Curated Optogenetic Publication Database

Abstract: Light is essential for various biochemical processes in all domains of life. In its presence certain proteins inside a cell are excited, which either stimulates or inhibits subsequent cellular processes. The artificial photocontrol of specifically proteins is of growing interest for the investigation of scientific questions on the organismal, cellular and molecular level as well as for the development of medicinal drugs or biocatalytic tools. For the targeted design of photocontrol in proteins, three major methods have been developed over the last decades, which employ either chemical engineering of small-molecule photosensitive effectors (photopharmacology), incorporation of photoactive non-canonical amino acids by genetic code expansion (photoxenoprotein engineering), or fusion with photoreactive biological modules (hybrid protein optogenetics). This review compares the different methods as well as their strategies and current applications for the light-regulation of proteins and provides background information useful for the implementation of each technique.

Abstract: The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline-arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.

Abstract: Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. We found that depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

Abstract: Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling edge pulse detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.

Abstract: Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

Abstract: Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, we find that during Drosophila mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that Drosophila mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.

Abstract: Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract: Cells reside in a dynamic microenvironment that presents them with regulatory signals that vary in time, space, and amplitude. The cell, in turn, interprets these signals and accordingly initiates downstream processes including cell proliferation, differentiation, migration, and self-organization. Conventional approaches to perturb and investigate signaling pathways (e.g., agonist/antagonist addition, overexpression, silencing, knockouts) are often binary perturbations that do not offer precise control over signaling levels, and/or provide limited spatial or temporal control. In contrast, optogenetics leverages light-sensitive proteins to control cellular signaling dynamics and target gene expression and, by virtue of precise hardware control over illumination, offers the capacity to interrogate how spatiotemporally varying signals modulate gene regulatory networks and cellular behaviors. Recent studies have employed various optogenetic systems in stem cell, embryonic, and somatic cell patterning studies, which have addressed fundamental questions of how cell-cell communication, subcellular protein localization, and signal integration affect cell fate. Other efforts have explored how alteration of signaling dynamics may contribute to neurological diseases and have in the process created physiologically relevant models that could inform new therapeutic strategies. In this review, we focus on emerging applications within the expanding field of optogenetics to study gene regulation, cell signaling, neurodevelopment, and neurological disorders, and we comment on current limitations and future directions for the growth of the field.

Abstract: Organoids derived from stem cells become increasingly important to study human development and to model disease. However, methods are needed to control and study spatio-temporal patterns of gene expression in organoids. To this aim, we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes, at single-cell resolution and in programmable spatio-temporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. High-resolution spatial transcriptomic and single-cell analyses showed that this local induction was sufficient to generate stereotypically patterned organoids in three dimensions and revealed new insights into SHH’s contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.

Abstract: Lives have acquired a variety of photoreceptive proteins which absorb light in the UV to far-red region during the evolution, such as many different types of rhodopsin, blue-light receptors including cryptochrome and phototropin, and red/far-red light photochromic phytochromes. After the long-time studies on the molecular mechanism of their action, they have been applied to various photobiological studies. Recent advancement in the research field is remarkable and brought many fruitful results especially in optogenetics. To introduce some of these results, we organized a symposium named “A variety of photoreceptors and the frontiers of optogenetics” at the 59th annual meeting of the Biological Society of Japan (BSJ) in November 2021. The symposium was co-organized by a research area of the Precursory Research for Embryonic Science and Technology Program (PRESTO) named “Optical Control”, directed by Prof. Shichida (Ritsumeikan University), sponsored by Japan Science and Technology Agency (JST). We invited 4 PRESTO members and 2 other researchers to cover the light absorption region from blue to far-red (Figure 1).

Abstract: The fibroblast growth factor (FGF) family is composed of 18 secreted signaling proteins consisting of canonical FGFs and endocrine FGFs that activate four receptor tyrosine kinases (FGFRs 1-4) and four intracellular proteins (intracellular FGFs or iFGFs) that primarily function to regulate the activity of voltage-gated sodium channels and other molecules. The canonical FGFs, endocrine FGFs, and iFGFs have been reviewed extensively by us and others. In this review, we briefly summarize past reviews and then focus on new developments in the FGF field since our last review in 2015. Some of the highlights in the past 6 years include the use of optogenetic tools, viral vectors, and inducible transgenes to experimentally modulate FGF signaling, the clinical use of small molecule FGFR inhibitors, an expanded understanding of endocrine FGF signaling, functions for FGF signaling in stem cell pluripotency and differentiation, roles for FGF signaling in tissue homeostasis and regeneration, a continuing elaboration of mechanisms of FGF signaling in development, and an expanding appreciation of roles for FGF signaling in neuropsychiatric diseases. This article is categorized under: Cardiovascular Diseases > Molecular and Cellular Physiology Neurological Diseases > Molecular and Cellular Physiology Congenital Diseases > Stem Cells and Development Cancer > Stem Cells and Development.

Abstract: Recent advances in optogenetics hold promise for vision restoration in degenerative eye diseases. Optogenetics refers to techniques that use light to control the cellular activity of targeted cells. Although optogenetics is a relatively new technology, multiple therapeutic options are already being explored in pre-clinical and phase I/II clinical trials with the aim of developing novel, safe, and effective treatments for major blinding eye diseases, such as glaucoma and retinitis pigmentosa. Optogenetic approaches to visual restoration are primarily aimed at replacing lost or dysfunctional photoreceptors by inserting light-sensitive proteins into downstream retinal neurons that have no intrinsic light sensitivity. Such approaches are attractive because they are agnostic to the genetic causes of retinal degeneration, which raises hopes that all forms of retinal dystrophic and degenerative diseases could become treatable. Optogenetic strategies can also have a far-reaching impact on translational research by serving as important tools to study the pathogenesis of retinal degeneration and to identify clinically relevant therapeutic targets. For example, the CRY-CIBN optogenetic system has been recently applied to animal models of glaucoma, suggesting a potential role of OCRL in the regulation of intraocular pressure in trabecular meshwork. As optogenetic strategies are being intensely investigated, it appears crucial to consider the opportunities and challenges such therapies may offer. Here, we review the more recent promising optogenetic molecules, vectors, and applications of optogenetics for the treatment of retinal degeneration and glaucoma. We also summarize the preliminary results of ongoing clinical trials for visual restoration.

Abstract: Drug resistance remains a central challenge towards durable cancer therapy, including for cancers driven by the EML4-ALK oncogene. EML4-ALK and related fusion oncogenes form cytoplasmic protein condensates that transmit oncogenic signals through the Ras/Erk pathway. However, whether such condensates play a role in drug response or resistance development is unclear. Here, we applied optogenetic functional profiling to examine how EML4-ALK condensates impact signal transmission through transmembrane receptor tyrosine kinases (RTKs), a common route of resistance signaling. We found that condensates dramatically suppress signaling through activated RTKs including EGFR. Conversely, ALK inhibition restored and hypersensitized RTK signals. Modulation of RTK sensitivity occurred because EML4-ALK condensates sequestered downstream adapters that are required to transduce signals from both EML4-ALK and ligand-stimulated RTKs. Strikingly, EGFR hypersensitization resulted in rapid and pulsatile Erk signal reactivation within 10s of minutes of drug addition. EGFR reactivation originated from paracrine signals from neighboring apoptotic cells, and reactivation could be blocked by inhibition of either EGFR or matrix metalloproteases. Paracrine signals promoted survival during ALK inhibition, and blockade of paracrine signals accelerated cell killing and suppressed drug tolerance. Our results uncover a regulatory role for protein condensates in cancer signaling and drug response and demonstrate the potential of optogenetic profiling for drug discovery based on functional biomarkers in cancer cells.

Abstract: Continuous and ubiquitous expression of foreign genes sometimes results in harmful effects on the growth, development and metabolic activities of plants. Tissue-specific promoters help to overcome this disadvantage, but do not allow one to precisely control transgene expression over time. Thus, inducible transgene expression systems have obvious benefits. In plants, transcriptional regulation is usually driven by chemical agents under the control of chemically-inducible promoters. These systems are diverse, but usually contain two elements, the chimeric transcription factor and the reporter gene. The commonly used chemically-induced expression systems are tetracycline-, steroid-, insecticide-, copper-, and ethanol-regulated. Unlike chemical-inducible systems, optogenetic tools enable spatiotemporal, quantitative and reversible control over transgene expression with light, overcoming limitations of chemically-inducible systems. This review updates and summarizes optogenetic and chemical induction methods of transgene expression used in basic plant research and discusses their potential in field applications.

Abstract: Optogenetics combines light and genetics to enable precise control of living cells, tissues, and organisms with tailored functions. Optogenetics has the advantages of noninvasiveness, rapid responsiveness, tunable reversibility, and superior spatiotemporal resolution. Following the initial discovery of microbial opsins as light-actuated ion channels, a plethora of naturally occurring or engineered photoreceptors or photosensitive domains that respond to light at varying wavelengths has ushered in the next chapter of optogenetics. Through protein engineering and synthetic biology approaches, genetically encoded photoswitches can be modularly engineered into protein scaffolds or host cells to control a myriad of biological processes, as well as to enable behavioral control and disease intervention in vivo. Here, we summarize these optogenetic tools on the basis of their fundamental photochemical properties to better inform the chemical basis and design principles. We also highlight exemplary applications of opsin-free optogenetics in dissecting cellular physiology (designated "optophysiology") and describe the current progress, as well as future trends, in wireless optogenetics, which enables remote interrogation of physiological processes with minimal invasiveness. This review is anticipated to spark novel thoughts on engineering next-generation optogenetic tools and devices that promise to accelerate both basic and translational studies.

Abstract: Secretory trafficking from the endoplasmic reticulum (ER) is subject to regulation by extrinsic and intrinsic factors. While much of the focus has been on biochemical triggers, little is known whether and how the ER is subject to regulation by mechanical signals. Here, we show that COPII-dependent ER-export is regulated by mechanical strain. Mechanotransduction to the ER was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulate ER-to-Golgi transport. Altogether, we establish an unprecedented link between mechanical strain and export from the ER.

Abstract: Patients with Alzheimer's disease suffer from a decrease in brain mass and a prevalence of amyloid-β plaques. These plaques are thought to play a role in disease progression, but their exact role is not entirely established. We developed an optogenetic model to induce amyloid-β intracellular oligomerization to model distinct disease etiologies. Here, we examine the effect of Wnt signaling on amyloid in an optogenetic, Drosophila gut stem cell model. We observe that Wnt activation rescues the detrimental effects of amyloid expression and oligomerization. We analyze the gene expression changes downstream of Wnt that contribute to this rescue and find changes in aging related genes, protein misfolding, metabolism, and inflammation. We propose that Wnt expression reduces inflammation through repression of Toll activating factors. We confirm that chronic Toll activation reduces lifespan, but a decrease in the upstream activator Persephone extends it. We propose that the protective effect observed for lithium treatment functions, at least in part, through Wnt activation and the inhibition of inflammation.

Abstract: Optogenetics is widely used to interrogate the neural circuits underlying disease and has most recently been harnessed for therapeutic applications. The optogenetic toolkit consists of light-responsive proteins that modulate specific cellular functions, vectors for the delivery of the transgenes that encode the light-responsive proteins to targeted cellular populations, and devices for the delivery of light of suitable wavelengths at effective fluence rates. A refined toolkit with a focus towards translational uses would include efficient and safer viral and non-viral gene-delivery vectors, increasingly red-shifted photoresponsive proteins, nanomaterials that efficiently transduce near-infrared light deep into tissue, and wireless implantable light-delivery devices that allow for spatiotemporally precise interventions at clinically relevant tissue depths. In this Review, we examine the current optogenetics toolkit and the most notable preclinical and translational uses of optogenetics, and discuss future methodological and translational developments and bottlenecks.

Abstract: Advances in genetic engineering, combined with the development of optical technologies, have allowed optogenetics to broaden its area of possible applications in recent years. However, the application of optogenetic tools in industry, including biotechnology and the production of biomaterials, is still limited, because each practical task requires the engineering of a specific optogenetic system. In this review, we discuss recent advances in the use of optogenetic tools in the production of biofuels and valuable chemicals, the synthesis of biomedical and polymer materials, and plant agrobiology. We also offer a comprehensive analysis of the properties and industrial applicability of light-controlled and other smart biomaterials. These data allow us to outline the prospects for the future use of optogenetics in bioindustry.

Abstract: Owing to its ubiquity and easy availability in nature, light has been widely employed to control complex cellular behaviors. Light-sensitive proteins are the foundation to such diverse and multilevel adaptive regulations in a large range of organisms. Due to their remarkable properties and potential applications in engineered systems, exploration and engineering of natural light-sensitive proteins have significantly contributed to expand optogenetic toolboxes with tailor-made performances in synthetic genetic circuits. Progressively, more complex systems have been designed in which multiple photoreceptors, each sensing its dedicated wavelength, are combined to simultaneously coordinate cellular responses in a single cell. In this review, we highlight recent works and challenges on multiplexed optogenetic circuits in natural and engineered systems for a dynamic regulation breakthrough in biotechnological applications.

Abstract: RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.

Abstract: Optogenetics is a powerful technique that allows the control of protein function with high spatiotemporal precision using light. Here, we describe the application of this method to control tissue mechanics during Drosophila embryonic development. We detail optogenetic protocols to either increase or decrease cell contractility and analyze the interplay between cell-cell interaction, tissue geometry, and force transmission during gastrulation.

Abstract: Intracellular membrane trafficking is a dynamic and complex cellular process. To study membrane trafficking with a high spatiotemporal resolution, we present an optogenetic method based on a blue-light inducible oligomerization of Rab GTPases, termed light-activated reversible inhibition by assembly trap of intracellular membranes (IM-LARIAT). In this chapter, we focus on the optical disruption of the dynamics and functions of previously studied intracellular membrane trafficking events, including transferrin recycling and growth cone regulation in relation to specific Rab GTPases. To aid general application, we provide a detailed description of transfection, imaging with a confocal microscope, and analysis of data.

Abstract: Dynamic protein-protein interactions are essential for proper cell functioning. Homo-interaction events-physical interactions between the same type of proteins-represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways. Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms. The emerging optogenetic technology, based on genetically encoded light-sensitive proteins, provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision. Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.

Abstract: The EGFR/Erk pathway is triggered by extracellular ligand stimulation, leading to stimulus-dependent dynamics of pathway activity. Although mechanical properties of the microenvironment also affect Erk activity, their effects on Erk signaling dynamics are poorly understood. Here, we characterize how the stiffness of the underlying substratum affects Erk signaling dynamics in mammary epithelial cells. We find that soft microenvironments attenuate Erk signaling, both at steady state and in response to epidermal growth factor (EGF) stimulation. Optogenetic manipulation at multiple signaling nodes reveals that intracellular signal transmission is largely unaffected by substratum stiffness. Instead, we find that soft microenvironments decrease EGF receptor (EGFR) expression and alter the amount and spatial distribution of EGF binding at cell membranes. Our data demonstrate that the mechanical microenvironment tunes Erk signaling dynamics via receptor-ligand interactions, underscoring how multiple microenvironmental signals are jointly processed through a highly conserved pathway that regulates tissue development, homeostasis, and disease progression.