Curated Optogenetic Publication Database

Abstract: Background Biomass formation and product synthesis decoupling have been proven to be promising to increase the titer of desired value add products. Optogenetics provides a potential strategy to develop light-induced circuits that conditionally control metabolic flux redistribution for enhanced microbial production. However, the limited number of light-sensitive proteins available to date hinders the progress of light-controlled tools. Results To address these issues, two optogenetic systems (TPRS and TPAS) were constructed by reprogramming the widely used repressor TetR and protease TEVp to expand the current optogenetic toolkit. By merging the two systems, a bifunctional optogenetic switch was constructed to enable orthogonally regulated gene transcription and protein accumulation. Application of this bifunctional switch to decouple biomass formation and shikimic acid biosynthesis allowed 35 g/L of shikimic acid production in a minimal medium from glucose, representing the highest titer reported to date by E. coli without the addition of any chemical inducers and expensive aromatic amino acids. This titer was further boosted to 76 g/L when using rich medium fermentation. Conclusion The cost effective and light-controlled switch reported here provides important insights into environmentally friendly tools for metabolic pathway regulation and should be applicable to the production of other value-add chemicals.

Abstract: Drug resistance remains a central challenge towards durable cancer therapy, including for cancers driven by the EML4-ALK oncogene. EML4-ALK and related fusion oncogenes form cytoplasmic protein condensates that transmit oncogenic signals through the Ras/Erk pathway. However, whether such condensates play a role in drug response or resistance development is unclear. Here, we applied optogenetic functional profiling to examine how EML4-ALK condensates impact signal transmission through transmembrane receptor tyrosine kinases (RTKs), a common route of resistance signaling. We found that condensates dramatically suppress signaling through activated RTKs including EGFR. Conversely, ALK inhibition restored and hypersensitized RTK signals. Modulation of RTK sensitivity occurred because EML4-ALK condensates sequestered downstream adapters that are required to transduce signals from both EML4-ALK and ligand-stimulated RTKs. Strikingly, EGFR hypersensitization resulted in rapid and pulsatile Erk signal reactivation within 10s of minutes of drug addition. EGFR reactivation originated from paracrine signals from neighboring apoptotic cells, and reactivation could be blocked by inhibition of either EGFR or matrix metalloproteases. Paracrine signals promoted survival during ALK inhibition, and blockade of paracrine signals accelerated cell killing and suppressed drug tolerance. Our results uncover a regulatory role for protein condensates in cancer signaling and drug response and demonstrate the potential of optogenetic profiling for drug discovery based on functional biomarkers in cancer cells.

Abstract: Bispecific T-cell engagers (BiTEs), which have shown potent antitumor activity in humans, are emerging as one of the most promising immunotherapeutic strategies for cancer treatment in recent years. However, the clinical application of BiTEs nowadays has been hampered by their short half-life in the circulatory system due to their low molecular weight and rapid renal clearance. Inevitable continuous infusion of BiTEs has become a routine operation in order to achieve effective treatment, although it is costly, inconvenient, time-consuming, and even painful for patients in some cases. To develop an on-demand, tunable, and reversible approach to overcome these limitations, we assembled a transcription-control device into mammalian cells based on a bacterial far-red light (FRL) responsive signaling pathway to drive the expression of a BiTE against Glypican 3 (GPC3), which is a highly tumor-specific antigen expressed in most hepatocellular carcinomas (HCC). As demonstrated in in vitro experiments, we proved that the FRL sensitive device spatiotemporally responded to the control of FRL illumination and produced a therapeutic level of BiTEs that recruited and activated human T cells to eliminate GPC3 positive tumor cells. By functionally harnessing the power of optogenetics to remotely regulate the production of BiTEs from bioengineered cells and demonstrating its effectiveness in treating tumor cells, this study provides a novel approach to achieve an in vivo supply of BiTEs, which could be potentially applied to other formats of bispecific antibodies and facilitate their clinical applications.

Abstract: Continuous and ubiquitous expression of foreign genes sometimes results in harmful effects on the growth, development and metabolic activities of plants. Tissue-specific promoters help to overcome this disadvantage, but do not allow one to precisely control transgene expression over time. Thus, inducible transgene expression systems have obvious benefits. In plants, transcriptional regulation is usually driven by chemical agents under the control of chemically-inducible promoters. These systems are diverse, but usually contain two elements, the chimeric transcription factor and the reporter gene. The commonly used chemically-induced expression systems are tetracycline-, steroid-, insecticide-, copper-, and ethanol-regulated. Unlike chemical-inducible systems, optogenetic tools enable spatiotemporal, quantitative and reversible control over transgene expression with light, overcoming limitations of chemically-inducible systems. This review updates and summarizes optogenetic and chemical induction methods of transgene expression used in basic plant research and discusses their potential in field applications.

Abstract: Optogenetics, a technology to manipulate biological phenomena thorough light, has attracted much attention in neuroscience. Recently, the Magnet System, a photo-inducible protein dimerization system which can control the intracellular behavior of various biomolecules with high accuracy using light was developed. Furthermore, photoactivation systems for controlling biological phenomena are being developed by combining this technique with genome-editing technology (CRISPR/Cas9 System) or DNA recombination technology (Cre-loxP system). Herein, we review the history of optogenetics and the latest Magnet System technology and introduce our recently developed photoactivatable Cre knock-in mice with temporal-, spatial-, and cell-specific accuracy.

Abstract: The EAL-BLUF fragment from Magnetococcus marinus BldP1 (EB1) light-dependently hydrolyzes c-di-GMP. Herein, the photoreaction of the BLUF domain of EB1 (eBLUF) is studied. It is found for the first time that a monomeric BLUF domain forms a dimer upon illumination and its dark recovery is very slow. The dimer of light- and dark-state protomers (LD-dimer) is much more stable than that of two light-state protomers (LL-dimer), and the dark recovery of the LD-dimer is approximately 20 times slower than that of the LL-dimer, which is suitable for optogenetic tools. The secondary structure of the L-monomer is different from those of the D-monomer and the LD-dimer. The transient grating measurements reveal that this conformational change occurs simultaneously with dimerization. Although the W91A mutant exhibits a spectral red shift, it forms a heterodimer with the L-monomer of wild-type eBLUF with similar stability to the LD-dimer. This suggests that the conformation of the dimerization site of W91A is similar to that of the dark state (dark-mimic mutant); that is, the light-induced structural changes in the chromophore cavity are not transferred to the other part of the protein. The selective photoinduced dimerization of eBLUF is potentially useful to control interprotein interactions between two different effector domains bound to these proteins.

Abstract: The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.

Abstract: Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex.1,2 In mammalian cells, end-binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for diverse +TIPs that control microtubule dynamics and interactions with other intracellular structures.3 Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation,4-6 the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light-sensitive EB1 variant, π-EB1, that allows local, acute, and reversible inactivation of +TIP association with growing microtubule ends in live cells.7 We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase but instead increases astral microtubule length and number. Yet in the absence of EB1 activity, astral microtubules fail to engage the cortical dynein/dynactin machinery, and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments, likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

Abstract: Telomeres form unique nuclear compartments that prevent degradation and fusion of chromosome ends by recruiting shelterin proteins and regulating access of DNA damage repair factors. To understand how these dynamic components protect chromosome ends, we combine in vivo biophysical interrogation and in vitro reconstitution of human shelterin. We show that shelterin components form multicomponent liquid condensates with selective biomolecular partitioning on telomeric DNA. Tethering and anomalous diffusion prevent multiple telomeres from coalescing into a single condensate in mammalian cells. However, telomeres coalesce when brought into contact via an optogenetic approach. TRF1 and TRF2 subunits of shelterin drive phase separation, and their N-terminal domains specify interactions with telomeric DNA in vitro. Telomeric condensates selectively recruit telomere-associated factors and regulate access of DNA damage repair factors. We propose that shelterin mediates phase separation of telomeric chromatin, which underlies the dynamic yet persistent nature of the end-protection mechanism.

Abstract: Optogenetics combines light and genetics to enable precise control of living cells, tissues, and organisms with tailored functions. Optogenetics has the advantages of noninvasiveness, rapid responsiveness, tunable reversibility, and superior spatiotemporal resolution. Following the initial discovery of microbial opsins as light-actuated ion channels, a plethora of naturally occurring or engineered photoreceptors or photosensitive domains that respond to light at varying wavelengths has ushered in the next chapter of optogenetics. Through protein engineering and synthetic biology approaches, genetically encoded photoswitches can be modularly engineered into protein scaffolds or host cells to control a myriad of biological processes, as well as to enable behavioral control and disease intervention in vivo. Here, we summarize these optogenetic tools on the basis of their fundamental photochemical properties to better inform the chemical basis and design principles. We also highlight exemplary applications of opsin-free optogenetics in dissecting cellular physiology (designated "optophysiology") and describe the current progress, as well as future trends, in wireless optogenetics, which enables remote interrogation of physiological processes with minimal invasiveness. This review is anticipated to spark novel thoughts on engineering next-generation optogenetic tools and devices that promise to accelerate both basic and translational studies.

Abstract: Secretory trafficking from the endoplasmic reticulum (ER) is subject to regulation by extrinsic and intrinsic factors. While much of the focus has been on biochemical triggers, little is known whether and how the ER is subject to regulation by mechanical signals. Here, we show that COPII-dependent ER-export is regulated by mechanical strain. Mechanotransduction to the ER was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulate ER-to-Golgi transport. Altogether, we establish an unprecedented link between mechanical strain and export from the ER.

Abstract: 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.

Abstract: Patients with Alzheimer's disease suffer from a decrease in brain mass and a prevalence of amyloid-β plaques. These plaques are thought to play a role in disease progression, but their exact role is not entirely established. We developed an optogenetic model to induce amyloid-β intracellular oligomerization to model distinct disease etiologies. Here, we examine the effect of Wnt signaling on amyloid in an optogenetic, Drosophila gut stem cell model. We observe that Wnt activation rescues the detrimental effects of amyloid expression and oligomerization. We analyze the gene expression changes downstream of Wnt that contribute to this rescue and find changes in aging related genes, protein misfolding, metabolism, and inflammation. We propose that Wnt expression reduces inflammation through repression of Toll activating factors. We confirm that chronic Toll activation reduces lifespan, but a decrease in the upstream activator Persephone extends it. We propose that the protective effect observed for lithium treatment functions, at least in part, through Wnt activation and the inhibition of inflammation.

Abstract: Optogenetics is widely used to interrogate the neural circuits underlying disease and has most recently been harnessed for therapeutic applications. The optogenetic toolkit consists of light-responsive proteins that modulate specific cellular functions, vectors for the delivery of the transgenes that encode the light-responsive proteins to targeted cellular populations, and devices for the delivery of light of suitable wavelengths at effective fluence rates. A refined toolkit with a focus towards translational uses would include efficient and safer viral and non-viral gene-delivery vectors, increasingly red-shifted photoresponsive proteins, nanomaterials that efficiently transduce near-infrared light deep into tissue, and wireless implantable light-delivery devices that allow for spatiotemporally precise interventions at clinically relevant tissue depths. In this Review, we examine the current optogenetics toolkit and the most notable preclinical and translational uses of optogenetics, and discuss future methodological and translational developments and bottlenecks.

Abstract: ConspectusLight activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the blue light using flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark- and light-adapted states. Thus, the driving force for photoactivity is unclear.To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time-resolved infrared (IR) experiments, coupled with chemical biology, provide important new insights into the signaling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labeling of the chromophore and protein separately. Further, a degree of control over BLUF photochemistry is achieved through mutagenesis, while unnatural amino acid substitution allows us to both fine-tune the photochemistry and time resolve protein dynamics with spatial resolution.Ultrafast studies of BLUF domains reveal non-single-exponential relaxation of the flavin excited state. That relaxation leads within one nanosecond to the original flavin ground state bound in a modified hydrogen-bonding network, as seen in transient and steady-state IR spectroscopy. The change in H-bond configuration arises from formation of an unusual enol (imine) form of a critical glutamine residue. The dynamics observed, complemented by quantum mechanical calculations, suggest a unique sequential electron then double proton transfer reaction as the driving force, followed by rapid reorganization in the binding site and charge recombination. Importantly, studies of several BLUF domains reveal an unexpected diversity in their dynamics, although the underlying structure appears highly conserved. It is suggested that this diversity reflects structural dynamics in the ground state at standard temperature, leading to a distribution of structures and photochemical outcomes. Time resolved IR measurements were extended to the millisecond regime for one BLUF domain, revealing signaling state formation on the microsecond time scale. The mechanism involves reorganization of a β-sheet connected to the chromophore binding pocket via a tryptophan residue. The potential of site-specific labeling amino acids with IR labels as a tool for probing protein structural dynamics was demonstrated.In summary, time-resolved IR studies of BLUF domains (along with related studies at visible wavelengths and quantum and molecular dynamics calculations) have resolved the photoactivation mechanism and real-time dynamics of signaling state formation. These measurements provide new insights into protein structural dynamics and will be important in optimizing the potential of BLUF domains in optobiology.

Abstract: Advances in genetic engineering, combined with the development of optical technologies, have allowed optogenetics to broaden its area of possible applications in recent years. However, the application of optogenetic tools in industry, including biotechnology and the production of biomaterials, is still limited, because each practical task requires the engineering of a specific optogenetic system. In this review, we discuss recent advances in the use of optogenetic tools in the production of biofuels and valuable chemicals, the synthesis of biomedical and polymer materials, and plant agrobiology. We also offer a comprehensive analysis of the properties and industrial applicability of light-controlled and other smart biomaterials. These data allow us to outline the prospects for the future use of optogenetics in bioindustry.

Abstract: Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells.

Abstract: 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.

Abstract: Owing to its ubiquity and easy availability in nature, light has been widely employed to control complex cellular behaviors. Light-sensitive proteins are the foundation to such diverse and multilevel adaptive regulations in a large range of organisms. Due to their remarkable properties and potential applications in engineered systems, exploration and engineering of natural light-sensitive proteins have significantly contributed to expand optogenetic toolboxes with tailor-made performances in synthetic genetic circuits. Progressively, more complex systems have been designed in which multiple photoreceptors, each sensing its dedicated wavelength, are combined to simultaneously coordinate cellular responses in a single cell. In this review, we highlight recent works and challenges on multiplexed optogenetic circuits in natural and engineered systems for a dynamic regulation breakthrough in biotechnological applications.

Abstract: RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.

Abstract: We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.

Abstract: 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.

Abstract: Dynamic protein-protein interactions are essential for proper cell functioning. Homo-interaction events-physical interactions between the same type of proteins-represent a pivotal subset of protein-protein interactions that are widely exploited in activating intracellular signaling pathways. Capacities of modulating protein-protein interactions with spatial and temporal resolution are greatly desired to decipher the dynamic nature of signal transduction mechanisms. The emerging optogenetic technology, based on genetically encoded light-sensitive proteins, provides promising opportunities to dissect the highly complex signaling networks with unmatched specificity and spatiotemporal precision. Here we review recent achievements in the development of optogenetic tools enabling light-inducible protein-protein homo-interactions and their applications in optical activation of signaling pathways.

Abstract: Intracellular antibody fragments such as nanobodies and scFvs are powerful tools for imaging and for modulating and neutralizing endogenous target proteins. Optogenetically activated intracellular antibodies (optobodies) constitute a light-inducible system to directly control intrabody activities in cells, with greater spatial and temporal resolution than intracellular antibodies alone. Here, we describe optogenetic and microscopic methods to activate optobodies in cells using a confocal microscope and an automated fluorescence microscope. In the protocol, we use the examples of an optobody targeting green fluorescent protein and an optobody that inhibits the endogenous gelsolin protein.

Abstract: Optogenetics is a powerful technique that allows the control of protein function with high spatiotemporal precision using light. Here, we describe the application of this method to control tissue mechanics during Drosophila embryonic development. We detail optogenetic protocols to either increase or decrease cell contractility and analyze the interplay between cell-cell interaction, tissue geometry, and force transmission during gastrulation.