Curated Optogenetic Publication Database

Abstract: To understand cell biological processes, like signalling pathways, protein movements, or metabolic processes, precise tools for manipulation are desired. Optogenetics allows to control cellular processes by light and can be applied at a high temporal and spatial resolution. In the last three decades, various optogenetic applications have been developed for animal, fungal, and prokaryotic cells. However, using optogenetics in plants has been difficult due to biological and technical issues, like missing cofactors, the presence of endogenous photoreceptors, or the necessity of light for photosynthesis, which potentially activates optogenetic tools constitutively. Recently developed tools overcome these limitations, making the application of optogenetics feasible also in plants. Here, we highlight the most useful recent applications in plants and give a perspective for future optogenetic approaches in plants science.

Abstract: The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

Abstract: The expulsion of dying epithelial cells requires well-orchestrated remodelling steps to maintain tissue sealing. This process, named cell extrusion, has been mostly analysed through the study of actomyosin regulation. Yet, the mechanistic relationship between caspase activation and cell extrusion is still poorly understood. Using the Drosophila pupal notum, a single layer epithelium where extrusions are caspase-dependent, we showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration and dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion, while microtubule stabilisation strongly impairs cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting step of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

Abstract: Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Abstract: Immune cells activate in binary, switch-like fashion via large protein assemblies known as signalosomes, but the molecular mechanism of the switch is not yet understood. Here, we employed an in-cell biophysical approach to dissect the assembly mechanism of the CARD-BCL10-MALT1 (CBM) signalosome, which governs nuclear transcription factor-κB activation in both innate and adaptive immunity. We found that the switch consists of a sequence-encoded and deeply conserved nucleation barrier to ordered polymerization by the adaptor protein BCL10. The particular structure of the BCL10 polymers did not matter for activity. Using optogenetic tools and single-cell transcriptional reporters, we discovered that endogenous BCL10 is functionally supersaturated even in unstimulated human cells, and this results in a predetermined response to stimulation upon nucleation by activated CARD multimers. Our findings may inform on the progressive nature of age-associated inflammation, and suggest that signalosome structure has evolved via selection for kinetic rather than equilibrium properties of the proteins.

Abstract: Optogenetics combines genetics and biophotonics to enable noninvasive control of biological processes with high spatiotemporal precision. When engineered into protein machineries that govern the cellular information flow as depicted in the central dogma, multiple genetically encoded non-opsin photosensory modules have been harnessed to modulate gene transcription, DNA or RNA modifications, DNA recombination, and genome engineering by utilizing photons emitting in the wide range of 200-1000 nm. We present herein generally applicable modular strategies for optogenetic engineering and highlight latest advances in the broad applications of opsin-free optogenetics to program transcriptional outputs and precisely manipulate the mammalian genome, epigenome, and epitranscriptome. We also discuss current challenges and future trends in opsin-free optogenetics, which has been rapidly evolving to meet the growing needs in synthetic biology and genetics research.

Abstract: We review fundamental mechanisms and applications of OptoGels: hydrogels with light-programmable properties endowed by photoswitchable proteins ("optoproteins") found in nature. Light, as the primary source of energy on earth, has driven evolution to develop highly-tuned functionalities, such as phototropism and circadian entrainment. These functions are mediated through a growing family of optoproteins that respond to the entire visible spectrum ranging from ultraviolet to infrared by changing their structure to transmit signals inside of cells. In a recent series of articles, engineers and biochemists have incorporated optoproteins into a variety of extracellular systems, endowing them with photocontrollability. While other routes exist for dynamically controlling material properties, light-sensitive proteins have several distinct advantages, including precise spatiotemporal control, reversibility, substrate selectivity, as well as biodegradability and biocompatibility. Available conjugation chemistries endow OptoGels with a combinatorially large design space determined by the set of optoproteins and polymer networks. These combinations result in a variety of tunable material properties. Despite their potential, relatively little of the OptoGel design space has been explored. Here, we aim to summarize innovations in this emerging field and highlight potential future applications of these next generation materials. OptoGels show great promise in applications ranging from mechanobiology, to 3D cell and organoid engineering, and programmable cell eluting materials.

Abstract: Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

Abstract: Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.

Abstract: Gene and cell therapies are widely recognized as future cancer therapeutics but poor controllability limits their clinical applications. Optogenetics, the use of light-controlled proteins to precisely spatiotemporally regulate the activity of genes and cells, opens up new possibilities for cancer treatment. Light of specific wavelength can activate the immune response, oncolytic activity and modulate cell signaling in tumor cells non-invasively, in dosed manner, with tissue confined action and without side effects of conventional therapies. Here, we review optogenetic approaches in cancer research, their clinical potential and challenges of incorporating optogenetics in cancer therapy. We critically discuss beneficial combinations of optogenetic technologies with therapeutic nanobodies, T-cell activation and CAR-T cell approaches, genome editors and oncolytic viruses. We consider viral vectors and nanoparticles for delivering optogenetic payloads and activating light to tumors. Finally, we highlight herein the prospects for integrating optogenetics into immunotherapy as a novel, fast, reversible and safe approach to cancer treatment.

Abstract: Harnessing the potential of optogenetics in biology requires methodologies from different disciplines ranging from biology, to mechatronics engineering, to control engineering. Light stimulation of a synthetic optogenetic construct in a given biological species can only be achieved via a suitable light stimulation platform. Emerging optogenetic applications entail a consistent, reproducible, and regulated delivery of light adapted to the application requirement. In this review, we explore the evolution of light-induction hardware-software platforms from simple illumination set-ups to sophisticated microscopy, microtiter plate and bioreactor designs, and discuss their respective advantages and disadvantages. Here, we examine design approaches followed in performing optogenetic experiments spanning different cell types and culture volumes, with induction capabilities ranging from single cell stimulation to entire cell culture illumination. The development of automated measurement and stimulation schemes on these platforms has enabled researchers to implement various in silico feedback control strategies to achieve computer-controlled living systems-a theme we briefly discuss in the last part of this review.

Abstract: Combining single-cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single-cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK-RAF and the ERK-RSK2-SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2-mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2-dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2-mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.

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

Abstract: Many types of regulated cell death exist, however the non-cell autonomous effects of specific forms of cell death remain poorly understood. Addressing this, Shkarina et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109038) describe an optogenetic method to activate distinct modes of cell death in select cells.

Abstract: Proper embryonic development requires directional axes to pattern cells into embryonic structures. In Drosophila, spatially discrete expression of transcription factors determines the anterior to posterior organization of the early embryo, while the Toll and TGFβ signalling pathways determine the early dorsal to ventral pattern. Embryonic MAPK/ERK signaling contributes to both anterior to posterior patterning in the terminal regions and to dorsal to ventral patterning during oogenesis and embryonic stages. Here we describe a novel loss of function mutation in the Raf kinase gene, which leads to loss of ventral cell fates as seen through the loss of the ventral furrow, the absence of Dorsal/NFκB nuclear localization, the absence of mesoderm determinants Twist and Snail, and the expansion of TGFβ. Gene expression analysis showed cells adopting ectodermal fates much like loss of Toll signaling. Our results combine novel mutants, live imaging, optogenetics and transcriptomics to establish a novel role for Raf, that appears to be independent of the MAPK cascade, in embryonic patterning.

Abstract: Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation.

Abstract: Early embryogenesis requires rapid division of pluripotent blastomeres, regulated genome activation, precise spatiotemporal signaling to pattern cell fate, and morphogenesis to shape primitive tissue architectures. The complexity of this process has inspired researchers to move beyond simple genetic perturbation into engineered devices and synthetic biology tools to permit temporal and spatial manipulation of the control systems guiding development. By precise alteration of embryo organization, it is now possible to advance beyond basic analytical strategies and directly test the sufficiency of models for developmental regulation. Separately, advances in micropatterning and embryoid culture have facilitated the bottom-up construction of complex embryo tissues allowing ex vivo systems to recapitulate even later stages of development. Embryos fertilized and grown ex vivo offer an excellent opportunity to exogenously perturb fundamental pathways governing embryogenesis. Here we review the technologies developed to thermally modulate the embryo cell cycle, and optically regulate morphogen and signaling pathways in space and time, specifically in the blastula embryo. Additionally, we highlight recent advances in cell patterning in two and three dimensions that have helped reveal the self-organizing properties and gene regulatory networks guiding early embryo organization.

Abstract: Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a protein’s subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.

Abstract: Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.

Abstract: Optogenetics has great potential for biotechnology and metabolic engineering due to the cost-effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light-delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low-caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light-controlled interaction between Cry2 and Cib1 was used for sweet-protein production. Optimization of the fermenter process was achieved by increasing the light-flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet-protein were produced in a pre-stationary growth phase, whereas at later stages, a decay in protein abundance was observable. Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic-controlled production in a fermenter is highly cost-effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step.

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

Abstract: Peroxisome proliferator-activated receptor (PPAR)-γ is a key transcription activator controlling adipogenesis and lipid metabolism. PPARγ binds PPAR response elements (PPREs) as the obligate heterodimer with retinoid X receptor (RXR) α, but exactly how PPARγ orchestrates the transcriptional response is unknown. This study demonstrates that PPARγ forms phase-separated droplets in vitro and solid-like nuclear condensates in cell, which is intriguingly mediated by its DNA binding domain characterized by the zinc finger motif. Furthermore, PPARγ forms nuclear condensates at PPREs sites through phase separation to compartmentalize its heterodimer partner RXRα to initiate PPARγ-specific transcriptional activation. Finally, using an optogenetic approach, the enforced formation of PPARγ/RXRα condensates leads to preferential enrichment at PPREs sites and significantly promotes the expression of PPARγ target genes. These results define a novel mechanism by which PPARγ engages the phase separation principles for efficient and specific transcriptional activation.

Abstract: Technological advances have driven many recent advances in developmental biology. Light sheet imaging can reveal single-cell dynamics in living three-dimensional tissues, whereas single-cell genomic methods open the door to a complete catalogue of cell types and gene expression states. An equally powerful but complementary set of approaches are also becoming available to define development processes from the bottom up. These synthetic approaches aim to reconstruct the minimal developmental patterns, signaling processes, and gene networks that produce the basic set of developmental operations: spatial polarization, morphogen interpretation, tissue movement, and cellular memory. In this review we discuss recent approaches at the intersection of synthetic biology and development, including synthetic circuits to deliver and record signaling stimuli and synthetic reconstitution of pattern formation on multicellular scales.

Abstract: Cells migrate through crowded microenvironments within tissues during normal development, immune response, and cancer metastasis. Although migration through pores and tracks in the extracellular matrix (ECM) has been well studied, little is known about cellular traversal into confining cell-dense tissues. We find that embryonic tissue invasion by Drosophila macrophages requires division of an epithelial ectodermal cell at the site of entry. Dividing ectodermal cells disassemble ECM attachment formed by integrin-mediated focal adhesions next to mesodermal cells, allowing macrophages to move their nuclei ahead and invade between two immediately adjacent tissues. Invasion efficiency depends on division frequency, but reduction of adhesion strength allows macrophage entry independently of division. This work demonstrates that tissue dynamics can regulate cellular infiltration.

Abstract: Engineered, light-sensitive protein switches are used to interrogate a broad variety of biological processes. These switches are typically constructed by genetically fusing naturally occurring light-responsive protein domains with functional domains from other proteins. Protein activity can be controlled using a variety of mechanisms including light-induced colocalization, caging, and allosteric regulation. Protein design efforts have focused on reducing background signaling, maximizing the change in activity upon light stimulation, and perturbing the kinetics of switching. It is common to combine structure-based modeling with experimental screening to identify ideal fusion points between domains and discover point mutations that optimize switching. Here, we introduce commonly used light-sensitive domains and summarize recent progress in using them to regulate protein activity.