A genetically encoded near-infrared fluorescent calcium ion indicator

Qian, Yong; Piatkevich, Kiryl D.; Larney, Benedict Mc; Abdelfattah, Ahmed S.; Mehta, Sohum; Murdock, Mitchell H.; Gottschalk, Sven; Molina, Rosana S.; Zhang, Wei; Chen, Yingche; Wu, Jiahui; Drobizhev, Mikhail; Hughes, Thomas E.; Zhang, Jin; Schreiter, Eric R.; Shoham, Sharon; Razansky, Daniel; Boyden, Edward S.; Campbell, Robert E.

doi:10.1038/s41592-018-0294-6

Cited by 177 publications

(184 citation statements)

References 27 publications

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“…Briefly, purified proteins were loaded into 384-well plates and then supplied with either 10 mM EGTA or 5 mM CaCl2 before measuring emission spectra. The extinction coefficients (EC), quantum yield (QY) and pKa for NIR-GECO variants were determined as previously described 8 . Ca 2+ titrations of NIR-GECO variants were performed with EGTA-buffered Ca 2+ solutions.…”

Section: Protein Purification and In Vitro Characterizationmentioning

confidence: 99%

Improved genetically encoded near-infrared fluorescent calcium ion indicators forin vivoimaging

Qian

Cosio

Piatkevich

et al. 2020

Preprint

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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 wavelengthGECIs, 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 Ca 2+ dynamics.monitor in vivo neuron activity in model organisms 1-4 . Over a time frame spanning two decades 5,6 , tremendous effort has been invested in the development of visible wavelength GECIs based on green and red fluorescent proteins (GFP and RFP, respectively). These efforts have produced a series of high-performance GECIs that are highly optimized in terms of brightness, kinetics, Ca 2+ affinities, cooperativity, and resting (baseline) fluorescence 2-4,7 . In contrast, efforts to develop GECIs with near-infrared (NIR) excitation and emission (>650 nm) are at a relatively nascent state 8,9 .We recently described the conversion of a NIR FP (mIFP, a biliverdin (BV)-binding NIR FP engineered from the PAS and GAF domains of Bradyrhizobium bacteriophytochrome) 10 into a GECI designated NIR-GECO1. NIR-GECO1 was engineered by genetic insertion of the Ca 2+ -responsive domain calmodulin (CaM)-RS20 into the protein loop close to the BV-binding site of mIFP 8 . NIR-GECO1 provides a robust inverted fluorescence response (that is, a fluorescence decrease upon Ca 2+ increase) in response to Ca 2+ concentration changes in cultured cells, primary neurons, and acute brain slices. Due to its NIR fluorescence, it has inherent advantages relative to visible wavelength GFP-and RFP-based GECIs including reduced phototoxicity, minimal spectral cross-talk with visible-light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption of excitation and emission light in mammalian tissues. However, as a first-generation GECI, NIR-GECO1 is suboptimal by several metrics including relatively low brightness and limited fluorescence response (that is, ΔF/F0 for a given change in Ca 2+ concentration), which limit its utility for in vivo imaging of neuronal activity. 4

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Section: Protein Purification and In Vitro Characterizationmentioning

confidence: 99%

Improved genetically encoded near-infrared fluorescent calcium ion indicators forin vivoimaging

Qian

Cosio

Piatkevich

et al. 2020

Preprint

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“…In particular, genetically encoded sensors are usually preferred due to their ability to precisely target cells of interest. A wide range of biomolecular sensors currently exists for detecting neural activity− calcium [2][3][4], voltage [3,5], potassium [6,7] and neurotransmitters [3]. While fluorescent reporters have been the mainstay, it suffers from tissue scattering, phototoxicity, photobleaching and autofluorescence affecting its long-term use [8].…”

Section: Introductionmentioning

confidence: 99%

“…While fluorescent reporters have been the mainstay, it suffers from tissue scattering, phototoxicity, photobleaching and autofluorescence affecting its long-term use [8]. Near-infrared indicators [4,9,10] and two-photon excitation [11,12] overcome some of these limitations, but the development of efficient indicators of neural activity remains an issue.…”

Section: Introductionmentioning

confidence: 99%

“…Intracellular calcium (Ca 2+ ) concentration and membrane potential are the two wellcharacterized proxies of neuronal activities. Several genetically encoded Ca 2+ indicators (GECIs) [2][3][4]13] were developed over the last two decades of which GCaMP variants [14] are the most widely used in the neuroscience community. However, the relationship between calcium signals and neuronal activity can vary among different neuronal types and may become completely uncoupled.…”

Section: Introductionmentioning

confidence: 99%

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An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Srinivasan

Griffin

Joshi

et al. 2019

Preprint

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1.AbstractGenetically encoded reporters have greatly increased our understanding of biology, especially in neuroscience. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach we have engineered Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential. Unlike other luciferase-luciferin bioluminescent systems, AMBER encodes the genes to express both the luciferase and luciferin. AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When AMBER is co-expressed with the luciferin producing genes it reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods we show that AMBER modulates its enzymatic activity as a function of the membrane potential. AMBER shows several-fold increase in the luminescent (ΔL/L) signal upon switching from the off to on state when the cell is depolarized. In vivo expression of AMBER in C. elegans allowed detecting pharyngeal pumping action and mechanosensory neural activity from multiple worms simultaneously. AMBER reports neural activity of multiple animals at the same time and can be used in social behavior assays to elucidate the role of membrane potential underlying behavior.2.Significance StatementThere have been many exciting advances in the development of genetically encoded voltage indicators to monitor intracelluar voltage changes. Most sensors employ fluorescence, which requires external light, potentially causing photobleaching or overheating. Consequently, there has been interest in developing luminescence reporters. However, they require addition of an exogenous substrate to produce light intracellularly. Here, we engineered a genetically encoded bioluminescent voltage indicator, AMBER, which unlike other bioluminescent activity indicators, does not require addition of an exogenous substrate. AMBER allows a large differential signal, a high signal-to-noise ratio, and causes minimal metabolic demand on cells. We used AMBER to record voltage activity in freely-moving C. elegans, demonstrating that AMBER is a important new tool for monitoring neuronal activity during social behavior.

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“…Although this approach has led to robust sensors excitable with shorter wavelengths (<550 nm), analogous sensors based on redshifted fluorescent proteins have proven much more difficult to optimize. 6,7 Here, we demonstrate a new hybrid 8,9 small-molecule:protein ('chemigenetic') sensor scaffold based on the self-labeling HaloTag protein 10,11 and fluorogenic rhodamine dyes [12][13][14][15] . These chemigenetic sensors combine the genetic targetability and exquisite molecular recognition of proteins with the improved photophysical properties of synthetic fluorophores.…”

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confidence: 99%

Bright and tunable far-red chemigenetic indicators

Deo¹,

Abdelfattah²,

Bhargava³

et al. 2020

Preprint

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Functional imaging using fluorescent indicators has revolutionized biology but additional sensor scaffolds are needed to access properties such as bright, far-red emission. We introduce a new platform for 'chemigenetic' fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. This approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in neurons. Fluorescent sensors enable noninvasive measurement of cellular function. Although this field began with small-molecule fluorescent dyes, functional imaging in biology rapidly switched to protein-based sensors after the discovery of green fluorescent protein (GFP). Protein-based indicators allow cell-specific expression and take advantage of evolved molecular recognition motifs found in nature. In particular, genetically encoded calcium and voltage indicators (GECIs and GEVIs) have become powerful tools for monitoring neuronal activity in living systems. 1, 2Commonly used GECIs and GEVIs, such as GCaMP 3, 4 and ASAP 5 , rely on the use of a circularly permuted (cp) GFP fused to sensor protein domains like calmodulin (CaM) or a voltage sensitive

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A genetically encoded near-infrared fluorescent calcium ion indicator

Cited by 177 publications

References 27 publications

Improved genetically encoded near-infrared fluorescent calcium ion indicators forin vivoimaging

Improved genetically encoded near-infrared fluorescent calcium ion indicators forin vivoimaging

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

Bright and tunable far-red chemigenetic indicators

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