Curated Optogenetic Publication Database

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: 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: Microtubules filaments are assembled into higher-order structures and machines critical for cellular processes using microtubule-associated proteins (MAPs). However, the design of synthetic MAPs that direct the formation of new structures in cells is challenging, as nanoscale biochemical activities must be organized across micron length-scales. Here we develop synthetic MAP-IDR condensates (synMAPs) that provide tunable and regulatable assembly of higher-order microtubule structures in vitro and in mammalian cells. synMAPs harness a small microtubule-binding domain from oligodendrocytes (TPPP) whose activity can be synthetically rewired by interaction with condensate-forming IDR sequences. This combination allows synMAPs to self-organize multivalent structures that bind and bridge microtubules into synthetic architectures. Regulating the connection between the microtubule-binding and condensate-forming components allows synMAPs to act as nodes in more complex cytoskeletal circuits in which the formation and dynamics of the microtubule structure can be controlled by small molecules or cell-signaling inputs. By systematically testing a panel of synMAP circuit designs, we define a two-level control scheme for dynamic assembly of microtubule architectures at the nanoscale (via microtubule-binding) and microscale (via condensate formation). synMAPs provide a compact and rationally engineerable starting point for the design of more complex microtubule architectures and cellular machines.

Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.

Abstract: Regulated cell death (RCD) controls the removal of dispensable, infected or malignant cells, and is thus essential for development, homeostasis and immunity of multicellular organisms. Over the last years different forms of RCD have been described (among them apoptosis, necroptosis, pyroptosis and ferroptosis), and the cellular signaling pathways that control their induction and execution have been characterized at the molecular level. It has also become apparent that different forms of RCD differ in their capacity to elicit inflammation or an immune response, and that RCD pathways show a remarkable plasticity. Biochemical and genetic studies revealed that inhibition of a given pathway often results in the activation of back-up cell death mechanisms, highlighting close interconnectivity based on shared signaling components and the assembly of multivalent signaling platforms that can initiate different forms of RCD. Due to this interconnectivity and the pleiotropic effects of 'classical' cell death inducers, it is challenging to study RCD pathways in isolation. This has led to the development of tools based on synthetic biology that allow the targeted induction of RCD using chemogenetic or optogenetic methods. Here we discuss recent advances in the development of such toolset, highlighting their advantages and limitations, and their application for the study of RCD in cells and animals.

Abstract: The concept of phase-separation-mediated formation of biomolecular condensates provides a new framework to understand cellular organization and cooperativity-dependent cellular functions. With growing understanding of how biological systems drive phase separation and how cellular functions are encoded by biomolecular condensates, opportunities have emerged for cellular control through engineering of synthetic biomolecular condensates. In this Review, we discuss how to construct synthetic biomolecular condensates and how they can regulate cellular functions. We first describe the fundamental principles by which biomolecular components can drive phase separation. Next, we discuss the relationship between the properties of condensates and their cellular functions, which informs the design of components to create programmable synthetic condensates. Finally, we describe recent applications of synthetic biomolecular condensates for cellular control and discuss some of the design considerations and prospective applications.

Abstract: Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Abstract: Molecular optogenetics is a highly dynamic research field. In the past two years, the field was characterized by the development of new allosteric switches as well as the forward integration of optogenetics research towards application. Further, two areas of research have significantly gathered momentum, the use of optogenetics to control liquid-liquid phase separation as well as the application of optogenetic tools in the extracellular space. Here, we review these areas and discuss future directions.

Abstract: Since the successful introduction of exogenous photosensitive proteins, channelrhodopsin, to neurons, optogenetics has enabled substantial understanding of profound brain function by selectively manipulating neural circuits. In an optogenetic system, optical stimulation can be precisely delivered to brain tissue to achieve regulation of cellular electrical activity with unprecedented spatio-temporal resolution in living organisms. In recent years, the development of various optical actuators and novel light-delivery techniques has greatly expanded the scope of optogenetics, enabling the control of other signal pathways in non-neuronal cells for different biomedical applications, such as phototherapy and immunotherapy. This review focuses on the recent advances in optogenetic regulation of cellular activities for photomedicine. We discuss emerging optogenetic tools and light-delivery platforms, along with a survey of optogenetic execution in mammalian and microbial cells.

Abstract: A comprehensive understanding of signaling mechanisms helps interpret fundamental biological processes and restore cell behavior from pathological conditions. Signaling outcome depends not only on the activity of each signaling component but also on their dynamic interaction in time and space, which remains challenging to probe by biochemical and cell-based assays. Opsin-based optogenetics has transformed neural science research with its spatiotemporal modulation of the activity of excitable cells. Motivated by this advantage, opsin-free optogenetics extends the power of light to a larger spectrum of signaling molecules. This review summarizes commonly used opsin-free optogenetic strategies, presents a historical overview of split protein complementation, and highlights the adaptation of split protein recombination as optogenetic sensors and actuators.

Abstract: Unraveling the transformative power of optogenetics in biology requires sophisticated engineering for the creation and optimization of light-regulatable proteins. In addition, diverse strategies have been used for the tuning of these light-sensitive regulators. This review highlights different protein engineering and synthetic biology approaches, which might aid in the development and optimization of novel optogenetic proteins (Opto-proteins). Focusing on non-neuronal optogenetics, chromophore availability, general strategies for creating light-controllable functions, modification of the photosensitive domains and their fusion to effector domains, as well as tuning concepts for Opto-proteins are discussed. Thus, this review shall not serve as an encyclopedic summary of light-sensitive regulators but aims at discussing important aspects for the engineering of light-controllable proteins through selected examples.

Abstract: Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.

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: ConspectusLight activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the blue light using flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark- and light-adapted states. Thus, the driving force for photoactivity is unclear.To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time-resolved infrared (IR) experiments, coupled with chemical biology, provide important new insights into the signaling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labeling of the chromophore and protein separately. Further, a degree of control over BLUF photochemistry is achieved through mutagenesis, while unnatural amino acid substitution allows us to both fine-tune the photochemistry and time resolve protein dynamics with spatial resolution.Ultrafast studies of BLUF domains reveal non-single-exponential relaxation of the flavin excited state. That relaxation leads within one nanosecond to the original flavin ground state bound in a modified hydrogen-bonding network, as seen in transient and steady-state IR spectroscopy. The change in H-bond configuration arises from formation of an unusual enol (imine) form of a critical glutamine residue. The dynamics observed, complemented by quantum mechanical calculations, suggest a unique sequential electron then double proton transfer reaction as the driving force, followed by rapid reorganization in the binding site and charge recombination. Importantly, studies of several BLUF domains reveal an unexpected diversity in their dynamics, although the underlying structure appears highly conserved. It is suggested that this diversity reflects structural dynamics in the ground state at standard temperature, leading to a distribution of structures and photochemical outcomes. Time resolved IR measurements were extended to the millisecond regime for one BLUF domain, revealing signaling state formation on the microsecond time scale. The mechanism involves reorganization of a β-sheet connected to the chromophore binding pocket via a tryptophan residue. The potential of site-specific labeling amino acids with IR labels as a tool for probing protein structural dynamics was demonstrated.In summary, time-resolved IR studies of BLUF domains (along with related studies at visible wavelengths and quantum and molecular dynamics calculations) have resolved the photoactivation mechanism and real-time dynamics of signaling state formation. These measurements provide new insights into protein structural dynamics and will be important in optimizing the potential of BLUF domains in optobiology.

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: Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Abstract: Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.

Abstract: Cells receive enormous amounts of information from their environment. How they act on this information-by migrating, expressing genes, or relaying signals to other cells-comprises much of the regulatory and self-organizational complexity found across biology. The "parts list" involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Abstract: Light has become established as a tool not only to visualize and investigate but also to steer biological systems. This review starts by discussing the unique features that make light such an effective control input in biology. It then gives an overview of how light‐control came to progress, starting with photoactivatable compounds and leading up to current genetic implementations using optogenetic approaches. The review then zooms in on optogenetics, focusing on photosensitive proteins, which form the basis for optogenetic engineering using synthetic biological approaches. As the regulation of transcription provides a highly versatile means for steering diverse biological functions, the focus of this review then shifts to transcriptional light regulators, which are presented in the biotechnologically highly relevant model organism Escherichia coli.

Abstract: Protein phase separation has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomolecular condensates. While the implication of this physical phenomenon within cell biology is providing us with novel ways for understanding how cells compartmentalize biochemical reactions and encode function in such liquid-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biological matter. Here, we propose that understanding the cell's instruction manual of phase separation will enable bioengineers to begin creating novel functionalized biological materials and unprecedented tools for synthetic biology. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the physical driving forces underlying phase separation, with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalogue and construction manual for the aspiring (membraneless) biomolecular architect. Our approach aims to inspire a new generation of bioengineers to rethink phase separation as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biological function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.

Abstract: Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

Abstract: Cybergenetic systems use computer interfaces to enable feed-back controls over biological processes in real time. The complex and dynamic nature of cellular metabolism makes cybergenetics attractive for controlling engineered metabolic pathways in microbial fermentations. Cybergenetics would not only create new avenues of research into cellular metabolism, it would also enable unprecedented strategies for pathway optimization and bioreactor operation and automation. Implementation of metabolic cybergenetics, however, will require new capabilities from actuators, biosensors, and control algorithms. The recent application of optogenetics in metabolic engineering, the expanding role of genetically encoded biosensors in strain development, and continued progress in control algorithms for biological processes suggest that this technology will become available in the not so distant future.

Abstract: Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models.

Abstract: Yeast has been a robust platform to manufacture a broad range of biofuels, commodity chemicals, natural products and pharmaceuticals. The membrane-bound organelles in yeast provide us the means to access the specialized metabolism for various biosynthetic applications. The separation and compartmentalization of genetic and metabolic events presents us the opportunity to precisely control and program gene expression for higher order biological functions. To further advance yeast synthetic biology platform, genetically encoded biosensors and actuators haven been engineered for in vivo monitoring and controlling cellular processes with spatiotemporal resolutions. The dynamic response, sensitivity and operational range of these genetically encoded sensors are determined by the regulatory architecture, dynamic assemly and interactions of the related proteins and genetic elements. This review provides an update of the basic design principles underlying the allosteric transcription factors, GPCR and optogenetics-based sensors, aiming to precisely analyze and control yeast cellular processes for various biotechnological applications.