Biology subjects

Drobizhev, M.

Publications and source records attributed to Drobizhev, M..

16 recordsLinked to original sources

bioRxiv2026

Calcium-independent biosensors for multiplexed in vivo imaging of extracellular lactate dynamics

Extracellular L-lactate reports cellular metabolism, but resolving its dynamics alongside intracellular metabolism and neuronal activity remains challenging. Here we develop eLACCO3 and R-eLACCO3, green and red cell-surface lactate biosensors with Ca2+-independent responses and faster response and recovery kinetics in cultured cells. Selected variants provide larger fluorescence responses than their predecessors in brain cells, slices, and living mice and improve detection of glucose-evoked extracellular lactate oscillations. Combining R-eLACCO3 with GCaMP6f enabled imaging of extracellular L-lactate simultaneously with odor-evoked activity in anesthetized mice and cortical activity in awake mice. These biosensors enable multiplexed measurements of extracellular lactate dynamics in relation to intracellular metabolism and neuronal activity.

molecular biology↗

bioRxiv2025

hpGRISZ: a high-performance fluorescent biosensor for in vivo imaging of synaptic zinc dynamics

Despite the crucial role of synaptic Zn2+ in neurotransmission and neural processing, direct in vivo measurement of Zn2+ transients has remained challenging due to the limited responsiveness of existing fluorescent indicators. Here we report hpGRISZ (high-performance green indicator for synaptic Zn2+), an ultraresponsive turn-on green fluorescent biosensor engineered through iterative linker optimization, mutagenesis, and directed evolution. hpGRISZ exhibits exceptional brightness, thermostability, and a large fluorescence response (F/F0 {approx} 25) with micromolar affinity suitable for detecting extracellular synaptic Zn2+ release. We comprehensively characterized hpGRISZ in vitro, in mammalian cells, and in vivo. A membrane-anchored version of hpGRISZ traffics robustly to the cell surface under physiological conditions, where it retains strong responsiveness and supports wide-field, two-photon, and fiber photometry recordings in awake mice. These assays revealed synaptic Zn2+ dynamics across distinct brain regions and neuronal circuits. Together, our findings establish hpGRISZ as a powerful tool for dissecting zinc signaling in neural circuits and as a prototype for next-generation genetically encoded biosensors for in vivo imaging.

neuroscience↗

bioRxiv2025

Directionality of two-photon absorption in representative fluorescent proteins

Molecules of fluorescent proteins (FPs) exhibit anisotropic optical properties. While the directionality of single-photon (1P) absorption and emission in the most commonly used FPs has already been characterized, directionality of their two-photon (2P) excitation remains poorly understood, even though it offers higher sensitivity to molecular orientation and promises to yield more information on molecular orientational distributions than the 1P processes. By measuring optical properties of FP crystals, we have now determined the two-photon absorptivity tensors (2PATs) of the FPs mTurquoise2, eGFP and mCherry. Our results demonstrate the feasibility of experimental 2PAT determinations, provide new insights into molecular mechanisms of 2P absorption in FPs, open a new avenue to rational development of novel biosensors and allow detailed structural interpretation of results obtained with such biosensors.

biophysics↗

bioRxiv2023

A blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption

SignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. AimWe describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices. ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

neuroscience↗

bioRxiv2022

Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

O_SCPLOWLC_SCPLOW-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics of O_SCPLOWLC_SCPLOW-Lactate, we develop a second-generation green fluorescent extracellular O_SCPLOWLC_SCPLOW-Lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular O_SCPLOWLC_SCPLOW-Lactate biosensor, designated R-iLACCO1. Compared to the first-generation eLACCO1.1 ({Delta}F/F = 1.5 in cultured neurons), eLACCO2.1 exhibits better membrane localization and fluorescence response ({Delta}F/F = 8.1 in cultured neurons) with faster response kinetics to extracellular O_SCPLOWLC_SCPLOW-Lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes in O_SCPLOWLC_SCPLOW-Lactate concentration in vitro ({Delta}F/F = 15 to 22) and in live mammalian cells ({Delta}F/F = 5.5 to 11). We demonstrate that these biosensors enable cellular-resolution imaging of extracellular and intracellular O_SCPLOWLC_SCPLOW-Lactate in cultured mammalian cells.

molecular biology↗

bioRxiv2022

High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate

L-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long been generally considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of L-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of L-lactate, we have developed a series of high-performance ({Delta}F/F = 15 to 30 in vitro), intensiometric, genetically-encoded green fluorescent protein (GFP)-based intracellular L-lactate biosensors with a range of affinities. We evaluated the performance of these biosensors by in vitro and live-cell characterization and demonstrated the utility with imaging applications in several cell lines.

bioengineering↗

bioRxiv2022

A red fluorescent genetically encoded biosensor for extracellular L-lactate

L-Lactate, traditionally recognized as a waste product of metabolism, is now appreciated as a key intercellular energy currency in mammals. To enable investigations of intercellular shuttling of L-lactate, we have previously reported eLACCO1.1, a green fluorescent genetically encoded biosensor for extracellular L-lactate. eLACCO1.1 enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue. However, eLACCO1.1 spectrally overlaps with commonly used optical biosensors and actuators, limiting its application for multiplexed imaging or combined use with optogenetic actuators. Here, we report a red fluorescent extracellular L-lactate biosensor, designated R-eLACCO2. R-eLACCO2 is the end-product of extensive directed evolution and exhibits a large fluorescence response to L-lactate with high molecular specificity in vitro. We demonstrate that R-eLACCO2 with optimized leader and anchor sequences shows a large fluorescence change in response to extracellular L-lactate on the membrane of live mammalian cells. R-eLACCO2 should enable multicolor imaging of extracellular L-lactate in combination with other fluorescent probes and optogenetic actuators.

bioengineering↗

bioRxiv2022

A Genetically Encoded Far-Red Fluorescent Indicator for Imaging Synaptically-Released Zn2+

Synaptic Zn2+ has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn2+ in the brain in live animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn2+. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn2+ dynamics in the nervous system. Our engineered far-red fluorescent indicator for synaptic Zn2+ (FRISZ) displayed a substantial Zn2+-specific turn-on response and low micromolar affinity. We genetically anchored FRISZ to the mammalian extracellular membrane via a transmembrane -helix. We further successfully used membrane-tethered FRISZ (FRISZ-TM) to image synaptic Zn2+ dynamics in response to sound in the primary auditory cortex (A1) in awake mice. This study thus establishes a new technology for studying the roles of synaptic Zn2+ in the nervous system.

neuroscience↗

bioRxiv2021

Voltage imaging in Drosophila using a hybrid chemical-genetic rhodamine voltage reporter

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila. Significance StatementVoltage imaging is a powerful method for interrogating neurobiology. Chemical indicators possess fast response kinetics, turn-on responses to membrane depolarization, and can be compatible with two-photon excitation. However, selective cell labeling in intact tissues and in vivo remains a challenge for completely synthetic fluorophores. Here, we show that a chemical - genetic hybrid approach in Drosophila enables cell-specific staining in vivo and voltage imaging in whole-brain explants and at neuromuscular junction synapses.

neuroscience↗

bioRxiv2021

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{middle dot}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 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.

molecular biology↗

bioRxiv2021

A genetically encoded fluorescent biosensor for extracellular L-lactate

To enable investigations of the emerging roles of cell-to-cell shuttling of L-lactate, we have developed an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. We demonstrate that this biosensor, designated eLACCO1.1, enables minimally invasive cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.

molecular biology↗

bioRxiv2020

Far-red fluorescent genetically encoded calcium ion indicators

Genetically encoded calcium ion (Ca2+) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca2+ dynamics and neuronal activities on a single cell level. Here we report the design and development of two new far-red fluorescent GECIs, FR-GECO1a and FR-GECO1c, based on the monomeric far-red fluorescent protein mKelly. We characterized these far-red GECIs as purified proteins and assessed their performance when expressed in cultured neurons. FR-GECOs have excitation and emission maxima at ~ 596 nm and ~ 644 nm, respectively, display large responses to Ca2+ ({Delta}F/F0 = 6 for FR-GECO1a, 18 for FR-GECO1c), and are bright under both one-photon and two-photon illumination. FR-GECOs also have high affinities (apparent Kd = 29 nM for FR-GECO1a, 83 nM for FR-GECO1c) for Ca2+, and they enable sensitive and fast detection of single action potentials in neurons.

neuroscience↗

bioRxiv2020

High throughput instrument to screen fluorescent proteins under two-photon excitation

Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

neuroscience↗

bioRxiv2020

A Fast High-Affinity Fluorescent Serotonin Biosensor Engineered from a Tick Lipocalin

The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. DetailsRegrettably, we, and several other researchers in the corresponding authors lab and not involved in the original study, have been unable to reproduce some of the reported key results under conditions originally described. We recently used the pDisplay-G-GESS-M plasmid to transfect HEK 293T cells and observed that the membrane localization of the biosensor was sensitive to FBS batches. By using an FBS batch from Gibco (Cat# 16000-044 and Lot #2103017RP), we were able to replicate the membrane localization similar to what was reported in Fig. 1e. However, the cells required the addition of several hundred micromolar of serotonin for robust fluorescence increase. These new results suggest that the affinity of G-GESS to serotonin is worse than that claimed in the initial article. We further used liquid chromatography-mass spectrometry (LC-MS) to analyze the serotonin stock solutions identified in our -80{degrees}C lab freezer and observed MS peaks suggesting a glutamate contamination in some of the analyzed samples. In addition, we used polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR) to amplify gene fragments from our viral stocks and the sequencing results indicate that the viral stocks were contaminated with a glutamate biosensor. While we are still performing further investigation, in light of the existing findings, all authors agree to retract this paper. In the initial article, the sensor was tested in several cell culture systems, brain slices and behaving mice. We are presently unable to fully explain the observed sensor responses described in the initial article, but it is highly possible that these responses could be caused by the contaminated chemical or viral stocks. We deeply apologize to the scientific community for not detecting these problems sooner.

neuroscience↗

bioRxiv2018

Understanding the Ca2+-dependent Fluorescence Change in Red Genetically Encoded Ca2+ Indicators

Genetically encoded Ca2+ indicators (GECIs) are widely used to illuminate dynamic Ca2+ signaling activity in living cells and tissues. Various fluorescence colors of GECIs are available, including red. Red GECIs are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a circularly permuted red fluorescent protein fused to a Ca2+ sensing domain, calmodulin and a calmodulin-binding peptide. A conformational change in the sensing domain upon binding Ca2+ causes a change in the fluorescence intensity of the fluorescent protein. Three factors could contribute to this change in fluorescence: 1) a shift in the protonation state of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient for one-photon excitation or the two-photon cross section for two-photon excitation. We conducted a systematic study of the photophysical properties of a select cohort of red GECIs in their Ca2+-free and Ca2+-saturated states to determine which factors are most important for the Ca2+-dependent change in fluorescence. In total, we analyzed nine red GECIs, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We found that these red GECIs could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases the magnitude of the change in fluorescence was different depending on one-photon excitation or two-photon excitation by up to a factor of two.

biophysics↗

bioRxiv2017

A genetically encoded Ca2+ indicator based on circularly permutated sea anemone red fluorescent protein

Genetically-encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs enable multicolor visualization with blue or cyan-excitable fluorophores and combined use with blue or cyan-excitable optogenetic actuators. Here we report the development, structure, and validation of a new red fluorescent Ca2+ indicator, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem cell derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo.

biophysics↗