Curated Optogenetic Publication Database

Abstract: Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other SSR systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.

Abstract: Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light-activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light-tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Genetic engineering strategy for the generation of modular light-activated protein dimerization units Support Protocol 1: Molecular cloning Basic Protocol 2: Cell culture and transfection Support Protocol 2: Production of dark containers for optogenetic samples Basic Protocol 3: Confocal microscopy and light-dependent activation of the dimerization system Alternate Protocol 1: Protein recruitment to intracellular compartments Alternate Protocol 2: Induction of organelles' membrane tethering Alternate Protocol 3: Optogenetic reconstitution of protein function Basic Protocol 4: Image analysis Support Protocol 3: Analysis of apparent on- and off-kinetics Support Protocol 4: Analysis of changes in organelle overlap over time.

Abstract: T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.

Abstract: Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of Caenorhabditis elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation, and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.

Abstract: Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light, without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and in human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses.

Abstract: The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light‐regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas‐related methods. However, these approaches lack the key characteristics and advantages provided by optical control. “Lockdown” introduces optical control of RNA levels utilizing a blue light‐dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence‐specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene‐expression and induce protein destabilization with blue light yields efficient triple‐controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin‐dependent kinase 1 (hCdk1) leads to blue light‐induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.

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: Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Abstract: The myocardin‐related transcription factor A (MRTF‐A) controls the transcriptional activity of the serum response factor (SRF) in a tightly controlled actin‐dependent manner. In turn, MRTF‐A is crucial for many actin‐dependent processes including adhesion, migration, and contractility and has emerged as novel targets for anti‐tumor strategies. MRTF‐A rapidly shuttles between cytoplasmic and nuclear compartment via dynamic actin interactions within its N‐terminal RPEL domain. Here, optogenetics is used to spatiotemporally control MRTF‐A nuclear localization by blue light using the light‐oxygen‐voltage‐sensing domain 2‐domain based system LEXY (light‐inducible nuclear export system). It is found that light‐regulated nuclear export of MRTF‐A occurs within 10–20 min. Importantly, MRTF‐A‐LEXY shuttling is independent of perturbations of actin dynamics. Furthermore, light‐regulation of MRTF‐A‐LEXY is reversible and repeatable for several cycles of illumination and its subcellular localization correlates with SRF transcriptional activity. As a consequence, optogenetic control of MRTF‐A subcellular localization determines subsequent cytoskeletal dynamics such as non‐apoptotic plasma membrane blebbing as well as invasive tumor‐cell migration through 3D collagen matrix. This data demonstrate robust optogenetic regulation of MRTF as a powerful tool to control SRF‐dependent transcription as well as cell motile behavior.

Abstract: As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. Herein, a generally applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively, is introduced. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By the paralleled insertion of two light-oxygen-voltage domain 2 modules into a single sunbody and the use of bivalent sunbodies, the range of dynamic changes of photoswitchable sunbodies is substantially enhanced. Furthermore, the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light is demonstrated.

Abstract: Epithelial cells possess the ability to change their shape in response to mechanical stress by remodelling their junctions and their cytoskeleton. This property lies at the heart of tissue morphogenesis in embryos. A key feature of embryonic cell shape changes is that they result from repeated mechanical inputs that make them partially irreversible at each step. Past work on cell rheology has rarely addressed how changes can become irreversible in a complex tissue. Here, we review new and exciting findings dissecting some of the physical principles and molecular mechanisms accounting for irreversible cell shape changes. We discuss concepts of mechanical ratchets and tension thresholds required to induce permanent cell deformations akin to mechanical plasticity. Work in different systems has highlighted the importance of actin remodelling and of E-cadherin endocytosis. We also list some novel experimental approaches to fine-tune mechanical tension, using optogenetics, magnetic beads or stretching of suspended epithelial tissues. Finally, we discuss some mathematical models that have been used to describe the quantitative aspects of accounting for mechanical cell plasticity and offer perspectives on this rapidly evolving field.

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: Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.

Abstract: Dynamic control of engineered microbes using light via optogenetics has been demonstrated as an effective strategy for improving the yield of biofuels, chemicals, and other products. An advantage of using light to manipulate microbial metabolism is the relative simplicity of interfacing biological and computer systems, thereby enabling in silico control of the microbe. Using this strategy for control and optimization of product yield requires an understanding of how the microbe responds in real-time to the light inputs. Toward this end, we present mechanistic models of a set of yeast optogenetic circuits. We show how these models can predict short- and long-time response to varying light inputs and how they are amenable to use with model predictive control (the industry standard among advanced control algorithms). These models reveal dynamics characterized by time-scale separation of different circuit components that affect the steady and transient levels of the protein under control of the circuit. Ultimately, this work will help enable real-time control and optimization tools for improving yield and consistency in the production of biofuels and chemicals using microbial fermentations.

Abstract: During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

Abstract: Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.

Abstract: To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.

Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.

Abstract: Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.

Abstract: The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis.

Abstract: There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.

Abstract: Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

Abstract: Phagophore closure is a critical step during macroautophagy. However, the proteins and mechanisms to regulate this step have been elusive for a long time. In 2017, Rab5 was affirmed to play a role in phagophore closure in yeast. Furthermore, in mammalian cells, ESCRT III was reported to have roles in phagophore closure and mitophagosome closure in vivo in 2018 and 2019, respectively. The role of ESCRT in phagophore closure was confirmed in yeast, both in vivo and in vitro, in 2019. Most importantly, the latter paper found that Atg17 recruited the ESCRT III subunit Snf7 to the phagophore to close it under the control of Rab5. To determine the closure characteristics of autophagosome-like membrane structures in ESCRT mutants, a traditional protease protection assay with immunoblotting was used, accompanied by new techniques that were developed, including immunofluorescence assays, autophagosome completion assays, and the optogenetic closure assay. This study delivered our current understanding of phagophore closure and provided more reference methods to detect membrane closure.

Abstract: The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.

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.