Curated Optogenetic Publication Database

Abstract: Optogenetics is a versatile and powerful tool for the control and analysis of cellular signaling processes. The activation of cellular receptors by light using optogenetic switches usually requires genetic manipulation of cells. However, this considerably limits the application in primary, nonengineered cells, which is crucial for the study of physiological signaling processes and for controlling cell fate and function for therapeutic purposes. To overcome this limitation, we developed a system for the light-dependent extracellular activation of cell surface receptors of nonengineered cells termed OptoREACT (Optogenetic Receptor Activation) based on the light-dependent protein interaction of A. thaliana phytochrome B (PhyB) with PIF6. In the OptoREACT system, a PIF6-coupled antibody fragment binds the T cell receptor (TCR) of Jurkat or primary human T cells, which upon illumination is bound by clustered phytochrome B to induce receptor oligomerization and activation. For clustering of PhyB, we either used tetramerization by streptavidin or immobilized PhyB on the surface of cells to emulate the interaction of a T cell with an antigen-presenting cell. We anticipate that this extracellular optogenetic approach will be applicable for the light-controlled activation of further cell surface receptors in primary, nonengineered cells for versatile applications in fundamental and applied research.

Abstract: Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.

Abstract: Alzheimer's disease (AD) is the most common form of dementia, resulting in disability and mortality. The global incidence of AD is consistently surging. Although numerous therapeutic agents with promising potential have been developed, none have successfully treated AD to date. Consequently, the pursuit of novel methodologies to address neurodegenerative processes in AD remains a paramount endeavor. A particularly promising avenue in this search is optogenetics, enabling the manipulation of neuronal activity. In recent years, research attention has pivoted from neurons to glial cells. This review aims to consider the potential of the optogenetic correction of astrocyte metabolism as a promising strategy for correcting AD-related disorders. The initial segment of the review centers on the role of astrocytes in the genesis of neurodegeneration. Astrocytes have been implicated in several pathological processes associated with AD, encompassing the clearance of β-amyloid, neuroinflammation, excitotoxicity, oxidative stress, and lipid metabolism (along with a critical role in apolipoprotein E function). The effect of astrocyte-neuronal interactions will also be scrutinized. Furthermore, the review delves into a number of studies indicating that changes in cellular calcium (Ca2+) signaling are one of the causes of neurodegeneration. The review's latter section presents insights into the application of various optogenetic tools to manipulate astrocytic function as a means to counteract neurodegenerative changes.

Abstract: Optogenetics offers a set of tools for the precise manipulation of signaling pathways. Here we exploit optogenetics to experimentally change the kinetics of protein-protein interactions on demand. We had developed a system in which the interaction of a modified T cell receptor (TCR) with an engineered ligand can be controlled by light. The ligand was the plant photoreceptor phytochrome B (PhyB) and the TCR included a TCRβ chain fused to GFP and a mutated PhyB-interacting factor (PIFS), resulting in the GFP-PIFS-TCR. We failed to engineer a nonfluorescent PIFS-fused TCR, since PIFS did not bind to PhyB when omitting GFP. Here we tested nine different versions of PIFS-fused TCRs. We found that the SNAP-PIFS-TCR was expressed well on the surface, bound to PhyB, and subsequently elicited activation signals. This receptor could be combined with a GFP reporter system in which the expression of GFP is driven by the transcription factor NF-AT.

Abstract: The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Abstract: Metabolic engineering approaches do not exclusively require fine-tuning of heterologous genes but oftentimes also modulation or even induction of host gene expression, e.g., in order to rewire metabolic fluxes. Here, we introduce the programmable red light switch PhiReX 2.0, which can rewire metabolic fluxes by targeting endogenous promoter sequences through single-guide RNAs (sgRNAs) and activate gene expression in Saccharomyces cerevisiae upon red light stimulation. The split transcription factor is built from the plant-derived optical dimer PhyB and PIF3, which is fused to a DNA-binding domain based on the catalytically dead Cas9 protein (dCas9) and a transactivation domain. This design combines at least two major advantages: first, the sgRNAs, guiding dCas9 to the promoter of interest, can be exchanged in an efficient and straightforward Golden Gate-based cloning approach, which allows for rational or randomized combination of up to four sgRNAs in a single expression array. Second, target gene expression can be rapidly upregulated by short red light pulses in a light dose-dependent manner and returned to the native expression level by applying far-red light without interfering with the cell culture. Using the native yeast gene CYC1 as an example, we demonstrated that PhiReX 2.0 can upregulate CYC1 gene expression by up to 6-fold in a light intensity-dependent and reversible manner using a single sgRNA.

Abstract: Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

Abstract: Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms.

Abstract: Gene- and cell-based therapies are the next frontiers in the field of medicine. Both are transformative and innovative therapies; however, a lack of safety data limits the translation of such promising technologies to the clinic. Improving the safety and promoting the clinical translation of these therapies can be achieved by tightly regulating the release and delivery of therapeutic outputs. In recent years, the rapid development of optogenetic technology has provided opportunities to develop precision-controlled gene- and cell-based therapies, in which light is introduced to precisely and spatiotemporally manipulate the behaviour of genes and cells. This review focuses on the development of optogenetic tools and their applications in biomedicine, including photoactivated genome engineering and phototherapy for diabetes and tumours. The prospects and challenges of optogenetic tools for future clinical applications are also discussed.

Abstract: To understand cell biological processes, like signalling pathways, protein movements, or metabolic processes, precise tools for manipulation are desired. Optogenetics allows to control cellular processes by light and can be applied at a high temporal and spatial resolution. In the last three decades, various optogenetic applications have been developed for animal, fungal, and prokaryotic cells. However, using optogenetics in plants has been difficult due to biological and technical issues, like missing cofactors, the presence of endogenous photoreceptors, or the necessity of light for photosynthesis, which potentially activates optogenetic tools constitutively. Recently developed tools overcome these limitations, making the application of optogenetics feasible also in plants. Here, we highlight the most useful recent applications in plants and give a perspective for future optogenetic approaches in plants science.

Abstract: Gene and cell therapies are widely recognized as future cancer therapeutics but poor controllability limits their clinical applications. Optogenetics, the use of light-controlled proteins to precisely spatiotemporally regulate the activity of genes and cells, opens up new possibilities for cancer treatment. Light of specific wavelength can activate the immune response, oncolytic activity and modulate cell signaling in tumor cells non-invasively, in dosed manner, with tissue confined action and without side effects of conventional therapies. Here, we review optogenetic approaches in cancer research, their clinical potential and challenges of incorporating optogenetics in cancer therapy. We critically discuss beneficial combinations of optogenetic technologies with therapeutic nanobodies, T-cell activation and CAR-T cell approaches, genome editors and oncolytic viruses. We consider viral vectors and nanoparticles for delivering optogenetic payloads and activating light to tumors. Finally, we highlight herein the prospects for integrating optogenetics into immunotherapy as a novel, fast, reversible and safe approach to cancer treatment.

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: 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: 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: 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: Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.

Abstract: Many organs and tissues have an intrinsic ability to regenerate from a dedicated, tissue-specific stem cell pool. As organisms age, the process of self-regulation or homeostasis begins to slow down with fewer stem cells available for tissue repair. Tissues become more fragile and organs less efficient. This slowdown of homeostatic processes leads to the development of cellular and neurodegenerative diseases. In this review, we highlight the recent use and future potential of optogenetic approaches to study homeostasis. Optogenetics uses photosensitive molecules and genetic engineering to modulate cellular activity in vivo, allowing precise experiments with spatiotemporal control. We look at applications of this technology for understanding the mechanisms governing homeostasis and degeneration as applied to widely used model organisms, such as Drosophila melanogaster, where other common tools are less effective or unavailable.

Abstract: Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Abstract: Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Abstract: Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vβ8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.

Abstract: Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.

Abstract: Dissection of cell signaling requires tools that can mimic spatiotemporal dynamics of individual pathways in living cells. Optogenetic methods enable manipulation of signaling processes with precise timing and local control. In this review, we describe recent optogenetic approaches for regulation of cell signaling, highlight their advantages and limitations, and discuss examples of their application.

Abstract: The nucleus is a very complex organelle present in eukaryotic cells. Having the crucial task to safeguard, organize and manage the genetic information, it must tightly control its molecular constituents, its shape and its internal architecture at any given time. Despite our vast knowledge of nuclear cell biology, much is yet to be unraveled. For instance, only recently we came to appreciate the existence of a dynamic nuclear cytoskeleton made of actin filaments that regulates processes such as gene expression, DNA repair and nuclear expansion. This suggests further exciting discoveries ahead of us. Modern cell biologists embrace a new methodology relying on precise perturbations of cellular processes that require a reversible, highly spatially-confinable, rapid, inexpensive and tunable external stimulus: light. In this review, we discuss how optogenetics, the state-of-the-art technology that uses genetically-encoded light-sensitive proteins to steer biological processes, can be adopted to specifically investigate nuclear cell biology.

Abstract: Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.