resonance energy transfer)-based family of sensors, which is composed of two fluorescent proteins, one acting as a donor and another as an acceptor, with a Ca 2+-binding domain located between them 1. The latter can be represented by calmodulin (CaM) in combination with the M13 peptide from myosin light chain kinase (CaM/M13) or by a minimal Ca 2+-binding motif from the C-terminal domain of troponin C (TnC). In the first type of FRET sensor, CaM carries four calcium ion-binding

Nature biotechnology, Sep 4, 2016

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on dif... more Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (∼70 nm) imaging of cells and mammalian tissues on conventional microscopes.

Chemistry & Biology, 2014

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by incre... more A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Methods in Cell Biology, 2011

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide... more Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and METHODS IN CELL BIOLOGY, VOL 102

Scientific Reports, 2013

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the ... more Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties. G FP-like fluorescent proteins (FPs) are indispensable imaging tools for all areas of biomedical research 1-3. Three dimensional structures of GFP-like proteins are highly conserved, consisting of a beta-barrel formed by about 220-240 amino acids. A chromophore is buried inside the barrel, shielded by the protein matrix from stochastic interactions with solvent molecules. The rigid environment provided by protein scaffold prevents thermal isomerization and nonfluorescent relaxation of the chromophore. A wide variety of FP spectral phenotypes originate from two major contributing factors: the chemical structure of the chromophore, and interactions occurring between the chromophore, both in the ground and excited states, and its immediate environment. Based on the chemical structure, which to a large extent determines the spectral properties of FPs, the chromophores can be classified into several groups 4. Most of the red and far-red FPs contain so called DsRedlike chromophores 5 , which can exist in either neutral or anionic states. Neutral DsRed-like chromophores absorb blue-cyan light and emit green-yellow, whereas the anionic forms possess excitation and emission maxima at ,560-580 and 570-610 nm, respectively 4,6,7. However, fluorescence spectra can be significantly perturbed by changes in the immediate chromophore environment. Spectroscopic studies combined with high resolution crystal structures revealed the interactions responsible for the bathochromic shift of fluorescence in several red and far-red FPs. Among the most common modifications of the chromophore environment in far-red FPs is the introduction of a hydrogen bond between the chromophore and its immediate environment. An important example is the hydrogen bond between the N-acylimine oxygen of the DsRed-like chromophore and a water molecule or a side chain of an amino acid (Figure 1A). This type of interaction has been observed in mNeptune 8 , eqFP650 (Ref. 9,10), eqFP670 (Ref. 9,10), mRojoA 11 , mRouge 11 , which possess water-mediated hydrogen bonds. In mPlum 12 and its variant mPlum/E16Q 13 , the N-acylimine oxygen of the chromophore forms direct hydrogen bonds with the side chain functionalities of Glu16 and Gln16, respectively 13. A hydrogen bond between the protonated Glu215 carboxyl group and the imidazolinone ring nitrogen was proposed to account for the