Optogenetic System Research Articles

Development of Optogenetic Dual-Switch System for Rewiring Metabolic Flux for Polyhydroxybutyrate Production.

Several strategies, including inducer addition and biosensor use, have been developed for dynamical regulation. However, the toxicity, cost, and inflexibility of existing strategies have created a demand for superior technology. In this study, we designed an optogenetic dual-switch system and applied it to increase polyhydroxybutyrate (PHB) production. First, an optimized chromatic acclimation sensor/regulator (RBS10–CcaS#10–CcaR) system (comprising an optimized ribosomal binding site (RBS), light sensory protein CcaS, and response regulator CcaR) was selected for a wide sensing range of approximately 10-fold between green-light activation and red-light repression. The RBS10–CcaS#10–CcaR system was combined with a blue light-activated YF1–FixJ–PhlF system (containing histidine kinase YF1, response regulator FixJ, and repressor PhlF) engineered with reduced crosstalk. Finally, the optogenetic dual-switch system was used to rewire the metabolic flux for PHB production by regulating the sequences and intervals of the citrate synthase gene (gltA) and PHB synthesis gene (phbCAB) expression. Consequently, the strain RBS34, which has high gltA expression and a time lag of 3 h, achieved the highest PHB content of 16.6 wt%, which was approximately 3-fold that of F34 (expressed at 0 h). The results indicate that the optogenetic dual-switch system was verified as a practical and convenient tool for increasing PHB production.

Design and Characterization of an Optogenetic System in Pichia pastoris.

Pichia pastoris (P. pastoris) is the workhorse in the commercial production of many valuable proteins. Traditionally, the regulation of gene expression in P. pastoris is achieved through induction by methanol which is toxic and flammable. The emerging optogenetic technology provides an alternative and cleaner gene regulation method. Based on the photosensitive protein EL222, we designed a novel "one-component" optogenetic system. The highest induction ratio was 79.7-fold under blue light compared to the group under darkness. After switching cells from dark to blue illumination, the system induced expression in just 1 h. Only 2 h after the system was switched back to the darkness from blue illumination, the target gene expression was inactivated 5-fold. The induction intensity of the optogenetic system is positively correlated with the dose and periodicity of blue illumination, and it has good spatial control. These results provide the first credible case of optogenetically induced protein expression in P. pastoris.

Red Light Optogenetics in Neuroscience.

Optogenetics, a field concentrating on controlling cellular functions by means of light-activated proteins, has shown tremendous potential in neuroscience. It possesses superior spatiotemporal resolution compared to the surgical, electrical, and pharmacological methods traditionally used in studying brain function. A multitude of optogenetic tools for neuroscience have been created that, for example, enable the control of action potential generation via light-activated ion channels. Other optogenetic proteins have been used in the brain, for example, to control long-term potentiation or to ablate specific subtypes of neurons. In in vivo applications, however, the majority of optogenetic tools are operated with blue, green, or yellow light, which all have limited penetration in biological tissues compared to red light and especially infrared light. This difference is significant, especially considering the size of the rodent brain, a major research model in neuroscience. Our review will focus on the utilization of red light-operated optogenetic tools in neuroscience. We first outline the advantages of red light for in vivo studies. Then we provide a brief overview of the red light-activated optogenetic proteins and systems with a focus on new developments in the field. Finally, we will highlight different tools and applications, which further facilitate the use of red light optogenetics in neuroscience.

Optimal control and dynamic modulation of the ATPase gene expression for enforced ATP wasting in batch fermentations

Competitive biotechnological processes need to operate over various conditions and adapt to changing economic contexts. Dynamic ATP turnover allows trading off declines in biomass formation and volumetric productivity for enhancements of product yields in fermentations where the product pathway is linked to ATP synthesis. To facilitate its practical implementation, we propose to dynamically manipulate the cellular ATP turnover by putting the ATPase enzyme, which hydrolyzes ATP into ADP, under the control of an optogenetic gene expression system. This allows achieving dynamic control of the ATP wasting online via a tunable external input. While light as control input is promising because it is easily tunable, generally non-invasive/non-toxic, and more affordable than classical chemical inducers, it makes the overall control task challenging. Thus, we derive an expanded version of dynamic enzyme-cost flux balance analysis that takes into account the dynamics of the optogenetic actuator. We then formulate a suitable optimal control problem to find optimal inputs for achieving the desired process performance. We test our approach in simulations using the batch anaerobic lactate fermentation of glucose by Escherichia coli as a case study.

A Self-Powered Optogenetic System for Implantable Blood Glucose Control.

Diabetes treatment and rehabilitation are usually a lifetime process. Optogenetic engineered designer cell-therapy holds great promise in regulating blood glucose homeostasis. However, portable, sustainable, and long-term energy supplementation has previously presented a challenge for the use of optogenetic stimulation in vivo. Herein, we purpose a self-powered optogenetic system (SOS) for implantable blood glucose control. The SOS consists of a biocompatible far-red light (FRL) source, FRL-triggered transgene-expressing cells, a power management unit, and a flexible implantable piezoelectric nanogenerator (i-PENG) to supply long-term energy by converting biomechanical energy into electricity. Our results show that this system can harvest energy from body movement and power the FRL source, which then significantly enhanced production of a short variant of human glucagon-like peptide 1 (shGLP-1) in vitro and in vivo. Indeed, diabetic mice equipped with the SOS showed rapid restoration of blood glucose homeostasis, improved glucose, and insulin tolerance. Our results suggest that the SOS is sufficiently effective in self-powering the modulation of therapeutic outputs to control glucose homeostasis and, furthermore, present a new strategy for providing energy in optogenetic-based cell therapy.

Activation of UCP1-Independent Ca2+ Cycling Thermogenesis by Wireless Optogenetics.

The identification of non-canonical UCP1-independent thermogenic mechanisms offers new opportunities to target such pathways to improve metabolic health. Based on our recent studies on Ca2+ futile cycling thermogenesis in beige fat, we applied the newly developed implantable wireless optogenetic system to activate Ca2+ cycling in an adipocyte-specific manner without external stimuli, i.e., fat-specific cold mimetics. Here, we describe the detailed methodology and application to the prevention of obesity.

Optogenetic Control of TGF-β Signaling.

Cells employ signaling pathways to make decisions in response to changes in their immediate environment. The Transforming Growth Factor β (TGF-β) signaling pathway plays pivotal roles in regulating many cellular processes, including cell proliferation, differentiation, and migrations. In order to manipulate and explore the dynamic behavior of TGF-β signaling at high spatiotemporal resolution, we developed an optogenetic system (the optoTGFBRs system), in which light is used to control TGF-β signaling precisely in time and space. Here, we describe about experimental details of how to build the optoTGFBRs system and utilize it to manipulate TGF-β signaling in a single cell or a cell population using microscope or LED array, respectively.

Optogenetic control of cancer cell survival in ChR2‐transfected HeLa cells

Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467nm) were applied for up to 24h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p<0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.

Optogenetics in bacteria - applications and opportunities.

ABSTRACTOptogenetics holds the promise of controlling biological processes with superb temporal and spatial resolution at minimal perturbation. Although many of the light-reactive proteins used in optogenetic systems are derived from prokaryotes, applications were largely limited to eukaryotes for a long time. In recent years, however, an increasing number of microbiologists use optogenetics as a powerful new tool to study and control key aspects of bacterial biology in a fast and often reversible manner. After a brief discussion of optogenetic principles, this review provides an overview of the rapidly growing number of optogenetic applications in bacteria, with a particular focus on studies venturing beyond transcriptional control. To guide future experiments, we highlight helpful tools, provide considerations for successful application of optogenetics in bacterial systems, and identify particular opportunities and challenges that arise when applying these approaches in bacteria.

Mapping the dynamic transfer functions of eukaryotic gene regulation.

Biological information can be encoded within the dynamics of signaling components, which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions-modulating the pulse frequency, amplitude, and pulse width of light-regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed tunable gene expression and filtering behaviors and provided a quantification of the limit to the amount of information that can be reliably transferred across a single promoter as ∼1.7 bits. Using a library of over 100 orthogonal chromatin regulators, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic gene regulation.

A small and highly sensitive red/far-red optogenetic switch for applications in mammals.

Optogenetic technologies have transformed our ability to precisely control biological processes in time and space. Yet, current eukaryotic optogenetic systems are limited by large or complex optogenetic modules, long illumination times, low tissue penetration or slow activation and deactivation kinetics. Here, we report a red/far-red light-mediated and miniaturized Δphytochrome A (ΔPhyA)-based photoswitch (REDMAP) system based on the plant photoreceptor PhyA, which rapidly binds the shuttle protein far-red elongated hypocotyl 1 (FHY1) under illumination with 660-nm light with dissociation occurring at 730 nm. We demonstrate multiple applications of REDMAP, including dynamic on/off control of the endogenous Ras/Erk mitogen-activated protein kinase (MAPK) cascade and control of epigenetic remodeling using a REDMAP-mediated CRISPR-nuclease-deactivated Cas9 (CRISPR-dCas9) (REDMAPcas) system in mice. We also demonstrate the utility of REDMAP tools for in vivo applications by activating the expression of transgenes delivered by adeno-associated viruses (AAVs) or incorporated into cells in microcapsules implanted into mice, rats and rabbits illuminated by light-emitting diodes (LEDs). Further, we controlled glucose homeostasis in type 1 diabetic (T1D) mice and rats using REDMAP to trigger insulin expression. REDMAP is a compact and sensitive tool for the precise spatiotemporal control of biological activities in animals with applications in basic biology and potentially therapy.

Abstract MP20: Optogenetic Control Of Pip2 To Assess Mechanisms Regulating The Epithelial Sodium Channel

The epithelial Na + channel (ENaC) plays a key role in Na + transport in epithelial linings to include the lung, colon and kidney. In the distal kidney tubules, ENaC regulates Na + reabsorption and blood volume. Thus, dysfunctions in signaling pathways regulating ENaC activity are linked to hypertension or hypotension. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a target of the G protein coupled receptor P2Y2 pathway, and is necessary for the proper function of ENaC. This nonvoltage-gated trimeric channel is comprised of α, β, and γ subunits. We recently described two intracellular PIP 2 binding sites on the N termini of β-, and γ-ENaC, with moderate μM affinity. Here, we report the functional effects on ENaC containing a combination of mutations to those PIP 2 binding sites, by controlled depletion of PIP 2 . We used a CIBN/CRY2-5-ptase optogenetic dimerization system to deplete PIP 2 levels in HEK293 cells transiently expressing wild type (wt) ENaC or the mutant ENaC constructs. A fluorescent Na + indicator, was used to monitor ENaC activity by tracking the relative intracellular Na + levels. Upon optogenetic-controlled depletion of PIP 2 , Na + levels decreased in cells expressing wt ENaC. Mutations to the PIP 2 sites of ENaC were expected to have no change in Na + levels upon PIP 2 depletion due to the disruption of PIP 2 binding. As a control, mutations to non-PIP 2 binding sites were included, and were expected to have decreased Na + levels similar to wt ENaC. Interestingly, mutation of each independent PIP 2 site resulted in only a small decrease of intracellular Na + , compared to wt ENaC. However, mutations throughout the entire N-terminus of β-ENaC, including the PIP 2 binding site, resulted in a significant increase of Na + upon PIP 2 depletion. We performed patch clamp electrophysiology and found that the ENaC recordings corresponded to the Na + fluctuations. These data suggest that the residues surrounding the PIP 2 binding sites play a significant role in the affinity of PIP 2 for ENaC. The role of these other domains in PIP 2 binding is still under investigation.

Revisiting the Role of TGFβ Receptor Internalization for Smad Signaling: It is Not Required in Optogenetic TGFβ Signaling Systems.

Endocytosis is an important process by which many signaling receptors reach their intracellular effectors. Accumulating evidence suggests that internalized receptors play critical roles in triggering cellular signaling, including transforming growth factor β (TGFβ) signaling. Despite intensive studies on the TGFβ pathway over the last decades, the necessity of TGFβ receptor endocytosis for downstream TGFβ signaling responses is a subject of debate. In this study, mathematical modeling and synthetic biology approaches are combined to re-evaluate whether TGFβ receptor internalization is indispensable for inducing Smad signaling. It is found that optogenetic systems with plasma membrane-tethered TGFβ receptors can induce fast and sustained Smad2 activation upon light stimulations. Modeling analysis suggests that endocytosis is precluded for the membrane-anchored optogenetic TGFβ receptors. Therefore, this study provides new evidence to support that TGFβ receptor internalization is not required for Smad2 activation.

An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans

BackgroundOptogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal, and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular, and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm.ResultsThe OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles, and light color. We demonstrate OptoArm’s power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit ageing.ConclusionWe have assembled an economical, reliable, and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research community.

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Near-Infrared-Light Remote-Controlled Activation of Cancer Immunotherapy Using Photothermal Conjugated Polymer Nanoparticles.

Remote control of the therapeutic process is an ideal strategy for maximizing efficacy and avoiding side effects, especially for cancer immunotherapy. Herein, a conjugated polymer nanoparticles (CPNs)-mediated optogenetic system for in situ activation of immunotherapy under near-infrared laser irradiation is reported. This system is composed of photothermal CPNs and interferon-gamma (IFN-γ) plasmid driven by heat shock promoter HSP70. The photothermally responsive CPNs serve as a photo-heat nanotransducer to trigger the gene transcription of IFN-γ cytokine. The secreted IFN-γ from cancer cells can sufficiently elicit surrounding tumor-associated macrophages activation through IFN-γ-JAK-STAT1 transcription-factor signaling pathway and finally induce cancer cell killing by immunotherapy. Therefore, this synergetic optogenetic system provides a promising approach to remotely control the process of cancer immunotherapy.

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Duplex optogenetic stimulation system modulates compulsive behaviors bidirectionally

Decisions enable us to consider our actions while adjusting behaviors to a relentlessly changing environment. Since conscious decision making requires our full attention and is therefore expensive, we use a cheaper system for everyday and repetitive tasks that run automatically without conscious evaluations, commonly described as habits. The combination of these two systems is highly adaptive. However, if there is a disruption in these systems’ balance, mental illnesses such as Obsessive-Compulsive Disorders (OCD) or addiction may arise. Now, how does a newly acquired memory or behavior transition to a habit? We know that the striatum’s dorsolateral region (DLS) plays a significant role in sustaining successful behaviors and habits manifestation. Furthermore, it has been shown that the micro-circuitry of the DLS is organized into two functional opposing pathways that consist of the direct pathway striatal spiny projection neurons (dSPNs), which facilitate movement, and the indirect pathway SPNs, which inhibit actions. Already Freud wrote in the letters to his friend Fliess that memory and motive are inseparable, and its recollection would have no force or meaning unless it would be coupled to a motive or emotion. Therefore, we hypothesize that emotional-associated cues would directly prime the DLS through the amygdala to turn newly acquired behaviors into habits. We are adding new supporting insight for this hypothesis by using a duplex optogenetic stimulation system of the amygdala input in the DLS, modulating specific compulsive behaviors bidirectionally. These behavior modulations were accompanied by spine density modulations and intrinsic excitability changes in the SPNs of the DLS. Keywords: Compulsions; Habits; Optogenetics; Neuromodulation; Dorsolateral striatum; Amygdala.

Single-component near-infrared optogenetic systems for gene transcription regulation

Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.

Temporal induction of Lhx8 by optogenetic control system for efficient bone regeneration

BackgroundThe spatiotemporal regulation of essential genes is crucial for controlling the growth and differentiation of cells in a precise manner during regeneration. Recently, optogenetics was considered as a potent technology for sophisticated regulation of target genes, which might be a promising tool for regenerative medicine. In this study, we used an optogenetic control system to precisely regulate the expression of Lhx8 to promote efficient bone regeneration.MethodsQuantitative real-time PCR and western blotting were used to detect the expression of Lhx8 and osteogenic marker genes. Alkaline phosphatase staining and alizarin red staining were used to detect alkaline phosphatase activity and calcium nodules. A customized optogenetic expression system was constructed to regulate Lhx8, of which the expression was activated in blue light but not in dark. We also used a critical calvarial defect model for the analysis of bone regeneration in vivo. Moreover, micro-computed tomography (micro-CT), three-dimensional reconstruction, quantitative bone measurement, and histological and immunohistochemistry analysis were performed to investigate the formation of new bone in vivo.ResultsDuring the osteogenic differentiation of BMSCs, the expression levels of Lhx8 increased initially but then decreased thereafter. Lhx8 promoted the early proliferation of BMSCs but inhibited subsequent osteogenic differentiation. The optogenetic activation of Lhx8 in BMSCs in the early stages of differentiation by blue light stimulation led to a significant increase in cell proliferation, thus allowing a sufficient number of differentiating BMSCs to enter the later osteogenic differentiation stage. Analysis of the critical calvarial defect model revealed that the pulsed optogenetic activation of Lhx8 in transplanted BMSCs over a 5-day period led to a significant increase in the generation of bone in vivo.ConclusionsLhx8 plays a critical role in balancing proliferation and osteogenic differentiation in BMSCs. The optogenetic activation of Lhx8 expression at early stage of BMSCs differentiation led to better osteogenesis, which would be a promising strategy for precise bone regeneration.