Curated Optogenetic Publication Database

Abstract: Phosphatidylinositol (3,4,5)‐triphosphate (PIP3), acts as a fundamental second messenger, is emerging as a promising biomarker for disease diagnosis and prognosis. However, the real time analysis of phosphoinositide in living cells remains key challenge owing to the low basal abundance and its fast metabolic rate. Herein, we design an optogenetic system that uses light sensitive protein‐protein interaction between Arabidopsis cryptochrome 2 (CRY2) and CIB1 to spatiotemporally visualize the PIP3 production with sub‐second timescale. In this system, a CIBN is anchored on the plasma membrane, whereas a CRY2 fused with a constitutively active PI3‐kinase (acPI3K) would be driven from the cytosol to the membrane by the blue‐light‐activated CRY2‐CIB1 interaction upon light irradiation. The PIP3 production is visualized via a fused fluorescent protein by the translocation of a Pleckstrin Homology (PH) domain(GRP1) from the cytosol to the plasma membrane with high specificity. We demonstrated the fast dynamics and reversibility of the optogenetic system initiated PIP3 synthesis on the plasma membrane. Notably, the real‐time cell movements were also observed upon localized light stimulation. The established optogenetic method provides a novel spatiotemporal strategy for specific PIP3 visualization, which is beneficial to improve the understanding of PIP3 functions.

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: ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

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: In Drosophila embryos, genes expressed along the dorsal-ventral axis are responsive to concentration of the Dorsal (Dl) transcription factor, which varies in space; however, levels of this morphogen also build over time. Since expression of high-threshold Dl target genes such as snail (sna) is supported before Dl levels peak, it is unclear what role increasing levels have if any. Here we investigated action of two enhancers that control sna expression in embryos, demonstrating using genome editing that Dl binding sites within one enhancer located promoter proximally, sna.prox, can limit the ability of the other distally-located enhancer, sna.dis, to increase sna levels. In addition, MS2-MCP live imaging was used to study sna transcription rate in wildtype, dl heterozygote, and a background in which a photo-sensitive degron is fused to Dl (dl-BLID). The results demonstrate that, when Dl levels are high, Dl acts through sna.prox to limit the activity of sna.dis and thereby influence sna transcription rate. In contrast, when Dl levels are kept low using dl-BLID, sna.prox positively influences sna transcription rate. Collectively, our data support the view that Dl’s effect on gene expression changes over time, switching from promoting sna expression at low concentration to dampening sna expression at high concentration by regulating enhancer interactions. We propose this differential action of the Dl morphogen is likely supported by occupancy of this factor first to high and then low affinity binding sites over time as Dl levels rise to coordinate action of these two co-acting enhancers.

Abstract: Biological systems assemble living materials that are autonomously patterned, can self-repair and can sense and respond to their environment. The field of engineered living materials aims to create novel materials with properties similar to those of natural biomaterials using genetically engineered organisms. Here, we describe an approach to fabricating functional bacterial cellulose-based living materials using a stable co-culture of Saccharomyces cerevisiae yeast and bacterial cellulose-producing Komagataeibacter rhaeticus bacteria. Yeast strains can be engineered to secrete enzymes into bacterial cellulose, generating autonomously grown catalytic materials and enabling DNA-encoded modification of bacterial cellulose bulk properties. Alternatively, engineered yeast can be incorporated within the growing cellulose matrix, creating living materials that can sense and respond to chemical and optical stimuli. This symbiotic culture of bacteria and yeast is a flexible platform for the production of bacterial cellulose-based engineered living materials with potential applications in biosensing and biocatalysis.

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: Optogenetic systems have been increasingly investigated in the field of biomedicine. Previous studies had found the inhibitory effect of the light-inducible genetic circuits on cancer cell growth. In our study, we applied an AND logic gates to the light-inducible genetic circuits to inhibit the cancer cells more specifically. The circuit would only be activated in the presence of both the human telomerase reverse transcriptase (hTERT) and the human uroplakin II (hUPII) promoter. The activated logic gate led to the expression of the p53 or E-cadherin protein, which could inhibit the biological function of tumor cells. In addition, we split the dCas9 protein to reduce the size of the synthetic circuit compared to the full-length dCas9. This light-inducible system provides a potential therapeutic strategy for future bladder cancer.

Abstract: Membrane receptors play a crucial role in transmitting external signals inside cells. Signal molecule-bound receptors activate multiple downstream pathways, the dynamics of which are modulated by intracellular trafficking. A significant contribution of β-arrestin to intracellular trafficking has been suggested, but the underlying mechanism is poorly understood. Here, we describe a protocol for manipulating β-arrestin-regulated membrane receptor trafficking using photo-induced dimerization of cryptochrome-2 from Arabidopsis thaliana and its binding partner CIBN. Additionally, the protocol guides analytical methods to quantify the changes in localization and modification of membrane receptors during trafficking.

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: 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 extracellular matrix (ECM) comprises a meshwork of biomacromolecules whose composition, architecture, and macroscopic properties, such as mechanics, instruct cell fate decisions during development and disease progression. Current methods implemented in mechanotransduction studies either fail to capture real-time mechanical dynamics or utilize synthetic polymers that lack the fibrillar nature of their natural counterparts. Here we present an optogenetic-inspired tool to construct light-responsive ECM mimetic hydrogels comprised exclusively of natural ECM proteins. Optogenetic tools offer seconds temporal resolution and submicron spatial resolution, permitting researchers to probe cell signaling dynamics with unprecedented precision. Here we demonstrated our approach of using SNAP-tag and its thiol-targeted substrate, benzylguanine-maleimide, to covalently attach blue-light-responsive proteins to collagen hydrogels. The resulting material (OptoGel), in addition to encompassing the native biological activity of collagen, stiffens upon exposure to blue light and softens in the dark. Optogels have immediate use in dissecting the cellular response to acute mechanical inputs and may also have applications in next-generation biointerfacing prosthetics.

Abstract: In nature, photoreceptor proteins undergo molecular responses to light, that exhibit supreme fidelity in time and space and generally occur under mild reaction conditions. To unlock these traits for material science, the light‐induced homodimerization of light‐oxygen‐voltage (LOV) photoreceptors is leveraged to control the assembly of gold nanoparticles. Conjugated to genetically encodable LOV proteins, the nanoparticles are monodispersed in darkness but rapidly assemble into large aggregates upon blue‐light exposure. The work establishes a new modality for reaction control in macromolecular chemistry and thus augurs enhanced precision in space and time in diverse applications of gold nanoparticles.

Abstract: The spatiotemporal organization of oligomeric protein complexes, such as the supramolecular organizing centers (SMOCs) made of MyDDosome and MAVSome, is essential for transcriptional activation of host inflammatory responses and immunometabolism. Light‐inducible assembly of MyDDosome and MAVSome is presented herein to induce activation of nuclear factor‐kB and type‐I interferons. Engineering of SMOCs and the downstream transcription factor permits programmable and customized innate immune operations in a light‐dependent manner. These synthetic molecular tools will likely enable optical and user‐defined modulation of innate immunity at a high spatiotemporal resolution to facilitate mechanistic studies of distinct modes of innate immune activations and potential intervention of immune disorders and cancer.

Abstract: Microbial synthesis of chemicals typically requires the redistribution of metabolic flux toward the synthesis of targeted products. Dynamic control is emerging as an effective approach for solving the hurdles mentioned above. As light could control the cell behavior in a spatial and temporal manner, the optogenetic-CRISPR interference (opto-CRISPRi) technique that allocates the metabolic resources according to different optical signal frequencies will enable bacteria to be controlled between the growth phase and the production stage. In this study, we applied a blue light-sensitive protein EL222 to regulate the expression of the dCpf1-mediated CRISPRi system that turns off the competitive pathways and redirects the metabolic flux toward the heterologous muconic acid synthesis in Escherichia coli. We found that the opto-CRISPRi system dynamically regulating the suppression of the central metabolism and competitive pathways could increase the muconic acid production by 130%. These results demonstrated that the opto-CRISPRi platform is an effective method for enhancing chemical synthesis with broad utilities.

Abstract: Irf6 and Esrp1 are important for palate development across vertebrates. In zebrafish, we found that irf6 regulates the expression of esrp1 We detailed overlapping Irf6 and Esrp1/2 expression in mouse orofacial epithelium. In zebrafish, irf6 and esrp1/2 share expression in periderm, frontonasal ectoderm and oral epithelium. Genetic disruption of irf6 and esrp1/2 in zebrafish resulted in cleft of the anterior neurocranium. The esrp1/2 mutant also developed cleft of the mouth opening. Lineage tracing of cranial neural crest cells revealed that the cleft resulted not from migration defect, but from impaired chondrogenesis. Analysis of aberrant cells within the cleft revealed expression of sox10, col1a1 and irf6, and these cells were adjacent to krt4 + and krt5 + cells. Breeding of mouse Irf6; Esrp1; Esrp2 compound mutants suggested genetic interaction, as the triple homozygote and the Irf6; Esrp1 double homozygote were not observed. Further, Irf6 heterozygosity reduced Esrp1/2 cleft severity. These studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish, and identify a unique aberrant cell population in zebrafish expressing sox10, col1a1 and irf6 Future work characterizing this cell population will yield additional insight into cleft pathogenesis.

Abstract: Living organisms have evolved sophisticated cell-mediated biomineralization mechanisms to build structurally ordered, environmentally adaptive composite materials. Despite advances in biomimetic mineralization research, it remains difficult to produce mineralized composites that integrate the structural features and 'living' attributes of their natural counterparts. Here, inspired by natural graded materials, we developed living patterned and gradient composites by coupling light-inducible bacterial biofilm formation with biomimetic hydroxyapatite (HA) mineralization. We showed that both the location and the degree of mineralization could be regulated by tailoring functional biofilm growth with spatial and biomass density control. The cells in the composites remained viable and could sense and respond to environmental signals. Additionally, the composites exhibited a maximum 15-fold increase in Young's modulus after mineralization and could be applied to repair damage in a spatially controlled manner. Beyond insights into the mechanism of formation of natural graded composites, our study provides a viable means of fabricating living composites with dynamic responsiveness and environmental adaptability.

Abstract: Plants measure light quality, intensity, and duration to coordinate growth and development with daily and seasonal changes in environmental conditions; however, the molecular details linking photochemistry to signal transduction remain incomplete. Two closely related light, oxygen, or voltage (LOV) domain-containing photoreceptor proteins, ZEITLUPE (ZTL) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), divergently regulate the protein stability of circadian clock and photoperiodic flowering components to mediate daily and seasonal development. Using structural approaches, we identified that mutations at the Gly46 position led to global rearrangements of the ZTL dimer interface in the isolated ZTL-LOV domain. Specifically, G46S and G46A variants induce a 180° rotation about the ZTL-LOV dimer interface that is coupled to ordering of N- and C-terminal signaling elements. These conformational changes hinge upon rotation of a C-terminal Gln residue (Gln154) analogous to that present in light-state structures of ZTL. In contrast to other LOV proteins, a Q154L variant retains light-state interactions with GIGANTEA (GI), thereby indicating N5 protonation is not required for ZTL signaling. The results presented herein confirm a divergent signaling mechanism within ZTL, whereby steric and electronic effects following adduct formation can be sufficient for signal propagation in LOV proteins containing a Gly residue at position 46. Examination of bacterial LOV structures with Gly residues at the equivalent position suggests that mechanisms of signal transduction in LOV proteins may be fluid across the LOV protein family.

Abstract: Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.