Genetically encoded Ca(2+) indicators; expanded affinity range, color hue and compatibility with optogenetics.
Ontology highlight
ABSTRACT:
SUBMITTER: Nagai T
PROVIDER: S-EPMC4243560 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
Publications
Genetically encoded Ca(2+) indicators; expanded affinity range, color hue and compatibility with optogenetics.
Nagai Takeharu T Horikawa Kazuki K Saito Kenta K Matsuda Tomoki T
Frontiers in molecular neuroscience 20141125
Similar Datasets
Project description:Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.
| S-EPMC3560286 | biostudies-literature
Project description:Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, "RCaMPs," engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca(2+)-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca(2+)]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca(2+) affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.
| S-EPMC3586699 | biostudies-literature
Project description:Cyclic AMP is a ubiquitous second messenger, which mediates many cellular responses mainly initiated by activation of cell surface receptors. Various Förster resonance energy transfer-based ratiometric cAMP indicators have been created for monitoring the spatial and temporal dynamics of cAMP at the single-cell level. However, single fluorescent protein-based cAMP indicators have been poorly developed, with improvement required for dynamic range and brightness. Based on our previous yellow fluorescent protein-based cAMP indicator, Flamindo, we developed an improved yellow fluorescent cAMP indicator named Flamindo2. Flamindo2 has a 2-fold expanded dynamic range and 8-fold increased brightness compared with Flamindo by optimization of linker peptides in the vicinity of the chromophore. We found that fluorescence intensity of Flamindo2 was decreased to 25% in response to cAMP. Live-cell cAMP imaging of the cytosol and nucleus in COS7 cells using Flamindo2 and nlsFlamindo2, respectively, showed that forskolin elevated cAMP levels in each compartment with different kinetics. Furthermore, dual-color imaging of cAMP and Ca2+ with Flamindo2 and a red fluorescent Ca2+ indicator, R-GECO, showed that cAMP and Ca2+ elevation were induced by noradrenaline in single HeLa cells. Our study shows that Flamindo2, which is feasible for multi-color imaging with other intracellular signaling molecules, is useful and is an alternative tool for live-cell imaging of intracellular cAMP dynamics.
| S-EPMC4069001 | biostudies-literature
Project description:Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.
| S-EPMC5557009 | biostudies-other
Project description:Imaging voltage using fluorescent-based sensors could be an ideal technique to probe neural circuits with high spatiotemporal resolution. However, due to insufficient signal-to-noise ratio (SNR), imaging membrane potential in mammalian preparations is still challenging. In recent years, many genetically encoded voltage indicators (GEVIs) have been developed. To compare them and guide decisions on which GEVI to use, we have characterized side by side the performance of eight GEVIs that represent different families of molecular constructs. We tested GEVIs in vitro with 1-photon imaging and in vivo with 1-photon wide-field imaging and 2-photon imaging. We find that QuasAr2 exhibited the best performance in vitro, whereas only ArcLight-MT could be used to reliably detect electrical activity in vivo with 2-photon excitation. No single GEVI was ideal for every experiment. These results provide a guide for choosing optimal GEVIs for specific applications.
| S-EPMC7075032 | biostudies-literature
Project description:Coupling of excitation to secretion, contraction and transcription often relies on Ca(2+) computations within the nanodomain-a conceptual region extending tens of nanometers from the cytoplasmic mouth of Ca(2+) channels. Theory predicts that nanodomain Ca(2+) signals differ vastly from the slow submicromolar signals routinely observed in bulk cytoplasm. However, direct visualization of nanodomain Ca(2+) far exceeds optical resolution of spatially distributed Ca(2+) indicators. Here we couple an optical, genetically encoded Ca(2+) indicator (TN-XL) to the carboxy tail of Ca(V)2.2 Ca(2+) channels, enabling near-field imaging of the nanodomain. Under total internal reflection fluorescence microscopy, we detect Ca(2+) responses indicative of large-amplitude pulses. Single-channel electrophysiology reveals a corresponding Ca(2+) influx of only 0.085 pA, and fluorescence resonance energy transfer measurements estimate TN-XL distance to the cytoplasmic mouth at ~55?Å. Altogether, these findings raise the possibility that Ca(2+) exits the channel through the analogue of molecular portals, mirroring the crystallographic images of side windows in voltage-gated K channels.
| S-EPMC3615648 | biostudies-literature
Project description:Genetically encoded optical indicators of neuronal activity enable unambiguous recordings of input-output activity patterns from identified cells in intact circuits. Among them, genetically encoded voltage indicators (GEVIs) offer additional advantages over calcium indicators as they are direct sensors of membrane potential and can adeptly report subthreshold events and hyperpolarization. Here, we outline the major GEVI designs and give an account of properties that need to be carefully optimized during indicator engineering. While designing the ideal GEVI, one should keep in mind aspects such as membrane localization, signal size, signal-to-noise ratio, kinetics and voltage dependence of optical responses. Using ArcLight and derivatives as prototypes, we delineate how a probe should be optimized for the former properties and developed along other areas in a need-based manner. Finally, we present an overview of the GEVI engineering process and lend an insight into their discovery, delivery and diagnosis.
| S-EPMC6399427 | biostudies-literature
Project description:Genetically encoded calcium indicators (GECIs), based on recombinant fluorescent proteins, have been engineered to observe calcium transients in living cells and organisms. Through observation of calcium, these indicators also report neural activity. We review progress in GECI construction and application, particularly toward in vivo monitoring of sparse action potentials (APs). We summarize the extrinsic and intrinsic factors that influence GECI performance. A simple model of GECI response to AP firing demonstrates the relative significance of these factors. We recommend a standardized protocol for evaluating GECIs in a physiologically relevant context. A potential method of simultaneous optical control and recording of neuronal circuits is presented.
| S-EPMC2755531 | biostudies-literature
Project description:Local adaptation is critical in speciation and evolution, yet comprehensive studies on proximate and ultimate causes of local adaptation are generally scarce. Here, we integrated field ecological experiments, genome sequencing, and genetic verification to demonstrate both driving forces and molecular mechanisms governing local adaptation of body coloration in a lizard from the Qinghai-Tibet Plateau. We found dark lizards from the cold meadow population had lower spectrum reflectance but higher melanin contents than light counterparts from the warm dune population. Additionally, the colorations of both dark and light lizards facilitated the camouflage and thermoregulation in their respective microhabitat simultaneously. More importantly, by genome resequencing analysis, we detected a novel mutation in Tyrp1 that underpinned this color adaptation. The allele frequencies at the site of SNP 459# in the gene of Tyrp1 are 22.22% G/C and 77.78% C/C in dark lizards and 100% G/G in light lizards. Model-predicted structure and catalytic activity showed that this mutation increased structure flexibility and catalytic activity in enzyme TYRP1, and thereby facilitated the generation of eumelanin in dark lizards. The function of the mutation in Tyrp1 was further verified by more melanin contents and darker coloration detected in the zebrafish injected with the genotype of Tyrp1 from dark lizards. Therefore, our study demonstrates that a novel mutation of a major melanin-generating gene underpins skin color variation co-selected by camouflage and thermoregulation in a lizard. The resulting strong selection may reinforce adaptive genetic divergence and enable the persistence of adjacent populations with distinct body coloration.
| S-EPMC10835340 | biostudies-literature
Project description:Chronic monitoring of neuronal activity in the living brain with optical imaging techniques became feasible owing to the continued development of genetically encoded calcium indicators (GECIs). Here we report for the first time the successful generation of transgenic marmosets (Callithrix jacchus), an important nonhuman primate model in neurophysiological research, which were engineered to express the green fluorescent protein (GFP)-based family of GECIs, GCaMP, under control of either the CMV or the hSyn promoter. High titer lentiviral vectors were produced, and injected into embryos collected from donor females. The infected embryos were then transferred to recipient females. Eight transgenic animals were born and shown to have stable and functional GCaMP expression in several different tissues. Germline transmission of the transgene was confirmed in embryos generated from two of the founder transgenic marmosets that reached sexual maturity. These embryos were implanted into six recipient females, three of which became pregnant and are in advanced stages of gestation. We believe these transgenic marmosets will be invaluable non-human primate models in neuroscience, allowing chronic in vivo monitoring of neural activity with functional confocal and multi-photon optical microscopy imaging of intracellular calcium dynamics.
| S-EPMC5057151 | biostudies-literature