Optogenetic Tools Research Articles

Initiation of lumen formation from junctions via differential actomyosin contractility regulated by dynamic recruitment of Rasip1

De novo lumen formation necessitates the precise segregation of junctional proteins from apical surfaces, yet the underlying mechanisms remain unclear. Using a zebrafish model, we develop a series of molecular reporters, photo-convertible and optogenetic tools to study the establishment of apical domains. Our study identifies Rasip1 as one of the earliest apical proteins recruited, which suppresses actomyosin contractility at junctional patches by inhibiting NMII, thereby allowing for the sustained outward flow of junctional complexes. Following the establishment of apical compartments, Rasip1 shuttles between junctions and the apical compartments in response to local high tension. Rasip1 confines Cdh5 to junctions by suppressing apical contractility. Conversely, the recruitment of Rasip1 to junctions is regulated by Heg1 and Krit1 to modulate contractility along junctions. Overall, de novo lumen formation and maintenance depend on the precise control of contractility within apical compartments and junctions, orchestrated by the dynamic recruitment of Rasip1.

Integrated Bioelectronic and Optogenetic Methods to Study Brain-Body Circuits.

The peripheral nervous system, consisting of somatic sensory circuits and autonomic effector circuits, enables communication between the body's organs and the brain. Dysregulation in these circuits is implicated in an array of disorders and represents a potential target for neuromodulation therapies. In this Perspective, we discuss recent advances in the neurobiological understanding of these brain-body pathways and the expansion of neurotechnologies beyond the brain to the viscera. We focus primarily on the development of integrated technologies that leverage bioelectronic devices with optogenetic tools. We highlight the discovery and application of ultrapotent and red-shifted channelrhodopsins for minimally invasive optogenetics and as tools to study brain-body circuits. These innovations enable studies of freely behaving animals and have enhanced our understanding of the role physiological signals play in brain states and behavior.

A Non-Invasive and DNA-free Approach to Upregulate Mammalian Voltage-Gated Calcium Channels and Neuronal Calcium Signaling via Terahertz Stimulation.

Mammalian voltage-gated calcium channels (CaV) play critical roles in cardiac excitability, synaptic transmission, and gene transcription. Dysfunctions in CaV are implicated in a variety of cardiac and neurodevelopmental disorders. Current pharmacological approaches to enhance CaV activity are limited by off-target effects, drug metabolism issues, cytotoxicity, and imprecise modulation. Additionally, genetically-encoded channel activators and optogenetic tools are restricted by gene delivery challenges and biosafety concerns. Here a novel terahertz (THz) wave-based method to upregulate CaV1.2, a key subtype of CaV, and boost CaV1-mediated Ca2+ signaling in neurons without introducing exogenous DNA is presented. Using molecular dynamics simulations, it is shown that 42.5 THz (7.05µm, 1418cm-1) waves enhance Ca2+ conductance in CaV1.2 by resonating with the stretching mode of the -COO- group in the selectivity filter. Electrophysiological recordings and Ca2+ imaging confirm that these waves rapidly, reversibly, and non-thermally increase calcium influx of CaV1.2 in HEK293 cells and induce acute Ca2+ signals in neurons. Furthermore, this irradiation upregulates critical CaV1 signals, including CREB phosphorylation and c-Fos expression, in vitro and in vivo, without raising significant biosafety risks. This DNA-free, non-invasive approach offers a promising approach for modulating CaV gating and Ca2+ signaling and treating diseases characterized by deficits in CaV functions.

Advanced deep-tissue imaging and manipulation enabled by biliverdin reductase knockout.

We developed near-infrared (NIR) photoacoustic and fluorescence probes, as well as optogenetic tools from bacteriophytochromes, and enhanced their performance using biliverdin reductase-A knock-out model (Blvra-/-). Blvra-/- elevates endogenous heme-derived biliverdin chromophore for bacteriophytochrome-derived NIR constructs. Consequently, light-controlled transcription with IsPadC-based optogenetic tool improved up to 25-fold compared to wild-type cells, with 100-fold activation in Blvra-/- neurons. In vivo , light-induced insulin production in Blvra-/- reduced blood glucose in diabetes by ∼60%, indicating high potential for optogenetic therapy. Using 3D photoacoustic, ultrasound, and two-photon fluorescence imaging, we overcame depth limitations of recording NIR probes. We achieved simultaneous photoacoustic imaging of DrBphP in neurons and super-resolution ultrasound localization microscopy of blood vessels ∼7 mm deep in the brain, with intact scalp and skull. Two-photon microscopy provided cell-level resolution of miRFP720-expressing neurons ∼2.2 mm deep. Blvra-/- significantly enhances efficacy of biliverdin-dependent NIR systems, making it promising platform for interrogation and manipulation of biological processes.

Three-Color Protein Photolithography with Green, Red, and Far-Red Light.

Protein photolithography is an invaluable tool for generating protein microchips and regulating interactions between cells and materials. However, the absence of light-responsive molecules that allow for the copatterning of multiple functional proteins with biocompatible visible light poses a significant challenge. Here, a new approach for photopatterning three distinct proteins on a single surface by using green, red, and far-red light is reported. The cofactor of the green light-sensitive protein CarH is engineered such that it also becomes sensitive to red and far-red light. These new cofactors are shown to be compatible with two CarH-based optogenetic tools to regulate bacterial cell-cell adhesions and gene expression in mammalian cells with red and far-red light. Further, by incorporating different CarH variants with varying light sensitivities in layer-by-layer (LbL) multiprotein films, specific layers within the films, along with other protein layers on top are precisely removed by using different colors of light, all with high spatiotemporal accuracy. Notably, with these three distinct colors of visible light, it is possible to incorporate diverse proteins under mild conditions in LbL films based on the reliable interaction between Ni2+- nitrilotriacetic acid (NTA) groups and polyhistidine-tags (His-tags)on the proteins and their subsequent photopatterning. This approach has potential applications spanning biofabrication, material engineering, and biotechnology.

Active state structures of a bistable visual opsin bound to G proteins

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the Gi and Gq heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways.

Optogenetic Control of the Mitochondrial Protein Import in Mammalian Cells.

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

Aberrant tau condensates as catalytic microcompartments propel tau fibrillation

In this issue of Structure, Soeda etal.1 employed optogenetic tools and demonstrate that an N-terminal truncation of tau and microtubule-binding deficiency lead to the formation of tau condensates, accelerating its fibrillation.

The Roles of an Extended N-terminal Region and ETD Motif in a Pump-like Cation Channelrhodopsin Discovered in a Lake Microbiome

Channelrhodopsins are light-gated ion channels consisting of seven-transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the “DTD motif” in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.

Molecular Diversity of Photosensitive Protein Opsins and Their High Potential for Optogenetic Applications.

Because G protein coupled receptors (GPCRs) represent the largest family of drug targets in clinical trials, GPCR signaling cascades are closely related to various physiological phenomena, attracting significant attention in pharmaceutical science. Opsins (also known as animal rhodopsins) are photoreceptive proteins containing retinal as a chromophore, which function as GPCRs and underlie the molecular basis of photoreception in animals. Recently, opsins have been progressively applied in an innovative technology called optogenetics to regulate biological activities using light. A wide variety of opsins have been identified in metazoans and characterized at the molecular and physiological levels, providing a foundation for their optogenetic applications. In this review, I briefly introduce the diversity of opsins in terms of their molecular functions, including G protein selectivity and photoreaction properties. This diversity provides a significant advantage for optically manipulating a wide variety of GPCR signaling cascades with high temporal resolution. Additionally, I discuss the rich array of opsin-based optogenetic tools used to control various physiological processes and their potential as therapeutic tools for vision restoration. Based on the introduction, I expect that the optogenetic approach will offer powerful tools to provide valuable insights into the molecular mechanisms of various physiological phenomena and next-generation treatment options for diseases beyond the capacity of traditional drugs.

Controlling spatial optical illuminations in optogenetics using an angled optical fiber tip

The advent of optogenetic tools has revolutionized neuroscience research through its spatiotemporally precise activation of specific neurons by illuminating light on opsin-expressing neurons. A long-standing challenge of in vivo optogenetics remains in delivering light to multiple brain sites simultaneously and maintaining high spatial resolution. Optical fiber-based technologies have been proposed to address these challenges. This work presents the fabrication and characterization of an innovative angled optical fiber probe based on a double-sided angled tip (DSAT) structure. A custom griding setup was used for the reproducible fabrication of a smooth DSAT probe. The designed probe enables precise spatial control of light propagation in brain tissue in which DSAT at angled tip 55° achieving a maximum lateral illumination position of ± 420 μm away from the optical axis and a peak irradiance of 478.5 mW/mm2, using a 5 mW of 473 nm laser light. Also, the designed DAST probe was simulated based on ray tracing method and obtained the practical tip angle to evaluate the propagation of light rays emitted from the DSAT at various input optical angles ranging from 0° to 12.5° to predict their irradiance and positions in the modelled tissue. The results indicate the probe generates two elliptical rings each containing two spots with higher optical concentration. Consequently, this device provides four optical spots with irradiance peaks that enable simultaneous illumination of four different locations in the brain tissue. Obtained experiment results are in good agreement with simulation results which can be used for multipoint illumination of brain tissue in optogenetics applications.

Neuroethology: A Review of Studies on Combat Behavior in Mice

Optogenetics has revolutionized neuroscience, enabling precise control and monitoring of neural circuits. This study focuses on the neural mechanisms underlying combat behaviors in mice, particularly the roles of the ventromedial hypothalamus (VMH) in freezing, jumping escape, and defensive attack behaviors. The research employed a combination of optogenetic tools, behavioral assays, and high-resolution imaging techniques. Genetically modified mice expressing channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR) were used. Viral vectors were injected into specific brain regions, and optogenetic stimulation was performed to activate or inhibit neuronal populations. Behavioral responses were documented and analyzed using specialized software, and brain tissues were prepared for histological examination. Activation of VMH neurons increased jumping escape behavior, while inhibition reduced defensive attack behaviors, highlighting the VMH's critical role in these responses. High-resolution video analysis provided detailed metrics on behavior, and histological examination confirmed precise targeting and expression of optogenetic proteins in the brain. The results demonstrated the direct influence of specific neural circuits on combat behaviors. The study elucidated the roles of VMH and associated neural circuits in mediating combat behaviors in mice. Optogenetic techniques provided precise control over neuronal activity, revealing critical insights into the neural mechanisms underlying these behaviors. The findings support the VMH's central role in regulating instinctual defensive actions and offer potential applications for developing therapeutic strategies for anxiety and pain disorders.

Optogenetic Tools for Regulating RNA Metabolism and Functions.

RNA molecules play a vital role in linking genetic information with various cellular processes. In recent years, a variety of optogenetic tools have been engineered for regulating cellular RNA metabolism and functions. These highly desirable tools can offer non-intrusive control with spatial precision, remote operation, and biocompatibility. Here, we would like to review these currently available approaches that can regulate RNAs with light: from non-genetically encodable chemically modified oligonucleotides to genetically encoded RNA aptamers that recognize photosensitive small-molecule or protein ligands. Some key applications of these optogenetic tools will also be highlighted to illustrate how they have been used for regulating all aspects of the RNA life cycle: from RNA synthesis, maturation, modification, and translation to their degradation, localization, and phase separation control. Some current challenges and potential practical utilizations of these RNA optogenetic tools will also be discussed.

Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy

Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven Cl− inward pump that is widely used as an optogenetic tool. Although NpHR is previously extensively studied, its Cl− uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl− uptake process. In this study, we used solid-state NMR to analyze NpHR both in the Cl−-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl− depletion is widespread over the NpHR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl− uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on NpHR point mutants for the suggested residues, we confirmed their importance in the Cl− uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl−-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known NpHR crystal structures, we proposed a model that this helix movement is required for creating the Cl− entrance path on the extracellular surface of the protein and is crucial to the Cl− uptake process.

Simultaneous spectral illumination of microplates for high-throughput optogenetics and photobiology.

The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis ofaction spectra. Current methods to determine action spectraonly allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validatedthe RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

Dye-Based Fluorescent Organic Nanoparticles, New Promising Tools for Optogenetics.

Dye-based fluorescent organic nanoparticles are a specific class of nanoparticles obtained by nanoprecipitation in water of pure dyes only. While the photophysical and colloidal properties of the nanoparticles strongly depend on the nature of the aggregated dyes, their excellent brightness in the visible and in the near infrared make these nanoparticles a unique and versatile platform for in vivo application. This article examines the promising utilization of these nanoparticles for in vivo optogenetics applications. Their photophysical properties as well as their biocompatibility and their capacity to activate Chrimson opsin in vivo through the fluorescence reabsorption process are demonstrated. Additionally, an illustrative example of employing these nanoparticles in fear reduction in mice through closed-loop stimulation is presented. Through an optogenetic methodology, the nanoparticles demonstrate an ability to selectively manipulate neurons implicated in the fear response and diminish the latter. Dye-based fluorescent organic nanoparticles represent a promising and innovative strategy for optogenetic applications, holding substantial potential in the domain of translational neuroscience. This work paves the way for novel therapeutic modalities for neurological and neuropsychiatricdisorders.

Optogenetic control of early embryos labeling using photoactivatable Cre recombinase 3.0.

Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

In vivo optogenetic manipulations of endogenous proteins reveal spatiotemporal roles of microtubule and kinesin in dendrite patterning.

During animal development, the spatiotemporal properties of molecular events largely determine the biological outcomes. Conventional gene analysis methods lack the spatiotemporal resolution for precise dissection of developmental mechanisms. Although optogenetic tools exist for manipulating designer proteins in cultured cells, few have been successfully applied to endogenous proteins in live animals. Here, we report OptoTrap, a light-inducible clustering system for manipulating endogenous proteins of diverse sizes, subcellular locations, and functions in Drosophila. This system turns on fast, is reversible in minutes or hours, and contains variants optimized for neurons and epithelial cells. By using OptoTrap to disrupt microtubules and inhibit kinesin-1 in neurons, we show that microtubules support the growth of highly dynamic dendrites and that kinesin-1 is required for patterning of low- and high-order dendritic branches in differential spatiotemporal domains. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Optogenetic manipulation of lysosomal physiology and autophagic activity.

Lysosomes are essential degradative organelles and signaling hubs within cells, playing a crucial role in the regulation of macroautophagy/autophagy. Dysfunction of lysosomes and impaired autophagy are closely associated with the development of various neurodegenerative diseases. Enhancing lysosomal activity and boosting autophagy levels holds great promise as effective strategies for treating these diseases. However, there remains a lack of methods to dynamically regulate lysosomal activity and autophagy levels in living cells or animals. In our recent work, we applied optogenetics to manipulate lysosomal physiology and function, developing three lysosome-targeted optogenetic tools: lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2. These new actuators enable light-dependent regulation of key aspects such as lysosomal membrane potential, lumenal pH, hydrolase activity, degradation processes, and Ca2+ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy via the MTOR pathway while it promotes Aβ clearance through autophagy induction in cellular models of Alzheimer disease. Furthermore, lyso-ChR2 activation reduces Aβ deposition and alleviates Aβ-induced paralysis in Caenorhabditis elegans models of Alzheimer disease. Our lysosomal optogenetic actuators offer a novel method for dynamically regulating lysosomal physiology and autophagic activity in living cells and animals.