Curated Optogenetic Publication Database

Abstract: Optogenetics has become a key tool to manipulate biological processes with high spatio-temporal resolution. Recently, a number of commercial and open-source multi-well illumination devices have been developed to provide throughput in optogenetics experiments. However, available commercial devices remain expensive and lack flexibility, while open-source solutions require programming knowledge and/or include complex assembly processes. We present a LED Illumination Tool for Optogenetic Stimulation (LITOS) based on an assembled printed circuit board controlling a commercially available 32 × 64 LED matrix as illumination source. LITOS can be quickly assembled without any soldering, and includes an easy-to-use interface, accessible via a website hosted on the device itself. Complex light stimulation patterns can easily be programmed without coding expertise. LITOS can be used with different formats of multi-well plates, petri dishes, and flasks. We validated LITOS by measuring the activity of the MAPK/ERK signaling pathway in response to different dynamic light stimulation regimes using FGFR1 and Raf optogenetic actuators. LITOS can uniformly stimulate all the cells in a well and allows for flexible temporal stimulation schemes. LITOS's affordability and ease of use aims at democratizing optogenetics in any laboratory.

Abstract: Mitochondria are highly dynamic organelles whose fragmentation by fission is critical to their functional integrity and cellular homeostasis. Here, we develop a method via optogenetic control of mitochondria-lysosome contacts (MLCs) to induce mitochondrial fission with spatiotemporal accuracy. MLCs can be achieved by blue-light-induced association of mitochondria and lysosomes through various photoactivatable dimerizers. Real-time optogenetic induction of mitochondrial fission is tracked in living cells to measure the fission rate. The optogenetic method partially restores the mitochondrial functions of SLC25A46-/- cells, which display defects in mitochondrial fission and hyperfused mitochondria. The optogenetic MLCs system thus provides a platform for studying mitochondrial fission and treating mitochondrial diseases.

Abstract: The H2 + H2 system has long been considered a benchmark system for ro-vibrational energy transfer in bimolecular collisions. However, most studies thus far have focused on collisions involving H2 molecules in the ground vibrational level or in the first excited vibrational state. While H2 + H2/HD collisions have received wide attention due to the important role they play in astrophysics, D2 + D2 collisions have received much less attention. Recently, Zhou et al. [ Nat. Chem. 2022, 14, 658-663, DOI: 10.1038/s41557-022-00926-z] examined stereodynamic aspects of rotational energy transfer in collisions of two aligned D2 molecules prepared in the v = 2 vibrational level and j = 2 rotational level. Here, we report quantum calculations of rotational and vibrational energy transfer in collisions of two D2 molecules prepared in vibrational levels up to v = 2 and identify key resonance features that contribute to the angular distribution in the experimental results of Zhou et al. The quantum scattering calculations were performed in full dimensionality and using the rigid-rotor approximation using a recently developed highly accurate six-dimensional potential energy surface for the H4 system that allows descriptions of collisions involving highly vibrationally excited H2 and its isotopologues.

Abstract: Since the successful introduction of exogenous photosensitive proteins, channelrhodopsin, to neurons, optogenetics has enabled substantial understanding of profound brain function by selectively manipulating neural circuits. In an optogenetic system, optical stimulation can be precisely delivered to brain tissue to achieve regulation of cellular electrical activity with unprecedented spatio-temporal resolution in living organisms. In recent years, the development of various optical actuators and novel light-delivery techniques has greatly expanded the scope of optogenetics, enabling the control of other signal pathways in non-neuronal cells for different biomedical applications, such as phototherapy and immunotherapy. This review focuses on the recent advances in optogenetic regulation of cellular activities for photomedicine. We discuss emerging optogenetic tools and light-delivery platforms, along with a survey of optogenetic execution in mammalian and microbial cells.

Abstract: How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.

Abstract: Gene- and cell-based therapies are the next frontiers in the field of medicine. Both are transformative and innovative therapies; however, a lack of safety data limits the translation of such promising technologies to the clinic. Improving the safety and promoting the clinical translation of these therapies can be achieved by tightly regulating the release and delivery of therapeutic outputs. In recent years, the rapid development of optogenetic technology has provided opportunities to develop precision-controlled gene- and cell-based therapies, in which light is introduced to precisely and spatiotemporally manipulate the behaviour of genes and cells. This review focuses on the development of optogenetic tools and their applications in biomedicine, including photoactivated genome engineering and phototherapy for diabetes and tumours. The prospects and challenges of optogenetic tools for future clinical applications are also discussed.

Abstract: Nine mammalian adenylyl cyclases (AC) are pseudoheterodimers with two hexahelical membrane domains, which are isoform-specifically conserved. Previously we proposed that these membrane domains are orphan receptors (https://doi.org/10.7554/eLife.13098; https://doi.org/10.1016/j.cellsig.2020.109538). Lipids extracted from fetal bovine serum at pH 1 inhibited several mAC activities. Guided by a lipidomic analysis we tested glycerophospholipids as potential ligands. Contrary to expectations we surprisingly discovered that 1-stearoyl-2-docosahexaenoyl-phosphatidic acid (SDPA) potentiated Gsα-activated activity of human AC isoform 3 seven-fold. The specificity of fatty acyl esters at glycerol positions 1 and 2 was rather stringent. 1-Stearoyl-2-docosahexaenoyl-phosphatidylserine and 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine significantly potentiated several Gsα-activated mAC isoforms to different extents. SDPA appears not interact with forskolin activation of AC isoform 3. SDPA enhanced Gsα-activated AC activities in membranes from mouse brain cortex. The action of SDPA was reversible. Unexpectedly, SDPA did not affect cAMP generation in HEK293 cells stimulated by isoproterenol, PGE2 and adenosine, virtually excluding a role as an extracellular ligand and, instead, suggesting an intracellular role. In summary, we discovered a new dimension of intracellular AC regulation by chemically defined glycerophospholipids.

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 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: 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: 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: 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: 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: 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.

Abstract: Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.

Abstract: Chemical induction is one of the most common modalities used to manipulate gene expression in living systems. However, chemical induction can be toxic or expensive that compromise the economic feasibility when it comes to industrial-scale synthetic biology applications. These complications have driven the pursuit of better induction systems. Optogenetics technique can be a solution as it not only enables dynamic control with unprecedented spatiotemporal precision but also is inexpensive and eco-friendlier. The optogenetic technique harnesses natural light-sensing modules that are genetically encodable and re-programmable in various hosts. By further engineering these modules to connect with the microbial regulatory machinery, gene expression and protein activity can be finely tuned simply through light irradiation. Recent works on applying optogenetics to microbial synthetic biology have yielded remarkable achievements. To further expand the usability of optogenetics, more optogenetic tools with greater portability that are compatible with different microbial hosts need to be developed. This review focuses on non-opsin optogenetic systems and the current state of optogenetic advancements in microbes, by showcasing the different designs and functions of optogenetic tools, followed by an insight into the optogenetic approaches used to circumvent challenges in synthetic biology.