Curated Optogenetic Publication Database

Abstract: The epithelial ion channel TRPV6 plays a pivotal role in calcium homeostasis. Channel function is intricately regulated at different stages, involving the lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Given that dysregulation of TRPV6 is associated with various diseases, including different types of cancer, there is a compelling need for its pharmacological targeting. Structural studies provide insights on how TRPV6 is affected by different inhibitors, with some binding to sites else occupied by lipids. These include the small molecule cis-22a, which, however, also binds to and thereby blocks the pore. By combining calcium imaging, electrophysiology and optogenetics, we identified residues within the pore and the lipid binding site that are relevant for regulation by cis-22a and PIP2 in a bidirectional manner. Yet, mutation of the cytosolic pore exit reduced inhibition by cis-22a but preserved sensitivity to PIP2 depletion. Our data underscore allosteric communication between the lipid binding site and the pore and vice versa for most sites along the pore.

Abstract: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system, and introduce a powerful new tool for bacterial optogenetics.

Abstract: Chimeric antigen receptor (CAR)-T cell immunotherapy emerges as an effective cancer treatment. However, significant safety concerns remain, such as cytokine release syndrome (CRS) and "on-target, off-tumor" cytotoxicity, due to a lack of precise control over conventional CAR-T cell activity. To address this issue, a nano-optogenetic approach has been developed to enable spatiotemporal control of CAR-T cell activity. This system is comprised of synthetic light-sensitive CAR-T cells and upconversion nanoparticles acting as an in situ nanotransducer, allowing near-infrared light to wirelessly control CAR-T cell immunotherapy.

Abstract: The ability of biological systems to convert inputs from their environment into information to guide future decisions is central to life and a matter of great importance. While we know the components of many of the signaling networks that make these decisions, our understanding of the dynamic flow of information between these parts remains far more limited. T cells are an essential white blood cell type of an adaptive immune response and can discriminate between healthy and infected cells with remarkable sensitivity. This chapter describes the use of a synthetic T-cell receptor (OptoCAR) that is optically tunable within cell conjugates, providing control over the duration, and intensity of intracellular T-cell signaling dynamics. Optical control can also provide control over signaling with high spatial precision, and the OptoCAR is likely to find application more generally when modulating T-cell function with imaging approaches.

Abstract: Mononegaviruses are promising tools as oncolytic and transgene vectors for gene therapy and regenerative medicine. However, when mononegaviruses are used for therapeutic applications, the viral activity must be strictly controlled due to concerns about toxicity and severe side effects. With this technology, mononegavirus vectors can be grown where they are intended and can be easily removed when they are no longer needed. In particular, a photoswitch protein called Magnet (consisting of two magnet domains) is incorporated into the hinge region between the connector and methyltransferase domains of the mononegavirus polymerase protein (L protein) to disrupt the L protein functions. Blue light (470 ± 20 nm) irradiation causes the dimerization of the two magnet domains, and the L protein is restored to activity, allowing viral gene expression and virus replication. Since the magnet domains' dimerization is reversible, viral gene expression and replication cease when blue light irradiation is stopped.

Abstract: A given protein can perform numerous roles in a cell with its participation in protein complexes and distinct localization within the cell playing a critical role in its diverse functions. Thus, the ability to artificially dimerize proteins and recruit proteins to specific locations in a cell has become a powerful tool for the investigation of protein function and the understanding of cell biology. Here, we discuss two systems that have been used to activate signal transduction pathways, a chemically inducible dimerization (CID) and a light-inducible (LI) system to control signaling and cytoskeletal regulation in a spatial and temporal manner.

Abstract: Optogenetic tools provide a means for controlling cellular processes that is rapid, noninvasive, and spatially and temporally precise. With the increase in available optogenetic systems, quantitative comparisons of their performances become important to guide experiments. In this chapter, we first discuss how photoreceptors can be repurposed for light-mediated control of transcription. Then, we provide a detailed protocol for characterizing light-regulated transcriptional systems in budding yeast using fluorescence time-lapse microscopy and mathematical modeling, expanding on our recent publication (Gligorovski et al., Nat Commun 14:3810, 2023).

Abstract: By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

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: Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

Abstract: Avena Sativa phototropin 1 Light-oxygen-voltage 2 domain (AsLOV2) is the model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change is initiated by the unfolding of the N-terminal helix in the dark state followed by the unfolding of the C-terminal helix. The light state is defined by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, β-sheets have been identified as crucial components for mediating allosteric signal transmission between the two termini. In this study, we combined microsecond all-atm molecular dynamics simulations and Markov state modeling of conformational states to quantify molecular basis of mutation-induced allostery in the AsLOV2 protein. Through a combination of computational investigations, we determine that the Hβ and Iβ strands are the most critical structural elements involved in the allosteric mechanism. To elucidate the role of these β-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive simulation analysis. The results highlighted the role of two hydrogen bond Asn482-Leu453 and Gln479-Val520 in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. The comprehensive atomistic-level analysis of the conformational landscapes revealed the critical functional role of β-sheet segments in the transmission of the allosteric signal upon the photoactivation of the light state.

Abstract: Mural cells are critical components of the cerebral vasculature. They are categorized into three primary subsets: arteriole smooth muscle cells (aSMCs), pericytes (PCs) and venule smooth muscle cells (vSMCs). It is well known that aSMCs can directly regulate cerebral blood flow (CBF) with their own contraction and dilation mechanisms. On the other hand, the direct involvement of PCs or vSMCs in CBF regulation is controversial. This ambiguity is largely due to the lack of specifically manipulable tools to isolate their function. To address this issue, we employed a set-subtraction approach by using a combination of tTA-mediated gene induction and Cre-mediated gene excision. We developed transgenic mice expressing optical actuators, channelrhodopsin-2 (ChR2) and photoactivated adenylyl cyclase (PAC) in smooth muscle actin (SMA)-negative mural cells that lack the machinery for SMA-mediated vasoregulation. Using these mouse models, we assessed CBF alterations in response to optical stimulation using laser Doppler techniques. Our results showed that optical stimulation induced notable CBF changes in both models. This study provides evidence for the potential regulatory role of PCs and vSMCs in cerebral hemodynamics and introduces powerful tools to specifically manipulate these cell types in vascular neurobiology.

Abstract: Presynaptic forms of plasticity occur throughout the nervous system and play an important role in learning and memory but the underlying molecular mechanisms are insufficiently understood. Here we show that the small GTPase Rab3 is a key mediator of cyclic AMP (cAMP)-induced presynaptic plasticity in Drosophila. Pharmacological and optogenetic cAMP production triggered concentration-dependent alterations of synaptic transmission, including potentiation and depression of evoked neurotransmitter release, as well as strongly facilitated spontaneous release. These changes correlated with a nanoscopic rearrangement of the active zone protein Unc13A and required Rab3. To link these results to animal behaviour, we turned to the established role of cAMP signalling in memory formation and demonstrate that Rab3 is necessary for olfactory learning. As Rab3 is dispensable for basal synaptic transmission, these findings highlight a molecular pathway specifically dedicated to tuning neuronal communication and adaptive behaviour.

Abstract: Optogenetics is an attractive synthetic biology tool for controlling the metabolic flux distribution. Here, we demonstrated optogenetic flux ratio control of glycolytic pathways consisting of the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP), and Entner-Doudoroff (ED) pathways by illuminating multicolor lights using blue light-responsive EL222 and green/red light-responsive CcaSR in Escherichia coli. EL222 forms a dimer and binds to a particular DNA sequence under blue light; therefore, target gene expression can be reduced or induced by inserting a recognition sequence into its promoter regions. First, a flux ratio between the PP and ED pathways was controlled by blue light using EL222. After blocking the EMP pathway, the EL222-recognition sequence was inserted between the -35 and -10 regions of gnd to repress the PP flux and was also inserted upstream of the -35 region of edd to induce ED flux. After adjusting light intensity, the PP:ED flux ratios were 60:39% and 29:70% under dark and blue light conditions, respectively. Finally, a CcaSR-based pgi expression system was implemented to control the flux ratio between the EMP and PP + ED pathways by illuminating green/red light. The EMP:PP:ED flux ratios were 80:9:11%, 14:35:51%, and 33:5:62% under green, red, and red and blue light, respectively.

Abstract: Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

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: Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington's Disease, and Alzheimer's Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.

Abstract: Microbial biosynthesis, as an alternative method for producing quantum dots (QDs), has gained attention because it can be conducted under mild and environmentally friendly conditions, distinguishing it from conventional chemical and physical synthesis approaches. However, there is currently no method to selectively control this biosynthesis process in a subset of microbes within a population using external stimuli. In this study, we have attained precise and selective control over the microbial biosynthesis of QDs through the utilization of an optogenetically engineered Escherichia coli (E. coli). The recombinant E. coli is designed to express smCSE enzyme, under the regulation of eLightOn system, which can be activated by blue light. The smCSE enzymes use L-cysteine and Cd2+ as substrates to form CdS QDs. This system enables light-inducible bacterial biosynthesis of QDs in precise patterns within a hydrogel for information encryption. As the biosynthesis progresses, the optical characteristics of the QDs change, allowing living materials containing the recombinant E. coli to display time-dependent patterns that self-destruct after reading. Compared to static encryption using fluorescent QD inks, dynamic information encryption based on living materials offers enhanced security.

Abstract: Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.

Abstract: Optogenetics has opened new possibilities in the remote control of diverse cellular functions with high spatiotemporal precision using light. However, delivering light to optically non-transparent systems remains a challenge. Here, we describe the photoactivation of light-oxygen-voltage-sensing domains (LOV domains) with in situ generated light from a chemiluminescence reaction between luminol and H2O2. This activation is possible due to the spectral overlap between the blue chemiluminescence emission and the absorption bands of the flavin chromophore in LOV domains. All four LOV domain proteins with diverse backgrounds and structures (iLID, BcLOV4, nMagHigh/pMagHigh, and VVDHigh) were photoactivated by chemiluminescence as demonstrated using a bead aggregation assay. The photoactivation with chemiluminescence required a critical light-output below which the LOV domains reversed back to their dark state with protein characteristic kinetics. Furthermore, spatially confined chemiluminescence produced inside giant unilamellar vesicles (GUVs) was able to photoactivate proteins both on the membrane and in solution, leading to the recruitment of the corresponding proteins to the GUV membrane. Finally, we showed that reactive oxygen species produced by neutrophil like cells can be converted into sufficient chemiluminescence to recruit the photoswitchable protein BcLOV4-mCherry from solution to the cell membrane. The findings highlight the utility of chemiluminescence as an endogenous light source for optogenetic applications, offering new possibilities for studying cellular processes in optically non-transparent systems.

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: Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double-stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off-target effects. Continuously expressed deaminases used for gene editing may lead to unwanted base alterations at unpredictable genomic locations. In the present study, blue-light-activated base editors (BLBEs) are engineered based on the distinct photoswitches magnets that can switch from a monomer to dimerization state in response to blue light. By fusing the N- and C-termini of split DNA deaminases with photoswitches Magnets, efficient A-to-G and C-to-T base editing is achieved in response to blue light in prokaryotic and eukaryotic cells. Furthermore, the results showed that BLBEs can realize precise blue light-induced gene editing across broad genomic loci with low off-target activity at the DNA- and RNA-level. Collectively, these findings suggest that the optogenetic utilization of base editing and optical base editors may provide powerful tools to promote the development of optogenetic genome engineering.

Abstract: RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR-Cas system to optogenetic control. The protocols typically take 2-3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR-Cas functions.

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.

Abstract: Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.