Curated Optogenetic Publication Database

Abstract: In adherent cells, actomyosin contractility is regulated mainly by the RhoA signaling pathway, which can be controlled by optogenetics. To model the mechanochemical coupling in such systems, we introduce a finite element framework based on the discontinuous Galerkin method, which allows us to treat cell doublets, chains of cells, and monolayers within the same conceptual framework. While the adherent cell layer is modeled as an actively contracting viscoelastic solid on an elastic foundation, different models are considered for the Rho pathway, starting with a simple linear chain that can be solved analytically and later including direct feedback that can be solved only numerically. Our model predicts signal propagation as a function of coupling strength and viscoelastic timescales and identifies the conditions for optimal cell responses and wave propagation. In general, it provides a systematic understanding of how biochemistry and mechanics simultaneously contribute to the communication of adherent cells.

Abstract: Dynamic regulation of cell-cell adhesion is fundamental to numerous biological processes and is the key to engineering multicellular structures. Optogenetic tools offer precise spatiotemporal control over cell-cell adhesions, but current methods often require the genetic modification of each participating cell type. To address this limitation, we engineered a single-component synthetic cell adhesion molecule based on the blue-light-responsive, plasma membrane-binding protein BcLOV4. We tagged BcLOV4 with a transmembrane domain to display it on the outer plasma membrane (BcLOV4-PM). Under blue light but not in the dark, BcLOV4-PM cells formed both homotypic adhesions with other BcLOV4-PM cells and heterotypic adhesions with a range of unmodified wild-type cells. While these adhesions were not reversed in the dark, they could be efficiently disrupted by increasing the temperature to 37 °C, leveraging BcLOV4's thermosensitivity. Using BcLOV4-PM-based adhesions, we demonstrated light-controlled compaction of spheroids in both monocultures and cocultures with wild-type cells. Altogether, BcLOV4-PM enables promiscuous, modular, light-dependent control of cell-cell adhesions without requiring genetic modification of all cell types involved, offering promising applications in tissue engineering and the study of multicellular process.

Abstract: Cancer cell invasion relies on dynamic cell shape changes, which originate from protrusive and contractile intracellular forces. Previous studies revealed that contractile forces are controlled by positive-feedback amplification of the contraction regulator Rho by Lbc GEFs. These GEFs were previously linked to tumor progression; however, the underlying mechanisms are poorly understood. Here, we generated a mouse melanoma model in which cytosolic levels of the Lbc GEF GEF-H1 are controlled by light. Using this model, we found that increased GEF-H1 levels strongly stimulate cell contraction dynamics. Interestingly, increased contraction dynamics rapidly induced expansion of tumor spheroids via a focal adhesion kinase-dependent mechanism. Furthermore, long-term stimulation led to the escape of individual cells from spheroids. These findings reveal new insights into the oncogenic roles of Lbc GEFs and how they might promote tumor cell invasion. We propose a mechanism in which increased cell contraction dynamics result in asymmetric pulling forces at the tumor border, promoting the detachment and escape of individual cells.

Abstract: In cell biology, optical techniques are increasingly used to measure cells' internal states (biosensors) and to stimulate cellular responses (optogenetics). Yet the design of all-optical experiments is often manual: a pre-determined stimulus pattern is applied to cells, biosensors are measured over time, and the resulting data is processed off-line. With the advent of machine learning for segmentation and tracking, it becomes possible to envision closed-loop experiments where real-time information about cells' positions and states are used to dynamically determine optogenetic stimuli to alter or control their behavior. Here, we develop PyCLM, a Python-based suite of tools to enable real-time measurement, image segmentation, and optogenetic control of thousands of cells per experiment. PyCLM is designed to be as simple for the end user as possible, and multipoint experiments can be set up that combine a wide variety of imaging, image processing, and stimulation modalities without any programming. We showcase PyCLM on diverse applications: studying the effect of epidermal growth factor receptor activity waves on epithelial tissue movement, simultaneously stimulating ~1,000 single cells to guide tissue flows, and performing real-time feedback control of cell-to-cell fluorescence heterogeneity. This tool will enable the next generation of dynamic experiments to probe cell and tissue properties, and provides a first step toward precise control of cell states at the tissue scale.

Abstract: Organ-on-a-chip platforms have emerged as promising human tissue models for drug screening and mechanistic studies, offering alternatives to traditional animal models. Integration of vascular structures into these platforms is pivotal for creating physiologically faithful models of individual organs and studying interorgan crosstalk. However, most vascular structures grown in vitro do not account for organ-specific endothelial permeability or its modulation in response to disease. Here, we present optoBarrier, an optogenetic organ-on-a-chip platform designed to modulate endothelial barrier permeability through light stimulation. By optically activating RhoA signaling in engineered optogenetic endothelial cells, we demonstrate the formation of stress fibers, disruption of vascular endothelial cadherin (VE-cadherin) and increased barrier permeability. We further show that permeability is tunable in a reversible and dose-dependent manner in response to light. We therefore propose that optoBarrier offers a user-defined, controlled and simple method to manipulate endothelial permeability for in vitro studies of human vasculature.

Abstract: Early detection of pollutants in water discharge is an integral part of environmental monitoring. Electroactive biofilm (EAB)-enabled, microbial fuel cell (MFC)-based biosensors facilitate self-powered online pollutant detection. However, as EABs are highly dynamic, naturally formed EABs as sensing and transducing elements limit the performance of MFC-based biosensors. Here, we report a fast-response and sensitive MFC-based biosensor enabled by enhancing Shewanella oneidensis biofilms on the electrode using an optogenetic approach. We incorporated a near-infrared (NIR) light-responsive synthetic bis(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) module into S. oneidensis to promote biofilm formation on the anode under NIR light. The biosensors with enhanced EABs exhibited a rapid and sensitive response to Cr(VI), reducing the sensing time from approximately 30 min to just 3 min. This improved sensing performance was maintained over three sensing cycles, even with fluctuating Cr(VI) concentrations. Based on the analyses of the electrode biofilms and extracellular polymeric substance matrices, different Cr(VI) response mechanisms for the normal and enhanced EABs were proposed; enhanced EAB's massive dispersal by Cr(VI) was the cause of the improved response of the biosensors. Such improved response still held in the natural water matrix. This proof-of-concept study provides valuable insights into controlling electrode biofilm dynamics for the rapid and robust early detection of pollutants using MFC-based biosensors.

Abstract: During mating in budding yeast, cells use pheromones to locate each other and fuse. This model system has shaped our current understanding of signal transduction and cell polarization in response to extracellular signals. The cell populations producing extracellular signal landscapes themselves are, however, less well understood, yet crucial for functionally testing quantitative models of cell polarization and for controlling cell behavior through bioengineering approaches. Here we engineered optogenetic control of pheromone landscapes in mating populations of budding yeast, hijacking the mating-pheromone pathway to achieve spatial control of growth, cell morphology, cell-cell fusion, and distance-dependent gene expression in response to light. Using our tool, we were able to spatially control and shape pheromone gradients, allowing the use of a biophysical model to infer the properties of large-scale gradients generated by mating populations in a single, quantitative experimental setup, predicting that the shape of such gradients depends quantitatively on population parameters. Spatial optogenetic control of diffusible signals and their degradation provides a controllable signaling environment for engineering artificial communication and cell-fate systems in gel-embedded cell populations without the need for physical manipulation.

Abstract: Flocculation in Saccharomyces cerevisiae is a critical phenotype with ecological and industrial significance. This study aimed to functionally dissect the contributions of individual FLO genes (FLO1, FLO5, FLO9, FLO10, FLO11) to flocculation by employing an optogenetic circuit (OptoQ-AMP5) for precise, light-inducible control of gene expression. A FLO-null platform yeast strain was engineered allowing the expression of individual FLO genes without native background interference. Each FLO gene was reintroduced into the FLO-null background under the control of OptoQ-AMP5. Upon light induction, strains expressing FLO1, FLO5, or FLO10 demonstrated strong flocculation, with FLO1 and FLO5 forming large and structurally distinct aggregates. FLO9 induced a weaker phenotype. Sugar inhibition assays revealed distinct sensitivities among flocculins, notably FLO9's novel sensitivity to fructose and maltotriose. Additionally, FLO-induced changes in cell surface hydrophobicity were quantified, revealing that FLO10 and FLO1 conferred the greatest hydrophobicity, correlating with their aggregation strength. This work establishes a robust platform for investigating flocculation mechanisms in yeast with temporal precision. It highlights the phenotypic diversity encoded within the FLO gene family and their differential responses to environmental cues. The optogenetic system provides a valuable tool for both fundamental studies and the rational engineering of yeast strains for industrial fermentation processes requiring controlled flocculation.

Abstract: The bacterial stress response is an intricately regulated system that plays a critical role in cellular resistance to drug treatment. The complexity of this response is further complicated by cell-to-cell heterogeneity in the expression of bacterial stress response genes. These genes are often organized into networks comprising one or more transcriptional regulators that control expression of a suite of downstream genes. While the expression heterogeneity of many of these upstream regulators has been characterized, the way in which this variability affects the larger downstream stress response remains hard to predict, prompting two key questions. First, how does heterogeneity and expression noise in stress response regulators propagate to the diverse downstream genes in their regulons. Second, when expression levels vary, how do multiple downstream genes act together to protect cells from stress. To address these questions, we focus on the transcription factor PhoP, a critical virulence regulator which coordinates pathogenicity in several gram-negative species. We use optogenetic stimulation to precisely control PhoP expression levels and examine how variations in PhoP affect the downstream activation of genes in the PhoP regulon. We find that these downstream genes exhibit differences both in mean expression level and sensitivity to increasing levels of PhoP. These response functions can also vary between individual cells, increasing heterogeneity in the population. We tie these variations to cell survival when bacteria are exposed to a clinically-relevant antimicrobial peptide, showing that high expression of the PhoP-regulon gene pmrD provides a protective effect against Polymyxin B. Overall, we demonstrate that even subtle heterogeneity in expression of a stress response regulator can have clear consequences for enabling bacteria to survive stress.

Abstract: Collective cell migration is key during development, wound healing, and metastasis and relies on coordinated cell behaviors at the group level. Src kinase is a key signalling protein for the physiological functions of epithelia, as it regulates many cellular processes, including adhesion, motility, and mechanotransduction. Its overactivation is associated with cancer aggressiveness. Here, we take advantage of optogenetics to precisely control Src activation in time and show that its pathological-like activation slows the collective rotation of epithelial cells confined into circular adhesive patches. We interpret velocity, force, and stress data during period of non-activation and period of activation of Src thanks to a hydrodynamic description of the cell assembly as a polar active fluid. Src activation leads to a 2-fold decrease in the ratio of polar angle to friction, which could result from increased adhesiveness at the cell-substrate interface. Measuring internal stress allows us to show that active stresses are subdominant compared to traction forces. Our work reveals the importance of fine-tuning the level of Src activity for coordinated collective behaviors.

Abstract: Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.

Abstract: Cell signaling through direct physical cell–cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with spatial responses. Herein, a light-activated contact-dependent communication scheme for synthetic cells is designed and demonstrated. A split luminescent protein is utilized to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. The modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

Abstract: Chimeric antigen receptor T cell therapies have achieved great success in eradicating some liquid tumors, whereas the preclinical results in treating solid tumors have proven less decisive. One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells. Here, we improve immune cell infiltration into solid tumors by manipulating septin-7 functions in cells. Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein. Blue light inhibits septin-7 function in live cells, inducing extended cell protrusions and cell polarization, enhancing cell transmigration efficiency through confining spaces. We genetically edited human natural killer cell line (NK92) and mouse primary CD8+ T-cells expressing the engineered protein, and we demonstrated improved penetration and cytotoxicity against various tumor spheroid models. Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.

Abstract: Cells use traction forces to sense mechanical cues in their environment. While the molecular clutch model effectively explains how cells exert more forces on stiffer substrates, it falls short in addressing their adaptation to dynamic mechanical fluctuations prevalent in tissues and organs. Here, using hydrogel with photo-responsive rigidity, we show that cells' response to rigidity changes is frequency dependent. Strikingly, at certain frequencies, cellular traction forces exceed those on static substrates 4-fold stiffer, challenging the established molecular clutch model. We discover that the discrepancy between the rapid adaptation of traction forces and the slower deactivation of mechanotransduction signaling proteins results in their accumulation, thereby enhancing long-term cellular traction in dynamic settings. Consequently, we propose a new model that melds immediate mechanosensing with extended mechanical signaling. Our study underscores the significance of dynamic rigidity in the development of synthetic biomaterials, emphasizing the importance of considering both immediate and prolonged cellular responses.

Abstract: Protein photolithography is an invaluable tool for generating protein microchips and regulating interactions between cells and materials. However, the absence of light-responsive molecules that allow for the copatterning of multiple functional proteins with biocompatible visible light poses a significant challenge. Here, a new approach for photopatterning three distinct proteins on a single surface by using green, red, and far-red light is reported. The cofactor of the green light-sensitive protein CarH is engineered such that it also becomes sensitive to red and far-red light. These new cofactors are shown to be compatible with two CarH-based optogenetic tools to regulate bacterial cell-cell adhesions and gene expression in mammalian cells with red and far-red light. Further, by incorporating different CarH variants with varying light sensitivities in layer-by-layer (LbL) multiprotein films, specific layers within the films, along with other protein layers on top are precisely removed by using different colors of light, all with high spatiotemporal accuracy. Notably, with these three distinct colors of visible light, it is possible to incorporate diverse proteins under mild conditions in LbL films based on the reliable interaction between Ni2+- nitrilotriacetic acid (NTA) groups and polyhistidine-tags (His-tags)on the proteins and their subsequent photopatterning. This approach has potential applications spanning biofabrication, material engineering, and biotechnology.

Abstract: Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

Abstract: The transition of bacteria from an individualistic to a biofilm lifestyle profoundly alters their biology. During biofilm development, the bacterial cell-cell adhesions are a major determinant of initial microcolonies, which serve as kernels for the subsequent microscopic and mesoscopic structure of the biofilm, and determine the resulting functionality. In this study, the significance of bacterial cell-cell adhesion dynamics on bacterial aggregation and biofilm maturation is elucidated. Using photoswitchable adhesins between bacteria, modifying the dynamics of bacterial cell-cell adhesions with periodic dark-light cycles is systematic. Dynamic cell-cell adhesions with liquid-like behavior improve bacterial aggregation and produce more compact microcolonies than static adhesions with solid-like behavior in both experiments and individual-based simulations. Consequently, dynamic cell-cell adhesions give rise to earlier quorum sensing activation, better intermixing of different bacterial populations, improved biofilm maturation, changes in the growth of cocultures, and higher yields in fermentation. The here presented approach of tuning bacterial cell-cell adhesion dynamics opens the door for regulating the structure and function of biofilms and cocultures with potential biotechnological applications.

Abstract: E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.

Abstract: Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

Abstract: Vitamin B12 (cobalamin) is essential for human health and its deficiency results in anemia and neurological damage. Vitamin B12 exists in different forms with various bioactivity but most sensors are unable to discriminate between them. Here, a whole-cell agglutination assay that is specific for adenosylcobalamin (AboB12), which is one of two bioactive forms, is reported. This biosensor consists of Escherichia coli that express the AdoB12 specific binding domain of CarH at their surface. In the presence of AdoB12, CarH forms tetramers, which leads to specific bacterial cell-cell adhesions and agglutination. These CarH tetramers disassemble upon green light illumination such that reversion of the bacterial aggregation can serve as internal quality control. The agglutination assay has a detection limit of 500 nм AdoB12, works in protein-poor biofluids such as urine, and has high specificity to AdoB12 over other forms of vitamin B12 as also demonstrated with commercially available supplements. This work is a proof of concept for a cheap and easy-to-readout AdoB12 sensor that can be implemented at the point-of-care to monitor high-dose vitamin B12 supplementation.

Abstract: The kinetics of a ligand-receptor interaction determine the responses of the receptor-expressing cell. One approach to experimentally and reversibly change this kinetics on demand is optogenetics. We have previously developed a system in which the interaction of a modified receptor with an engineered ligand can be controlled by light. In this system the ligand is a soluble Phytochrome B (PhyB) tetramer and the receptor is fused to a mutated PhyB-interacting factor (PIFS). However, often the natural ligand is not soluble, but expressed as a membrane protein on another cell. This allows ligand-receptor interactions in two dimensions. Here, we developed a strategy to generate cells that display PhyB as a membrane-bound protein by expressing the SpyCatcher fused to a transmembrane domain in HEK-293T cells and covalently coupling purified PhyB-SpyTag to these cells. As proof-of-principle, we use Jurkat T cells that express a GFP-PIFS-T cell receptor and show that these cells can be stimulated by the PhyB-coupled HEK-293T cells in a light dependent manner. Thus, we call the PhyB-coupled cells opto-antigen presenting cells (opto-APCs). Our work expands the toolbox of optogenetic technologies, allowing two-dimensional ligand-receptor interactions to be controlled by light.

Abstract: The mechanical properties of biological tissues are key to their physical integrity and function. Although external loading or biochemical treatments allow the estimation of these properties globally, it remains difficult to assess how such external stimuli compare with cell-generated contractions. Here we engineer microtissues composed of optogenetically-modified fibroblasts encapsulated within collagen. Using light to control the activity of RhoA, a major regulator of cellular contractility, we induce local contractions within microtissues, while monitoring microtissue stress and strain. We investigate the regulation of these local contractions and their spatio-temporal distribution. We demonstrate the potential of our technique for quantifying tissue elasticity and strain propagation, before examining the possibility of using light to create and map local anisotropies in mechanically heterogeneous microtissues. Altogether, our results open an avenue to guide the formation of tissues while non-destructively charting their rheology in real time, using their own constituting cells as internal actuators.

Abstract: Escherichia coli biofilm may form naturally on biotic and abiotic surfaces; this represents a promising approach for efficient biochemical production in industrial fermentation. Recently, industrial exploitation of the advantages of optogenetics, such as simple operation, high spatiotemporal control, and programmability, for regulation of biofilm formation has garnered considerable attention. In this study, we used the blue light signaling-induced optogenetic system Magnet in an E. coli biofilm-based immobilized fermentation system to produce l-threonine in sufficient quantity. Blue light signaling significantly affected the phenotype of E. coli W1688. A series of biofilm-related experiments confirmed the inhibitory effect of blue light signaling on E. coli W1688 biofilm. Subsequently, a strain lacking a blue light-sensing protein (YcgF) was constructed via genetic engineering, which substantially reduced the inhibitory effect of blue light signaling on biofilm. A high-efficiency biofilm-forming system, Magnet, was constructed, which enhanced bacterial aggregation and biofilm formation. Furthermore, l-threonine production was increased from 10.12 to 16.57 g/L during immobilized fermentation, and the fermentation period was shortened by 6 h. IMPORTANCE We confirmed the mechanism underlying the inhibitory effects of blue light signaling on E. coli biofilm formation and constructed a strain lacking a blue light-sensing protein; this mitigated the aforementioned effects of blue light signaling and ensured normal fermentation performance. Furthermore, this study elucidated that the blue light signaling-induced optogenetic system Magnet effectively regulates E. coli biofilm formation and contributes to l-threonine production. This study not only enriches the mechanism of blue light signaling to regulate E. coli biofilm formation but also provides a theoretical basis and feasibility reference for the application of optogenetics technology in biofilm-based immobilized fermentation systems.

Abstract: Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation drives yes-associated protein (YAP) nuclear localization within minutes and consequent mechanotransduction verified by YAP-transcriptional enhanced associate domain transcriptional activity. These single-transgene tools do not require protein binding partners for dynamic membrane localization and permit spatiotemporally precise control over RhoA signaling to advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

Abstract: Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.