Curated Optogenetic Publication Database

Abstract: Biomolecular condensates (BMCs), play significant roles in organizing cellular functions in the absence of membranes through phase separation events involving RNA, proteins, and RNA-protein complexes. These membrane-less organelles form dynamic multivalent weak interactions, often involving intrinsically disordered proteins or regions (IDPs/IDRs). However, the nature of these crucial interactions, how most of these organelles are organized and are functional, remains unknown. Aberrant condensates have been implicated in neurodegenerative diseases and various cancers, presenting novel therapeutic opportunities for small molecule condensate modulators. Recent advancements in optogenetic technologies, particularly Corelet, enable precise manipulation of BMC dynamics within living cells, facilitating high-throughput screening for small molecules that target these complex structures. By elucidating the molecular mechanisms governing BMC formation and function, this innovative approach holds promise to unlock therapeutic strategies against previously "undruggable" protein targets, paving the way for effective interventions in disease.

Abstract: Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Abstract: Biomolecular condensates appear throughout cell physiology and pathology, but the specific role of condensation or its dynamics is often difficult to determine. Optogenetics offers an expanding toolset to address these challenges, providing tools to directly control condensation of arbitrary proteins with precision over their formation, dissolution, and patterning in space and time. In this review, we describe the current state of the field for optogenetic control of condensation. We survey the proteins and their derivatives that form the foundation of this toolset, and we discuss the factors that distinguish them to enable appropriate selection for a given application. We also describe recent examples of the ways in which optogenetic condensation has been used in both basic and applied studies. Finally, we discuss important design considerations when engineering new proteins for optogenetic condensation, and we preview future innovations that will further empower this toolset in the coming years.

Abstract: 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.

Abstract: Recent advancements in plant bioprinting and optogenetic tools have unlocked new avenues to revolutionize plant tissue engineering. Bioprinting of plant cells has the potential to craft intricate 3D structures incorporating multiple cell types, replicating the complex microenvironments found in plants. Concurrently, optogenetic tools enable the control of biological events with spatial, temporal, and quantitative precision. Originally developed for human and microbial systems, these two cutting-edge methodologies are now being adapted for plant research. Although still in the early stages of development, we here review the latest progress in plant bioprinting and optogenetics and discuss compelling opportunities for plant biotechnology and research arising from the combination of the two technologies.

Abstract: In recent decades, the field of synthetic biology has witnessed remarkable progress, driving advances in both research and practical applications. One pivotal area of development involves the design of transgene switches capable of precisely regulating specified outputs and controlling cell behaviors in response to physical cues, which encompass light, magnetic fields, temperature, mechanical forces, ultrasound, and electricity. In this review, we delve into the cutting-edge progress made in the field of physically controlled protein expression in engineered mammalian cells, exploring the diverse genetic tools and synthetic strategies available for engineering targeting cells to sense these physical cues and generate the desired outputs accordingly. We discuss the precision and efficiency limitations inherent in these tools, while also highlighting their immense potential for therapeutic applications.

Abstract: The Islets of Langerhans reside within the endocrine pancreas as highly vascularised micro-organs that are responsible for the secretion of key hormones, such as insulin and glucagon. Islet function relies on a range of dynamic molecular processes that include calcium (Ca2+) waves, hormone pulses, and complex interactions between islet cell types. Dysfunction of these processes results in poor maintenance of blood glucose homeostasis and is a hallmark of diabetes. Very recently, the development of optogenetic methods that rely on light-sensitive molecular actuators has allowed perturbing islet function with near physiological spatio-temporal acuity. These actuators harness natural photoreceptor proteins and their engineered variants to manipulate mouse and human cells that are not normally light-responsive. Until recently, optogenetics in islet biology has primarily focused on hormone production and secretion; however, studies on further aspects of islet function, including paracrine regulation between islet cell types and dynamics within intracellular signaling pathways are emerging. Here, we discuss the applicability of optogenetics to islets cells and comprehensively review seminal as well as recent work on optogenetic actuators and their effects in islet function and diabetes mellitus (DM).

Abstract: Diabetes mellitus (DM) is a common chronic disease affecting humans globally. It is characterized by abnormally elevated blood glucose levels due to the failure of insulin production or reduction of insulin sensitivity and functionality. Insulin and glucagon-like peptide (GLP)-1 replenishment or improvement of insulin resistance are the two major strategies to treat diabetes. Recently, optogenetics that uses genetically encoded light-sensitive proteins to precisely control cell functions has been regarded as a novel therapeutic strategy for diabetes. Here, we summarize the latest development of optogenetics and its integration with synthetic biology approaches to produce light-responsive cells for insulin/GLP-1 production, amelioration of insulin resistance and neuromodulation of insulin secretion. In addition, we introduce the development of cell encapsulation and delivery methods and smart bioelectronic devices for the in vivo application of optogenetics-based cell therapy in diabetes. The remaining challenges for optogenetics-based cell therapy in the clinical translational study are also discussed.

Abstract: Hydrophilic and biocompatible hydrogels are widely applied as ideal scaffolds in tissue engineering. The "smart" gelation material can alter its structural, physiochemical, and functional features in answer to various endo/exogenous stimuli to better biomimic the endogenous extracellular matrix for the engineering of cells and tissues. Light irradiation owns a high spatial-temporal resolution, complete biorthogonal reactivity, and fine-tunability and can thus induce physiochemical reactions within the matrix of photoresponsive hydrogels with good precision, efficiency, and safety. Both gel structure (e.g., geometry, porosity, and dimension) and performance (like conductivity and thermogenic or mechanical properties) can hence be programmed on-demand to yield the biochemical and biophysical signals regulating the morphology, growth, motility, and phenotype of engineered cells and tissues. Here we summarize the strategies and mechanisms for encoding light-reactivity into a hydrogel and demonstrate how fantastically such responsive gels change their structure and properties with light irradiation as desired and thus improve their applications in tissue engineering including cargo delivery, dynamic three-dimensional cell culture, and tissue repair and regeneration, aiming to provide a basis for more and better translation of photoresponsive hydrogels in the clinic.

Abstract: Optogenetic, known as the method of 21 centuries, combines optic and genetic engineering to precisely control photosensitive proteins for manipulation of a broad range of cellular functions, such as flux of ions, protein oligomerization and dissociation, cellular intercommunication, and so on. In this technique, light is conventionally delivered to targeted cells through optical fibers or micro light-emitting diodes, always suffering from high invasiveness, wide-field illumination facula, strong absorption, and scattering by nontargeted endogenous substance. Light-transducing nanomaterials with advantages of high spatiotemporal resolution, abundant wireless-excitation manners, and easy functionalization for recognition of specific cells, recently have been widely explored in the field of optogenetics; however, there remain a few challenges to restrain its clinical applications. This review summarized recent progress on light-responsive genetically encoded proteins and the myriad of activation strategies by use of light-transducing nanomaterials and their disease-treatment applications, which is expected for sparking helpful thought to push forward its preclinical and translational uses.

Abstract: At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes-plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object.

Abstract: Protein engineering is important for creating novel variants from natural proteins, enabling a wide range of applications. Approaches such as rational design and directed evolution are routinely used to make new protein variants. Computational tools like de novo design can introduce new protein folds. Expanding the amino acid repertoire to include unnatural amino acids with non-canonical side chains in vitro by native chemical ligation and in vivo via codon expansion methods broadens sequence and structural possibilities. Circular permutation (CP) is an invaluable approach to redesigning a protein by rearranging the amino acid sequence, where the connectivity of the secondary structural elements is altered without changing the overall structure of the protein. Artificial CP proteins (CPs) are employed in various applications such as biocatalysis, sensing of small molecules by fluorescence, genome editing, ligand-binding protein switches, and optogenetic engineering. Many studies have shown that CP can lead to either reduced or enhanced stability or catalytic efficiency. The effects of CP on a protein's energy landscape cannot be predicted a priori. Thus, it is important to understand how CP can affect the thermodynamic and kinetic stability of a protein. In this review, we discuss the discovery and advancement of techniques to create protein CP, and existing reviews on CP. We delve into the plethora of biological applications for designed CP proteins. We subsequently discuss the experimental and computational reports on the effects of CP on the thermodynamic and kinetic stabilities of proteins of various topologies. An understanding of the various aspects of CP will allow the reader to design robust CP proteins for their specific purposes.

Abstract: Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.

Abstract: Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Abstract: The ORAI proteins serve as crucial pore-forming subunits of calcium-release-activated calcium (CRAC) channels, pivotal in regulating downstream calcium-related signaling pathways. Dysregulated calcium homeostasis arising from mutations and post-translational modifications in ORAI can lead to immune disorders, myopathy, cardiovascular diseases, and even cancers. Small molecules targeting ORAI present an approach for calcium signaling modulation. Moreover, emerging techniques like optogenetics and optochemistry aim to offer more precise regulation of ORAI. This review focuses on the role of ORAI in cancers, providing a concise overview of their significance in the initiation and progression of cancers. Additionally, it highlights state-of-the-art techniques for ORAI channel modulation, including advanced optical tools, potent pharmacological inhibitors, and antibodies. These novel strategies offer promising avenues for the functional regulation of ORAI in research and may inspire innovative approaches to cancer therapy targeting ORAI.

Abstract: Inteins are proteins found in nature that execute protein splicing. Among them, split inteins stand out for their versatility and adaptability, presenting creative solutions for addressing intricate challenges in various biological applications. Their exquisite attributes, including compactness, reliability, orthogonality, low toxicity, and irreversibility, make them of interest to various fields including synthetic biology, biotechnology and biomedicine. In this review, we delve into the inherent challenges of using inteins, present approaches for overcoming these challenges, and detail their reliable use for specific cellular tasks. We will discuss the use of conditional inteins in areas like cancer therapy, drug screening, patterning, infection treatment, diagnostics and biocontainment. Additionally, we will underscore the potential of inteins in executing basic logical operations with practical implications. We conclude by showcasing their potential in crafting complex genetic circuits for performing computations and feedback control that achieves robust perfect adaptation.

Abstract: Liquid-liquid phase separation (LLPS) is responsible for the emergence of intracellular membrane-less organelles and the development of coacervate protocells. Benefitting from the advantages of simplicity, precision, programmability, and noninvasiveness, light has become an effective tool to regulate the assembly dynamics of LLPS, and mediate various biochemical processes associated with LLPS. In this review, recent advances in optically controlling membrane-less organelles within living organisms are summarized, thereby modulating a series of biological processes including irreversible protein aggregation pathologies, transcription activation, metabolic flux, genomic rearrangements, and enzymatic reactions. Among these, the intracellular systems (i.e., optoDroplet, Corelet, PixELL, CasDrop, and other optogenetic systems) that enable the photo-mediated control over biomolecular condensation are highlighted. The design of photoactive complex coacervate protocells in laboratory settings by utilizing photochromic molecules such as azobenzene and diarylethene is further discussed. This review is expected to provide in-depth insights into phase separation-associated biochemical processes, bio-metabolism, and diseases.

Abstract: Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

Abstract: Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor-ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.

Abstract: Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Abstract: [This corrects the article DOI: 10.1002/mco2.226.].

Abstract: T cells regulate adaptive immune responses through complex signaling pathways mediated by T cell receptor (TCR). The functional domains of the TCR are combined with specific antibodies for the development of chimeric antigen receptor (CAR) T cell therapy. In this review, we first overview current understanding on the T cell signaling pathways as well as traditional methods that have been widely used for the T cell study. These methods, however, are still limited to investigating dynamic molecular events with spatiotemporal resolutions. Therefore, genetically encoded biosensors and optogenetic tools have been developed to study dynamic T cell signaling pathways in live cells. We review these cutting-edge technologies that revealed dynamic and complex molecular mechanisms at each stage of T cell signaling pathways. They have been primarily applied to the study of dynamic molecular events in TCR signaling, and they will further aid in understanding the mechanisms of CAR activation and function. Therefore, genetically encoded biosensors and optogenetic tools offer powerful tools for enhancing our understanding of signaling mechanisms in T cells and CAR-T cells.

Abstract: CRISPR-Cas effectors are powerful tools for genome and transcriptome targeting and editing. Naturally, these protein-RNA complexes are part of the microbial innate immune system, which emerged from the evolutionary arms race between microbes and phages. This coevolution has also given rise to so-called anti-CRISPR (Acr) proteins that counteract the CRISPR-Cas adaptive immunity. Acrs constitutively block cognate CRISPR-Cas effectors, e.g., by interfering with guide RNA binding, target DNA/RNA recognition, or target cleavage. In addition to their important role in microbiology and evolution, Acrs have recently gained particular attention for being useful tools and switches to regulate or fine-tune the activity of CRISPR-Cas effectors. Due to their commonly small size, high inhibition potency, and structural and mechanistic versatility, Acrs offer a wide range of potential applications for controlling CRISPR effectors in heterologous systems, including mammalian cells.Here, we review the diverse applications of Acrs in mammalian cells and organisms and discuss the underlying engineering strategies. These applications include (i) persistent blockage of CRISPR-Cas function to create write-protected cells, (ii) reduction of CRISPR-Cas off-target editing, (iii) focusing CRISPR-Cas activity to specific cell types and tissues, (iv) spatiotemporal control of CRISPR effectors based on engineered, opto-, or chemogenetic Acrs, and (v) the use of Acrs for selective binding and detection of CRISPR-Cas effectors in complex samples. We will also highlight potential future applications of Acrs in a biomedical context and point out present challenges that need to be overcome on the way.

Abstract: In the early 2000s, the field of neuroscience experienced a groundbreaking transformation with the advent of optogenetics. This innovative technique harnesses the properties of naturally occurring and genetically engineered rhodopsins to confer light sensitivity upon target cells. The remarkable spatiotemporal precision offered by optogenetics has provided researchers with unprecedented opportunities to dissect cellular physiology, leading to an entirely new level of investigation. Initially revolutionizing neuroscience, optogenetics quickly piqued the interest of the wider scientific community, and optogenetic applications were expanded to cardiovascular research. Over the past decade, researchers have employed various optical tools to observe, regulate, and steer the membrane potential of excitable cells in the heart. Despite these advancements, achieving control over specific signaling pathways within the heart has remained an elusive goal. Here, we review the optogenetic tools suitable to control cardiac signaling pathways with a focus on GPCR signaling, and delineate potential applications for studying these pathways, both in healthy and diseased hearts. By shedding light on these exciting developments, we hope to contribute to the ongoing progress in basic cardiac research to facilitate the discovery of novel therapeutic possibilities for treating cardiovascular pathologies.

Abstract: Optogenetics is a powerful approach that enables researchers to use light to dynamically manipulate cellular behavior. Since the first published use of optogenetics in synthetic biology, the field has expanded rapidly, yielding a vast array of tools and applications. Despite its immense potential for achieving high spatiotemporal precision, optogenetics has predominantly been employed as a substitute for conventional chemical inducers. In this short review, we discuss key features of microbial optogenetics and highlight applications for understanding biology, cocultures, bioproduction, biomaterials, and therapeutics, in which optogenetics is more fully utilized to realize goals not previously possible by other methods.