Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 25 of 182 results

Using Synthetic Biology to Engineer Spatial Patterns.

blue green red Cryptochromes Cyanobacteriochromes LOV domains Phytochromes Review
Adv Biosyst, 17 Dec 2018 DOI: 10.1002/adbi.201800280 Link to full text
Abstract: Synthetic biology has emerged as a multidisciplinary field that provides new tools and approaches to address longstanding problems in biology. It integrates knowledge from biology, engineering, mathematics, and biophysics to build—rather than to simply observe and perturb—biological systems that emulate natural counterparts or display novel properties. The interface between synthetic and developmental biology has greatly benefitted both fields and allowed to address questions that would remain challenging with classical approaches due to the intrinsic complexity and essentiality of developmental processes. This Progress Report provides an overview of how synthetic biology can help to understand a process that is crucial for the development of multicellular organisms: pattern formation. It reviews the major mechanisms of genetically encoded synthetic systems that have been engineered to establish spatial patterns at the population level. Limitations, challenges, applications, and potential opportunities of synthetic pattern formation are also discussed.

Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons.

blue cyan red Cryptochromes FKF1/G1 Fluorescent proteins LOV domains Phytochromes Review
Int J Mol Sci, 14 Dec 2018 DOI: 10.3390/ijms19124052 Link to full text
Abstract: Cellular activation of RAS GTPases into the GTP-binding "ON" state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson's disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets.

Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.

blue CRY2/CIB1 C3A Endogenous gene expression
Integr Biol (Camb), 6 Dec 2018 DOI: 10.1039/c8ib00109j Link to full text
Abstract: Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.

Development of a Wireless-Controlled LED Array for the Tunable Optogenetic Control of Cellular Activities.

blue CRY2/CIB1 HeLa Signaling cascade control Control of vesicular transport
Engineering, 6 Dec 2018 DOI: 10.1016/j.eng.2018.08.005 Link to full text
Abstract: Prohibitins (PHBs) are a highly conserved class of proteins first discovered as inhibitors of cellular proliferation. Since then PHBs have been found to have a significant role in transcription, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism, placing these proteins among the key regulators of pathologies such as cancer, neuromuscular degeneration, and other metabolic diseases. The human genome encodes two PHB proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2), which function not only as a heterodimeric complex, but also independently. While many previous reviews have focused on the better characterized prohibitin, PHB1, this review focuses on PHB2 and new data concerning its cellular functions both in complex with PHB1 and independent of PHB1.

Optogenetic control of morphogenesis goes 3D.

blue Cryptochromes Review
EMBO J, 21 Nov 2018 DOI: 10.15252/embj.2018100961 Link to full text
Abstract: The generation of form in living embryos, a process termed “morphogenesis” from the Greek word lοqφοcέmerg, is one of the most fascinating unsolved problems in biology. In embryonic epithelia, most attention has been paid to events occurring at the apical surface of epithelia, particularly the regulation of actomyosin contractility during morphogenetic change. In a new report, De Renzis and colleagues demonstrate a key role for regulated actomyosin contractility at the basal surface of the epithelium during formation of the first epithelial fold in Drosophila (the “ventral furrow”) (Krueger et al, 2018).

Optogenetic dissection of Rac1 and Cdc42 gradient shaping.

blue CRY2/CIB1 CRY2/CRY2 HeLa Control of cytoskeleton / cell motility / cell shape
Nat Commun, 16 Nov 2018 DOI: 10.1038/s41467-018-07286-8 Link to full text
Abstract: During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, β2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.

Downregulation of basal myosin-II is required for cell shape changes and tissue invagination.

blue CRY2/CIB1 D. melanogaster in vivo Control of cytoskeleton / cell motility / cell shape Developmental processes
EMBO J, 15 Nov 2018 DOI: 10.15252/embj.2018100170 Link to full text
Abstract: Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. Here we use optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. We show that while basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process.

Programming Bacteria With Light—Sensors and Applications in Synthetic Biology

blue cyan green near-infrared red UV violet Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Front Microbiol, 8 Nov 2018 DOI: 10.3389/fmicb.2018.02692 Link to full text
Abstract: Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.

Membrane dynamics induced by a PIP3 optogenetic tool.

blue CRY2/CIB1 Cos-7 HEK293 NIH/3T3 Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Anal Sci, 2 Nov 2018 DOI: 10.2116/analsci.18sdp06 Link to full text
Abstract: Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information of membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2–CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

Bringing Light to Transcription: The Optogenetics Repertoire.

blue red UV Cryptochromes LOV domains Phytochromes UV receptors Review
Front Genet, 2 Nov 2018 DOI: 10.3389/fgene.2018.00518 Link to full text
Abstract: The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.

Real-Time Genetic Compensation Defines the Dynamic Demands of Feedback Control.

blue CRY2/CIB1 S. cerevisiae Signaling cascade control
Cell, 18 Oct 2018 DOI: 10.1016/j.cell.2018.09.044 Link to full text
Abstract: Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments can often identify the existence of feedback interactions but are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed-loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors, in real time, the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver-on the fly-an optogenetically enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.

RalB directly triggers invasion downstream Ras by mobilizing the Wave complex.

blue CRY2/CIB1 HEK293T Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Elife, 15 Oct 2018 DOI: 10.7554/elife.40474 Link to full text
Abstract: The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anti-cancer strategies.

Light Control of the Tet Gene Expression System in Mammalian Cells.

blue CRY2/CIB1 EpH4 HEK293T mouse embryonic brain slices mouse in vivo primary mouse hippocampal neurons
Cell Rep, 9 Oct 2018 DOI: 10.1016/j.celrep.2018.09.026 Link to full text
Abstract: Gene expression and its network structure are dynamically altered in multicellular systems during morphological, functional, and pathological changes. To precisely analyze the functional roles of dynamic gene expression changes, tools that manipulate gene expression at fine spatiotemporal resolution are needed. The tetracycline (Tet)-controlled gene expression system is a reliable drug-inducible method, and it is used widely in many mammalian cultured cells and model organisms. Here, we develop a photoactivatable (PA)-Tet-OFF/ON system for precise temporal control of gene expression at single-cell resolution. By integrating the cryptochrome 2-cryptochrome-interacting basic helix-loop-helix 1 (Cry2-CIB1) light-inducible binding switch, expression of the gene of interest is tightly regulated under the control of light illumination and drug application in our PA-Tet-OFF/ON system. This system has a large dynamic range of downstream gene expression and rapid activation/deactivation kinetics. We also demonstrate the optogenetic regulation of exogenous gene expression in vivo, such as in developing and adult mouse brains.

Optogenetic Medicine: Synthetic Therapeutic Solutions Precision-Guided by Light.

blue cyan green near-infrared red UV Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Cold Spring Harb Perspect Med, 5 Oct 2018 DOI: 10.1101/cshperspect.a034371 Link to full text
Abstract: Gene- and cell-based therapies are well recognized as central pillars of next-generation medicine, but controllability remains a critical issue for clinical applications. In this context, optogenetics is opening up exciting new opportunities for precision-guided medicine by using illumination with light of appropriate intensity and wavelength as a trigger signal to achieve pinpoint spatiotemporal control of cellular activities, such as transgene expression. In this review, we highlight recent advances in optogenetics, focusing on devices for biomedical applications. We introduce the construction and applications of optogenetic-based biomedical tools to treat neurological diseases, diabetes, heart diseases, and cancer, as well as bioelectronic implants that combine light-interfaced electronic devices and optogenetic systems into portable personalized precision bioelectronic medical tools. Optogenetics-based technology promises the capability to achieve traceless, remotely controlled precision dosing of an enormous range of therapeutic outputs. Finally, we discuss the prospects for optogenetic medicine, as well as some emerging challenges.

Light‐Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.

blue cyan green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Adv Sci, 30 Sep 2018 DOI: 10.1002/advs.201800952 Link to full text
Abstract: The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology‐inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light‐controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non‐neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light‐sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light‐controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.

Optogenetic control of epithelial-mesenchymal transition in cancer cells.

blue CRY2/CIB1 A549 HeLa Signaling cascade control Control of cytoskeleton / cell motility / cell shape Cell differentiation
Sci Rep, 20 Sep 2018 DOI: 10.1038/s41598-018-32539-3 Link to full text
Abstract: Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the initiation and promotion of cancer cell metastasis. The phosphoinositide 3-kinase (PI3K) signaling pathway has been demonstrated to be involved in TGF-β induced EMT, but the complicated TGF-β signaling network makes it challenging to dissect the important role of PI3K on regulation of EMT process. Here, we applied optogenetic controlled PI3K module (named 'Opto-PI3K'), which based on CRY2 and the N-terminal of CIB1 (CIBN), to rapidly and reversibly control the endogenous PI3K activity in cancer cells with light. By precisely modulating the kinetics of PI3K activation, we found that E-cadherin is an important downstream target of PI3K signaling. Compared with TGF-β treatment, Opto-PI3K had more potent effect in down-regulation of E-cadherin expression, which was demonstrated to be regulated in a light dose-dependent manner. Surprisingly, sustained PI3K activation induced partial EMT state in A549 cells that is highly reversible. Furthermore, we demonstrated that Opto-PI3K only partially mimicked TGF-β effects on promotion of cell migration in vitro. These results reveal the importance of PI3K signaling in TGF-β induced EMT, suggesting other TGF-β regulated signaling pathways are necessary for the full and irreversible promotion of EMT in cancer cells. In addition, our study implicates the great promise of optogenetics in cancer research for mapping input-output relationships in oncogenic pathways.

A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells.

blue CRY2/CIB1 VVD S. cerevisiae Cell cycle control Transgene expression
ACS Synth Biol, 4 Sep 2018 DOI: 10.1021/acssynbio.8b00180 Link to full text
Abstract: Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology.

Synergistic Ensemble of Optogenetic Actuators and Dynamic Indicators in Cell Biology.

blue Cryptochromes Review
Mol Cells, 29 Aug 2018 DOI: 10.14348/molcells.2018.0295 Link to full text
Abstract: Discovery of the naturally evolved fluorescent proteins and their genetically engineered biosensors have enormously contributed to current bio-imaging techniques. These reporters to trace dynamic changes of intracellular protein activities have continuously transformed according to the various demands in biological studies. Along with that, light-inducible optogenetic technologies have offered scientists to perturb, control and analyze the function of intracellular machineries in spatiotemporal manner. In this review, we present an overview of the molecular strategies that have been exploited for producing genetically encoded protein reporters and various optogenetic modules. Finally, in particular, we discuss the current efforts for combined use of these reporters and optogenetic modules as a powerful tactic for the control and imaging of signaling events in cells and tissues.

Spatiotemporal control of zebrafish (Danio rerio) gene expression using a light-activated CRISPR activation system.

blue CRY2/CIB1 HEK293T ZF4 Transgene expression
Gene, 1 Aug 2018 DOI: 10.1016/j.gene.2018.07.077 Link to full text
Abstract: CRISPR activation (CRISPRa) system is the convenient tool for targeted-gene activation, it has been developed and combined with a lighting-based system that can control transcription initiation spatially and temporally by utilizing photoreceptor derived from plant Arabidopsis thaliana. A blue light photoreceptor the Cryptochrome 2 (CRY2), and its binding partner CIB1 will dimerize by exposure to the blue light and it has been applied to human cells. However, the application of a combination of these two systems to zebrafish cell is still not explored. We performed zebrafish gene activation using p65 and VP64 activators in the zebrafish cells (ZF4). Our study demonstrated that we have successfully controlled the transcription level of ASCL1a, BCL6a, and HSP70 genes using blue light-activated CRISPR activation system. The result showed that using this system, mRNA level expression of ASCL1a, BCL6a, and HSP70 genes increased after irradiated under blue light for several hours and significantly different to those which treated in the dark.

A compendium of chemical and genetic approaches to light-regulated gene transcription.

blue cyan green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Crit Rev Biochem Mol Biol, 24 Jul 2018 DOI: 10.1080/10409238.2018.1487382 Link to full text
Abstract: On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.

Illuminating pathogen-host intimacy through optogenetics.

blue green red BLUF domains Cryptochromes LOV domains Opsins Phytochromes Review
PLoS Pathog, 12 Jul 2018 DOI: 10.1371/journal.ppat.1007046 Link to full text
Abstract: The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host-pathogen interactions in a way that has not been feasible otherwise.

Blue-Light Receptors for Optogenetics.

blue green red UV BLUF domains Cryptochromes Fluorescent proteins LOV domains Opsins Phytochromes UV receptors Review
Chem Rev, 9 Jul 2018 DOI: 10.1021/acs.chemrev.8b00163 Link to full text
Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Optical activation of TrkA signaling.

blue CRY2/CIB1 CRY2/CRY2 NIH/3T3 PC-12 Signaling cascade control Cell differentiation
ACS Synth Biol, 5 Jul 2018 DOI: 10.1021/acssynbio.8b00126 Link to full text
Abstract: Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.

Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation.

blue red BphS CRY2/CIB1 HEK293 mouse in vivo Cell differentiation Endogenous gene expression Immediate control of second messengers
Proc Natl Acad Sci USA, 2 Jul 2018 DOI: 10.1073/pnas.1802448115 Link to full text
Abstract: The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.

Guided morphogenesis through optogenetic activation of Rho signalling during early Drosophila embryogenesis.

blue CRY2/CIB1 D. melanogaster in vivo Developmental processes
Nat Commun, 18 Jun 2018 DOI: 10.1038/s41467-018-04754-z Link to full text
Abstract: During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. Here we use optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. We show that precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes.
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