The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.
- MeSH
- akční potenciály fyziologie MeSH
- angiotensin-konvertující enzym 2 * MeSH
- mutace genetika MeSH
- myši MeSH
- neurony fyziologie MeSH
- rodopsin * genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
N-terminal P23H opsin mutation accounts for most of retinitis pigmentosa (RP) cases. P23H functions and folding can be rescued by small chaperone ligands, which contributes to validate mutant opsin as a suitable target for pharmacological treatment of RP. However, the lack of structural details on P23H mutant opsin strongly impairs drug design, and new chemotypes of effective chaperones of P23H opsin are in high demand. Here, a computational-boosted workflow combining homology modeling with molecular dynamics (MD) simulations and virtual screening was used to select putative P23H opsin chaperones among different libraries through a structure-based approach. In vitro studies corroborated the reliability of the structural model generated in this work and identified a number of novel chemotypes of safe and effective chaperones able to promote P23H opsin trafficking to the outer cell membrane.
- MeSH
- lidé MeSH
- molekulární chaperony genetika metabolismus terapeutické užití MeSH
- opsiny * genetika MeSH
- reprodukovatelnost výsledků MeSH
- retinopathia pigmentosa * farmakoterapie genetika metabolismus MeSH
- tyčinkové opsiny chemie genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.
- MeSH
- Archaea genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- fluorescenční protilátková technika MeSH
- gating iontového kanálu účinky záření MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- multigenová rodina MeSH
- mutace MeSH
- protonové pumpy chemie genetika metabolismus MeSH
- rodopsin chemie genetika metabolismus MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- světlo MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In deep-water animals, the visual sensory system is often challenged by the dim-light environment. Here, we focus on the molecular mechanisms involved in rapid deep-water adaptations. We examined visual system evolution in a small-scale yet phenotypically and ecologically diverse adaptive radiation, the species flock of cichlid fishes in deep crater lake Barombi Mbo in Cameroon, West Africa. We show that rapid adaptations of the visual system to the novel deep-water habitat primarily occurred at the level of gene expression changes rather than through nucleotide mutations, which is compatible with the young age of the radiation. Based on retinal bulk RNA sequencing of all eleven species, we found that the opsin gene expression pattern was substantially different for the deep-water species. The nine shallow-water species feature an opsin palette dominated by the red-sensitive (LWS) opsin, whereas the two unrelated deep-water species lack expression of LWS and the violet-sensitive (SWS2B) opsin, thereby shifting the cone sensitivity to the centre of the light spectrum. Deep-water species further predominantly express the green-sensitive RH2Aα over RH2Aβ. We identified one amino acid substitution in the RH2Aα opsin specific to the deep-water species. We finally performed a comparative gene expression analysis in retinal tissue of deep- vs. shallow-water species. We thus identified 46 differentially expressed genes, many of which are associated with functions in vision, hypoxia management or circadian clock regulation, with some of them being associated with human eye diseases.
- MeSH
- cichlidy genetika fyziologie MeSH
- čípky retiny - opsiny genetika MeSH
- druhová specificita MeSH
- ekosystém MeSH
- fylogeneze MeSH
- jezera MeSH
- molekulární evoluce * MeSH
- regulace genové exprese genetika MeSH
- retina metabolismus fyziologie MeSH
- sekvenční analýza RNA MeSH
- světlo MeSH
- zrak genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Kamerun MeSH
During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5-dopamine pathway, regulates optic axis clearance in preparation for visual function.
- MeSH
- cévní endotel metabolismus MeSH
- dopamin metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- oči krevní zásobení enzymologie růst a vývoj metabolismus MeSH
- opsiny genetika metabolismus MeSH
- proteiny přenášející dopamin přes plazmatickou membránu antagonisté a inhibitory chemie metabolismus MeSH
- retinální gangliové buňky metabolismus účinky záření MeSH
- sklivec metabolismus MeSH
- světlo * MeSH
- threonin metabolismus MeSH
- transportéry VIAAT fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The red fox (Vulpes vulpes) is the carnivore with the widest distribution in the world. Not much is known about the visual system of these predominantly forest-dwelling animals. The closely related Arctic fox (Vulpes lagopus) lives in more open tundra habitats. In search for corresponding adaptations, we examined the photoreceptors and retinal ganglion cells (RGCs), using opsin immunohistochemistry, lucifer yellow injections and Nissl staining. Both species possess a majority of middle-to-longwave-sensitive (M/L) and a minority of shortwave-sensitive (S) cones, indicating dichromatic color vision. Area centralis peak cone densities are 22,600/mm2 in the red fox and 44,800/mm2 in the Arctic fox. Both have a centro-peripheral density decrease of M/L cones, and a dorsoventrally increasing density of S cones. Rod densities and rod/cone ratios are higher in the red fox than the Arctic fox. Both species possess the carnivore-typical alpha and beta RGCs. The RGC topography shows a centro-peripheral density gradient with a distinct area centralis (mean peak density 7,900 RGCs/mm2 in the red fox and 10,000 RGCs/mm2 in the Arctic fox), a prominent visual streak of higher RGC densities in the Arctic fox, and a moderate visual streak in the red fox. Visual acuity and estimated sound localization ability were nearly identical between both species. In summary, the red fox retina shows adaptations to nocturnal activity in a forest habitat, while the Arctic fox retina is better adapted to higher light levels in the open tundra.
- MeSH
- čípky retiny fyziologie MeSH
- druhová specificita MeSH
- fotoreceptory obratlovců fyziologie MeSH
- imunohistochemie MeSH
- lišky fyziologie MeSH
- lokalizace zvuku fyziologie MeSH
- oči anatomie a histologie MeSH
- opsiny metabolismus MeSH
- retinální gangliové buňky fyziologie MeSH
- tyčinky retiny fyziologie MeSH
- vidění barevné fyziologie MeSH
- životní prostředí MeSH
- zraková ostrost fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Mammals usually possess a majority of medium-wavelength sensitive (M-) and a minority of short-wavelength sensitive (S-) opsins in the retina, enabling dichromatic vision. Unexpectedly, subterranean rodents from the genus Fukomys exhibit an S-opsin majority, which is exceptional among mammals, albeit with no apparent adaptive value. Because thyroid hormones (THs) are pivotal for M-opsin expression and metabolic rate regulation, we have, for the first time, manipulated TH levels in the Ansell's mole-rat (Fukomys anselli) using osmotic pumps. In Ansell's mole-rats, the TH thyroxine (T4) is naturally low, likely as an adaptation to the harsh subterranean ecological conditions by keeping resting metabolic rate (RMR) low. We measured gene expression levels in the eye, RMR, and body mass (BM) in TH-treated animals. T4 treatment increased both, S- and M-opsin expression, albeit M-opsin expression at a higher degree. However, this plasticity was only given in animals up to approximately 2.5 years. Mass-specific RMR was not affected following T4 treatment, although BM decreased. Furthermore, the T4 inactivation rate is naturally higher in F. anselli compared to laboratory rodents. This is the first experimental evidence that the S-opsin majority in Ansell's mole-rats is a side effect of low T4, which is downregulated to keep RMR low.
- MeSH
- bazální metabolismus účinky léků MeSH
- čípky retiny - opsiny genetika metabolismus MeSH
- mikroftalmičtí podzemní hlodavci krev metabolismus MeSH
- retina metabolismus MeSH
- thyroxin krev nedostatek MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transposable elements (TEs) are able to jump to new locations (transposition) in the genome, usually after replication. They constitute the so-called selfish or junk DNA and take over large proportions of some genomes. Due to their ability to move around they can change the DNA landscape of genomes and are therefore a rich source of innovation in genes and gene regulation. Surge of sequence data in the past years has significantly facilitated large scale comparative studies. Cephalochordates have been regarded as a useful proxy to ancestral chordate condition partially due to the comparatively slow evolutionary rate at morphological and genomic level. In this study, we used opsin gene family from three Branchiostoma species as a window into cephalochordate genome evolution. We compared opsin complements in terms of family size, gene structure and sequence allowing us to identify gene duplication and gene loss events. Furthermore, analysis of the opsin containing genomic loci showed that they are populated by TEs. In summary, we provide evidence of the way transposable elements may have contributed to the evolution of opsin gene family and to the shaping of cephalochordate genomes in general.
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.
- MeSH
- Archaea metabolismus MeSH
- buněčné linie MeSH
- Escherichia coli metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- lidé MeSH
- liposomy MeSH
- molekulární modely MeSH
- optogenetika * metody MeSH
- protonové pumpy metabolismus MeSH
- protony MeSH
- retina metabolismus MeSH
- rodopsin chemie metabolismus MeSH
- spektrální analýza MeSH
- světlo MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Retinal disorders represent the main cause of decreased quality of vision and even blindness worldwide. The loss of retinal cells causes irreversible damage of the retina, and there are currently no effective treatment protocols for most retinal degenerative diseases. A promising approach for the treatment of retinal disorders is represented by stem cell-based therapy. The perspective candidates are mesenchymal stem cells (MSCs), which can differentiate into multiple cell types and produce a number of trophic and growth factors. In this study, we show the potential of murine bone marrow-derived MSCs to differentiate into cells expressing retinal markers and we identify the key supportive role of interferon-γ (IFN-γ) in the differentiation process. MSCs were cultured for 7 days with retinal extract and supernatant from T-cell mitogen concanavalin A-stimulated splenocytes, simulating the inflammatory site of retinal damage. MSCs cultured in such conditions differentiated to the cells expressing retinal cell markers such as rhodopsin, S antigen, retinaldehyde-binding protein, calbindin 2, recoverin, and retinal pigment epithelium 65. To identify a supportive molecule in the supernatants from activated spleen cells, MSCs were cultured with retinal extract in the presence of various T-cell cytokines. The expression of retinal markers was enhanced only in the presence of IFN-γ, and the supportive role of spleen cell supernatants was abrogated with the neutralization antibody anti-IFN-γ. In addition, differentiated MSCs were able to express a number of neurotrophic factors, which are important for retinal regeneration. Taken together, the results show that MSCs can differentiate into cells expressing retinal markers and that this differentiation process is supported by IFN-γ.
- MeSH
- buněčná diferenciace * MeSH
- cis-trans-isomerasy genetika metabolismus MeSH
- interferon gama farmakologie MeSH
- kalbindin 2 genetika metabolismus MeSH
- kultivované buňky MeSH
- mezenchymální kmenové buňky cytologie účinky léků metabolismus MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- rekoverin genetika metabolismus MeSH
- retina cytologie metabolismus MeSH
- rodopsin genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
