Curated Optogenetic Publication Database

Abstract: The ability to independently control the expression of different genes is important for quantitative biology. Using budding yeast, we characterize GAL1pr, GALL, MET3pr, CUP1pr, PHO5pr, tetOpr, terminator-tetOpr, Z3EV, blue-light inducible optogenetic systems El222-LIP, El222-GLIP, and red-light inducible PhyB-PIF3. We report kinetic parameters, noise scaling, impact on growth, and the fundamental leakiness of each system using an intuitive unit, maxGAL1. We uncover disadvantages of widely used tools, e.g., nonmonotonic activity of MET3pr and GALL, slow off kinetics of the doxycycline- and estradiol-inducible systems tetOpr and Z3EV, and high variability of PHO5pr and red-light activated PhyB-PIF3 system. We introduce two previously uncharacterized systems: strongLOV, a more light-sensitive El222 mutant, and ARG3pr, which is induced in the absence of arginine or presence of methionine. To demonstrate fine control over gene circuits, we experimentally tune the time between cell cycle Start and mitosis, artificially simulating near-wild-type timing. All strains, constructs, code, and data ( https://promoter-benchmark.epfl.ch/ ) are made available.

Abstract: We developed a platform that utilizes a calcium-dependent luciferase to convert neuronal activity into activation of light sensing domains within the same cell. The platform is based on a Gaussia luciferase variant with high light emission split by calmodulin-M13 sequences that depends on influx of calcium ions (Ca2+) for functional reconstitution. In the presence of its luciferin, coelenterazine (CTZ), Ca2+ influx results in light emission that drives activation of photoreceptors, including optogenetic channels and LOV domains. Critical features of the converter luciferase are light emission low enough to not activate photoreceptors under baseline condition and high enough to activate photosensing elements in the presence of Ca2+ and luciferin. We demonstrate performance of this activity-dependent sensor and integrator for changing membrane potential and driving transcription in individual and populations of neurons in vitro and in vivo.

Abstract: Cells tend to divide along the direction in which they are longest, as famously stated by Oscar Hertwig in 1884 in his long axis rule. The orientation of the mitotic spindle determines the cell division axis, and the long axis rule is usually ensured by forces stemming from microtubules. Pulling on the spindle from the cell cortex can give rise to unstable behaviors, and we here set out to understand how the long axis rule is realized in early embryonic divisions where cortical pulling forces are prevalent. We focus on early C. elegans development, where we compressed embryos to reveal that cortical pulling forces favor an alignment of the spindle with the short axis of the cell. Strikingly, we find that this misalignment is corrected by an actomyosin-based mechanism that rotates the entire cell, including the mitotic spindle. We uncover that myosin-driven contractility in the cytokinetic ring generates inward forces that align it with the short axis, and thereby the spindle with the long axis. A theoretical model together with experiments using slightly compressed mouse zygotes suggest that a constricting cytokinetic ring can ensure the long axis rule in cells that are free to rotate inside a confining structure, thereby generalizing the underlying principle.

Abstract: The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.

Abstract: The cyclic recombinase (Cre)/loxP recombination system is a powerful technique for in vivo cell labeling and tracking. However, achieving high spatiotemporal precision in cell tracking using this system is challenging due to the requirement for reliable tissue-specific promoters. In contrast, light-inducible systems offer superior regional confinement, tunability and non-invasiveness compared to conventional lineage tracing methods. Here, we took advantage of the unique strengths of the zebrafish to develop an easy-to-use highly efficient, genetically encoded, Magnets-based, light-inducible transgenic Cre/loxP system. Our system relies on the reassembly of split Cre fragments driven by the affinity of the Magnets and is controlled by the zebrafish ubiquitin promoter. We demonstrate that our system does not exhibit phototoxicity or leakiness in the dark, and it enables efficient and robust Cre/loxP recombination in various tissues and cell types at different developmental stages through noninvasive illumination with blue light. Our newly developed tool is expected to open novel opportunities for light-controlled tracking of cell fate and migration in vivo.

Abstract: Fluorescent proteins, while essential for bioimaging, are limited to visualizing cellular localization without offering additional functionality. We report for the first time a strategy to expand the chemical, structural, and functional diversity of fluorescent proteins by harnessing light to induce red fluorescence in a previously non-fluorescent protein. We accomplish this by inducing the transfer of the genetically encoded chromophore from a photocleavable protein (PhoCl1) to a non-fluorescent kinase (MjRibK) inducing red fluorescence in the latter. We have employed analytical and spectroscopic techniques to validate the presence of red fluorescence in MjRibK. Furthermore, molecular dynamics simulations were carried out to investigate the amino acid residues of MjRibK involved in the generation of red fluorescence. Finally, we demonstrate the ability of the red fluorescent MjRibK to operate as a cyclable high-temperature sensor. We anticipate that this light-induced chromophore transfer strategy will open new possibilities for developing multifunctional genetically encoded fluorescent sensors.

Abstract: Wnt/β-catenin signaling and its dysregulation play critical roles in the fate determination of stem cells and the pathology of various diseases. However, the application of translated Wnt ligand in regenerative medicine is hampered by its hydrophobicity and cross-reactivity with Frizzled (FZD) receptors. Here, we generate an engineered water-soluble, FZD subtype-specific agonist, RRP-pbFn, for high-efficiency Wnt/β-catenin signaling activation. In the absence of direct binding to LRP5/6, RRP-pbFn stimulates Wnt/β-catenin signaling more potently than surrogate Wnt. RRP-pbFn supports the growth of a variety of mouse and human organoids, and induces the expansion of liver and intestine progenitors in vivo. Meanwhile, we develop a synthetic LRP antagonist, RRP-Dkk1c, which exhibits heightened effectiveness in attenuating Wnt/β-catenin signaling activity compared to Dkk1, thereby abolishing the formation of CT26-derived colon cancer xenograft in vivo. Together, these two paired Wnt/β-catenin signaling manipulators hold great promise for biomedical research and potential therapeutics.

Abstract: The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.

Abstract: In addition to biochemical and electrochemical signaling, cells also rely extensively on mechanical signaling to regulate their behavior. While a number of tools have been adapted from physics and engineering to manipulate cell mechanics, they typically require specialized equipment or lack spatiotemporal precision. Alternatively, a recent, more elegant approach is to use light itself to modulate the mechanical equilibrium inside the cell. This approach leverages the power of optogenetics, which can be controlled in a fully reversible manner in both time and space, to tune RhoA signaling, the master regulator of cellular contractility. We review here the fundamentals of this approach, including illustrating the tunability and flexibility that optogenetics offers, and demonstrate how this tool can be used to modulate both internal cytoskeletal flows and contractile force generation. Together these features highlight the advantages that optogenetics offers for investigating mechanical interactions in cells.

Abstract: Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.

Abstract: Hydrogels with adjustable mechanical properties have been engineered as matrices for mammalian cells and allow the dynamic, mechano-responsive manipulation of cell fate and function. Recent research yields hydrogels, where biological photoreceptors translated optical signals into a reversible and adjustable change in hydrogel mechanics. While their initial application provides important insights into mechanobiology, broader implementation is limited by a small dynamic range of addressable stiffness. Herein, this limitation is overcome by developing a photoreceptor-based hydrogel with reversibly adjustable stiffness from ≈800 Pa to the sol state. The hydrogel is based on star-shaped polyethylene glycol, functionalized with the red/far-red light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor 6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6, thus crosslinking the polymers and resulting in gelation. However, upon illumination with far-red light, the proteins dissociate and trigger a complete gel-to-sol transition. The hydrogel's light-responsive mechanical properties are comprehensively characterized and it is applied as a reversible extracellular matrix for the spatiotemporally controlled deposition of mammalian cells within a microfluidic chip. It is anticipated that this technology will open new avenues for the site- and time-specific positioning of cells and will contribute to overcome spatial restrictions.

Abstract: Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Abstract: Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.

Abstract: Gene deletion is one of the standard approaches in genetics to investigate the roles and functions of target genes. However, the influence of gene deletion on cellular phenotypes is usually analyzed sometime after the gene deletion was introduced. Such lags from gene deletion to phenotype evaluation could select only the fittest fraction of gene-deleted cells and hinder the detection of potentially diverse phenotypic consequences. Therefore, dynamic aspects of gene deletion, such as real-time propagation and compensation of deletion effects on cellular phenotypes, still need to be explored. To resolve this issue, we have recently introduced a new method that combines a photoactivatable Cre recombination system and microfluidic single-cell observation. This method enables us to induce gene deletion at desired timings in single bacterial cells and to monitor their dynamics for prolonged periods. Here, we detail the protocol for estimating the fractions of gene-deleted cells based on a batch-culture assay. The duration of blue light exposure significantly affects the fractions of gene-deleted cells. Therefore, gene-deleted and non-deleted cells can coexist in a cellular population by adjusting the duration of blue light exposure. Single-cell observations under such illumination conditions allow the comparison of temporal dynamics between gene-deleted and non-deleted cells and unravel phenotypic dynamics provoked by gene deletion.

Abstract: Angiogenesis and tissue repair in chronic non-healing diabetic wounds remain critical clinical problems. Engineered MSC-derived exosomes have significant potential for the promotion of wound healing. Here, we discuss the effects and mechanisms of eNOS-rich umbilical cord MSC exosomes (UCMSC-exo/eNOS) modified by genetic engineering and optogenetic techniques on diabetic chronic wound repair.

Abstract: Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach's utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

Abstract: The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.

Abstract: Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Abstract: Optogenetics is a technique employing natural or genetically engineered photoreceptors in transgene organisms to manipulate biological activities with light. Light can be turned on or off, and adjusting its intensity and duration allows optogenetic fine-tuning of cellular processes in a noninvasive and spatiotemporally resolved manner. Since the introduction of Channelrhodopsin-2 and phytochrome-based switches nearly 20 years ago, optogenetic tools have been applied in a variety of model organisms with enormous success, but rarely in plants. For a long time, the dependence of plant growth on light and the absence of retinal, the rhodopsin chromophore, prevented the establishment of plant optogenetics until recent progress overcame these difficulties. We summarize the recent results of work in the field to control plant growth and cellular motion via green light-gated ion channels and present successful applications to light-control gene expression with single or combined photoswitches in plants. Furthermore, we highlight the technical requirements and options for future plant optogenetic research.

Abstract: Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

Abstract: The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the optoCRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of optoCRISPRi often exhibit no more than a 10-fold dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light-activated CRISPRi system with a high dynamic range (40 fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, nonessential genes, or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further research involving complex gene networks, metabolic flux redirection, or bioprinting.

Abstract: During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.

Abstract: Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.

Abstract: Living cells utilize signaling pathways to sense, transduce, and process information. As the extracellular stimulation often has rich temporal characteristics which may govern dynamic cellular responses, it is important to quantify the rate of information flow through the signaling pathways. In this study, we used an epithelial cell line expressing a light-activatable FGF receptor and an ERK activity reporter to assess the ability of the MAPK/ERK pathway to transduce signal encoded in a sequence of pulses. By stimulating the cells with random light pulse trains, we demonstrated that the MAPK/ERK channel capacity is at least 6 bits per hour. The input reconstruction algorithm detects the light pulses with 1-min accuracy 5 min after their occurrence. The high information transmission rate may enable the pathway to coordinate multiple processes including cell movement and respond to rapidly varying stimuli such as chemoattracting gradients created by other cells.

Abstract: Immunotherapy emerged as a paradigm shift in cancer treatments, which can effectively inhibit cancer progression by activating the immune system. Remarkable clinical outcomes have been achieved through recent advances in cancer immunotherapy, including checkpoint blockades, adoptive cellular therapy, cancer vaccine, and tumor microenvironment modulation. However, extending the application of immunotherapy in cancer patients has been limited by the low response rate and side effects such as autoimmune toxicities. With great progress being made in nanotechnology, nanomedicine has been exploited to overcome biological barriers for drug delivery. Given the spatiotemporal control, light-responsive nanomedicine is of great interest in designing precise modality for cancer immunotherapy. Herein, we summarized current research utilizing light-responsive nanoplatforms to enhance checkpoint blockade immunotherapy, facilitate targeted delivery of cancer vaccines, activate immune cell functions, and modulate tumor microenvironment. The clinical translation potential of those designs is highlighted and challenges for the next breakthrough in cancer immunotherapy are discussed.