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
- Action Potentials physiology MeSH
- Angiotensin-Converting Enzyme 2 * MeSH
- Mutation genetics MeSH
- Mice MeSH
- Neurons physiology MeSH
- Rhodopsin * genetics MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural 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 genetics metabolism MeSH
- Cell Membrane metabolism MeSH
- Fluorescent Antibody Technique MeSH
- Ion Channel Gating radiation effects MeSH
- Protein Conformation MeSH
- Models, Molecular MeSH
- Multigene Family MeSH
- Mutation MeSH
- Proton Pumps chemistry genetics metabolism MeSH
- Rhodopsin chemistry genetics metabolism MeSH
- Spectroscopy, Fourier Transform Infrared MeSH
- Light MeSH
- Structure-Activity Relationship MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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 metabolism MeSH
- Cell Line MeSH
- Escherichia coli metabolism MeSH
- Hydrogen-Ion Concentration MeSH
- Protein Conformation MeSH
- Humans MeSH
- Liposomes MeSH
- Models, Molecular MeSH
- Optogenetics * methods MeSH
- Proton Pumps metabolism MeSH
- Protons MeSH
- Retina metabolism MeSH
- Rhodopsin chemistry metabolism MeSH
- Spectrum Analysis MeSH
- Light MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Chromatography, High Pressure Liquid MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Cell Differentiation * MeSH
- cis-trans-Isomerases genetics metabolism MeSH
- Interferon-gamma pharmacology MeSH
- Calbindin 2 genetics metabolism MeSH
- Cells, Cultured MeSH
- Mesenchymal Stem Cells cytology drug effects metabolism MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Recoverin genetics metabolism MeSH
- Retina cytology metabolism MeSH
- Rhodopsin genetics metabolism MeSH
- Carrier Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Photobiology MeSH
- Lizards MeSH
- Peptides MeSH
- Rhodopsin physiology MeSH
- Signal Transduction MeSH
- Rod Cell Outer Segment metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Comparative Study MeSH
- MeSH
- Receptors, Adrenergic, beta cytology physiology genetics MeSH
- Mutagens MeSH
- Rhodopsin physiology MeSH
- Protein Binding MeSH
- Publication type
- Review MeSH
