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 26 - 50 of 1085 results
26.

A CRISPR-Cas9-Based Near-Infrared Upconversion-Activated DNA Methylation Editing System.

blue CRY2/CIB1 HEK293T mouse in vivo TPC-1 Nucleic acid editing
ACS Appl Mater Interfaces, 1 Feb 2021 DOI: 10.1021/acsami.0c21223 Link to full text
Abstract: DNA methylation is a kind of a crucial epigenetic marker orchestrating gene expression, molecular function, and cellular phenotype. However, manipulating the methylation status of specific genes remains challenging. Here, a clustered regularly interspaced palindromic repeats-Cas9-based near-infrared upconversion-activated DNA methylation editing system (CNAMS) was designed for the optogenetic editing of DNA methylation. The fusion proteins of photosensitive CRY2PHR, the catalytic domain of DNMT3A or TET1, and the fusion proteins for CIBN and catalytically inactive Cas9 (dCas9) were engineered. The CNAMS could control DNA methylation editing in response to blue light, thus allowing methylation editing in a spatiotemporal manner. Furthermore, after combination with upconversion nanoparticles, the spectral sensitivity of DNA methylation editing was extended from the blue light to near-infrared (NIR) light, providing the possibility for remote DNA methylation editing. These results demonstrated a meaningful step forward toward realizing the specific editing of DNA methylation, suggesting the wide utility of our CNAMS for functional studies on epigenetic regulation and potential therapeutic strategies for related diseases.
27.

Optogenetic manipulation of cellular communication using engineered myosin motors.

blue CRY2olig Ambystoma mexicanum in vivo C3H/10T1/2 Cos-7 Control of cytoskeleton / cell motility / cell shape
Nat Cell Biol, 1 Feb 2021 DOI: 10.1038/s41556-020-00625-2 Link to full text
Abstract: Cells achieve highly efficient and accurate communication through cellular projections such as neurites and filopodia, yet there is a lack of genetically encoded tools that can selectively manipulate their composition and dynamics. Here, we present a versatile optogenetic toolbox of artificial multi-headed myosin motors that can move bidirectionally within long cellular extensions and allow for the selective transport of GFP-tagged cargo with light. Utilizing these engineered motors, we could transport bulky transmembrane receptors and organelles as well as actin remodellers to control the dynamics of both filopodia and neurites. Using an optimized in vivo imaging scheme, we further demonstrate that, upon limb amputation in axolotls, a complex array of filopodial extensions is formed. We selectively modulated these filopodial extensions and showed that they re-establish a Sonic Hedgehog signalling gradient during regeneration. Considering the ubiquitous existence of actin-based extensions, this toolbox shows the potential to manipulate cellular communication with unprecedented accuracy.
28.

A synthetic switch based on orange carotenoid protein to control blue light responses in chloroplasts.

blue OCP A. thaliana in vivo A. thaliana leaf protoplasts
bioRxiv, 30 Jan 2021 DOI: 10.1101/2021.01.27.428448 Link to full text
Abstract: Synthetic biology approaches to engineer light‐responsive system are widely used, but their applications in plants are still limited, due to the interference with endogenous photoreceptors. Cyanobacteria, such as Synechocystis spp., possess a soluble carotenoid associated protein named Orange Carotenoid binding Protein (OCP) that, when activated by blue‐green light, undergoes reversible conformational changes that enable photoprotection of the phycobilisomes. Exploiting this system, we developed a new chloroplast‐localized synthetic photoswitch based on a photoreceptor‐associated protein‐fragment complementation assay (PCA). Since Arabidopsis thaliana does not possess the prosthetic group needed for the assembly of the OCP2 protein, we implemented the carotenoid biosynthetic pathway with a bacterial β‐carotene ketolase enzyme (crtW), to generate keto‐carotenoids producing plants. The novel photoswitch was tested and characterized in Arabidopsis protoplasts with experiments aimed to uncover its regulation by light intensity, wavelength, and its conversion dynamics. We believe that this pioneer study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses, such as gene transcription or enzymatic activity, upon exposure to specific light spectra.
29.

Transcription activation is enhanced by multivalent interactions independent of liquid-liquid phase separation.

blue CRY2/CIB1 HeLa U-2 OS Transgene expression
bioRxiv, 28 Jan 2021 DOI: 10.1101/2021.01.27.428421 Link to full text
Abstract: Transcription factors (TFs) consist of a DNA binding and an activation domain (AD) that are considered to be independent and exchangeable modules. However, recent studies conclude that also the physico-chemical properties of the AD can control TF assembly at chromatin via driving a phase separation into “transcriptional condensates”. Here, we dissected the mechanism of transcription activation at a reporter gene array with real-time single-cell fluorescence microscopy readouts. Our comparison of different synthetic TFs reveals that the phase separation propensity of the AD correlates with high transcription activation capacity by increasing binding site occupancy, residence time and the recruitment of co-activators. However, we find that the actual formation of phase separated TF liquid-like droplets has a neutral or inhibitory effect on transcription induction. Thus, our study suggests that the ability of a TF to phase separate reflects the functionally important property of the AD to establish multivalent interactions but does not by itself enhance transcription.
30.

A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice.

blue CRY2/CIB1 LOVTRAP VVD A549 Cos-7 HEK293 HEK293T HeLa mouse in vivo NCI-H1299 PC-3 U-87 MG Transgene expression
Nat Commun, 27 Jan 2021 DOI: 10.1038/s41467-021-20913-1 Link to full text
Abstract: Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.
31.

Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage.

blue CRY2/CIB1 MDCK SYF Signaling cascade control Control of cytoskeleton / cell motility / cell shape
J Cell Sci, 25 Jan 2021 DOI: 10.1242/jcs.254599 Link to full text
Abstract: Upon activation by different transmembrane receptors, the same signaling protein can induce distinct cellular responses. A way to decipher the mechanisms of such pleiotropic signaling activity is to directly manipulate the decision-making activity that supports the selection between distinct cellular responses. We developed an optogenetic probe (optoSRC) to control SRC signaling, an example of a pleiotropic signaling node, and we demonstrated its ability to generate different acto-adhesive structures (lamellipodia or invadosomes) upon distinct spatio-temporal control of SRC kinase activity. The occurrence of each acto-adhesive structure was simply dictated by the dynamics of optoSRC nanoclusters in adhesive sites, which were dependent on the SH3 and Unique domains of the protein. The different decision-making events regulated by optoSRC dynamics induced distinct downstream signaling pathways, which we characterized using time-resolved proteomic and network analyses. Collectively, by manipulating the molecular mobility of SRC kinase activity, these experiments reveal the pleiotropy-encoding mechanism of SRC signaling.
32.

Dynamical Modeling of Optogenetic Circuits in Yeast for Metabolic Engineering Applications.

blue EL222 in silico
ACS Synth Biol, 25 Jan 2021 DOI: 10.1021/acssynbio.0c00372 Link to full text
Abstract: Dynamic control of engineered microbes using light via optogenetics has been demonstrated as an effective strategy for improving the yield of biofuels, chemicals, and other products. An advantage of using light to manipulate microbial metabolism is the relative simplicity of interfacing biological and computer systems, thereby enabling in silico control of the microbe. Using this strategy for control and optimization of product yield requires an understanding of how the microbe responds in real-time to the light inputs. Toward this end, we present mechanistic models of a set of yeast optogenetic circuits. We show how these models can predict short- and long-time response to varying light inputs and how they are amenable to use with model predictive control (the industry standard among advanced control algorithms). These models reveal dynamics characterized by time-scale separation of different circuit components that affect the steady and transient levels of the protein under control of the circuit. Ultimately, this work will help enable real-time control and optimization tools for improving yield and consistency in the production of biofuels and chemicals using microbial fermentations.
33.

Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.

blue iLID HeLa U-2 OS Control of cytoskeleton / cell motility / cell shape
Elife, 22 Jan 2021 DOI: 10.7554/elife.61170 Link to full text
Abstract: During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.
34.

Optogenetic Control of Phosphatidylinositol (3,4,5)‐triphosphate Production by Light‐sensitive Cryptochrome Proteins on the Plasma Membrane.

blue CRY2/CIB1 C2C12 CHO-K1 NIH/3T3
Chin J Chem, 21 Jan 2021 DOI: 10.1002/cjoc.202000648 Link to full text
Abstract: Phosphatidylinositol (3,4,5)‐triphosphate (PIP3), acts as a fundamental second messenger, is emerging as a promising biomarker for disease diagnosis and prognosis. However, the real time analysis of phosphoinositide in living cells remains key challenge owing to the low basal abundance and its fast metabolic rate. Herein, we design an optogenetic system that uses light sensitive protein‐protein interaction between Arabidopsis cryptochrome 2 (CRY2) and CIB1 to spatiotemporally visualize the PIP3 production with sub‐second timescale. In this system, a CIBN is anchored on the plasma membrane, whereas a CRY2 fused with a constitutively active PI3‐kinase (acPI3K) would be driven from the cytosol to the membrane by the blue‐light‐activated CRY2‐CIB1 interaction upon light irradiation. The PIP3 production is visualized via a fused fluorescent protein by the translocation of a Pleckstrin Homology (PH) domain(GRP1) from the cytosol to the plasma membrane with high specificity. We demonstrated the fast dynamics and reversibility of the optogenetic system initiated PIP3 synthesis on the plasma membrane. Notably, the real‐time cell movements were also observed upon localized light stimulation. The established optogenetic method provides a novel spatiotemporal strategy for specific PIP3 visualization, which is beneficial to improve the understanding of PIP3 functions.
35.

Optogenetics in Sinorhizobium meliloti Enables Spatial Control of Exopolysaccharide Production and Biofilm Structure.

blue EL222 S. meliloti Transgene expression Control of cell-cell / cell-material interactions
ACS Synth Biol, 19 Jan 2021 DOI: 10.1021/acssynbio.0c00498 Link to full text
Abstract: Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.
36.

Random sub-diffusion and capture of genes by the nuclear pore reduces dynamics and coordinates interchromosomal movement.

blue CRY2/CIB1 S. cerevisiae
bioRxiv, 17 Jan 2021 DOI: 10.1101/2021.01.16.426968 Link to full text
Abstract: Hundreds of genes interact with the yeast nuclear pore complex (NPC), localizing at the nuclear periphery and clustering with co-regulated genes. Dynamic tracking of peripheral genes shows that they cycle on and off the NPC and that interaction with the NPC slows their sub-diffusive movement. Furthermore, NPC-dependent inter-chromosomal clustering leads to coordinated movement of pairs of loci separated by hundreds of nanometers. We developed Fractional Brownian Motion simulations for chromosomal loci in the nucleoplasm and interacting with NPCs. These simulations predict the rate and nature of random sub-diffusion during repositioning from nucleoplasm to periphery and match measurements from two different experimental models, arguing that recruitment to the nuclear periphery is due to random subdiffusion, collision, and capture by NPCs. Finally, the simulations do not lead to inter-chromosomal clustering or coordinated movement, suggesting that interaction with the NPC is necessary, but not sufficient, to cause clustering.
37.

TopBP1 assembles nuclear condensates to switch on ATR signaling.

blue CRY2/CRY2 HEK293 Signaling cascade control
Mol Cell, 16 Jan 2021 DOI: 10.1016/j.molcel.2020.12.049 Link to full text
Abstract: ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.
38.

Dual Systems for Enhancing Control of Protein Activity through Induced Dimerization Approaches.

blue violet Cryptochromes Fluorescent proteins LOV domains Review
Adv Biol, 14 Jan 2021 DOI: 10.1002/adbi.202000234 Link to full text
Abstract: To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.
39.

Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell–Cell Interactions.

green TtCBD MDA-MB-231 Control of cell-cell / cell-material interactions
Adv Biol, 14 Jan 2021 DOI: 10.1002/adbi.202000199 Link to full text
Abstract: The regulation of cell–cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell–cell interactions with high precision are of great interest to a better understanding of their roles and building tissue‐like structures. Herein, the green light‐responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell–cell interactions form and lead to cell clustering in a concentration‐dependent manner. Upon green light illumination, the CarH based cell–cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell–cell interactions impact cell migration, as observed in a wound‐healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell–cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell–cell interactions in biological processes.
40.

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.

blue cyan green near-infrared red UV violet BLUF domains Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Adv Biol, 14 Jan 2021 DOI: 10.1002/adbi.202000180 Link to full text
Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
41.

Optogenetic control of small GTPases reveals RhoA mediates intracellular calcium signaling.

blue CRY2/CIB1 iLID HEK293T HeLa hTERT RPE-1 MDCK Signaling cascade control
J Biol Chem, 13 Jan 2021 DOI: 10.1016/j.jbc.2021.100290 Link to full text
Abstract: Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.
42.

High levels of Dorsal transcription factor downregulate, not promote, snail expression by regulating enhancer action.

blue AsLOV2 D. melanogaster in vivo Developmental processes
bioRxiv, 11 Jan 2021 DOI: 10.1101/2021.01.11.426256 Link to full text
Abstract: In Drosophila embryos, genes expressed along the dorsal-ventral axis are responsive to concentration of the Dorsal (Dl) transcription factor, which varies in space; however, levels of this morphogen also build over time. Since expression of high-threshold Dl target genes such as snail (sna) is supported before Dl levels peak, it is unclear what role increasing levels have if any. Here we investigated action of two enhancers that control sna expression in embryos, demonstrating using genome editing that Dl binding sites within one enhancer located promoter proximally, sna.prox, can limit the ability of the other distally-located enhancer, sna.dis, to increase sna levels. In addition, MS2-MCP live imaging was used to study sna transcription rate in wildtype, dl heterozygote, and a background in which a photo-sensitive degron is fused to Dl (dl-BLID). The results demonstrate that, when Dl levels are high, Dl acts through sna.prox to limit the activity of sna.dis and thereby influence sna transcription rate. In contrast, when Dl levels are kept low using dl-BLID, sna.prox positively influences sna transcription rate. Collectively, our data support the view that Dl’s effect on gene expression changes over time, switching from promoting sna expression at low concentration to dampening sna expression at high concentration by regulating enhancer interactions. We propose this differential action of the Dl morphogen is likely supported by occupancy of this factor first to high and then low affinity binding sites over time as Dl levels rise to coordinate action of these two co-acting enhancers.
43.

Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression.

blue CRY2/CIB1 EL222 S. cerevisiae Transgene expression
bioRxiv, 7 Jan 2021 DOI: 10.1101/2021.01.07.425755 Link to full text
Abstract: Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve novel dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling-edge pulse-detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile-signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.
44.

Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis.

blue TULIP C. elegans in vivo Developmental processes
bioRxiv, 7 Jan 2021 DOI: 10.1101/2021.01.04.425207 Link to full text
Abstract: Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of C. elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.
45.

Photoactivated Adenylyl Cyclases: Fundamental Properties and Applications.

blue violet BLUF domains Cyanobacteriochromes LOV domains Review
Adv Exp Med Biol, 6 Jan 2021 DOI: 10.1007/978-981-15-8763-4_7 Link to full text
Abstract: Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.
46.

Genetically Encoded Photosensitizer for Destruction of Protein or Cell Function.

blue LOV domains Review
Adv Exp Med Biol, 6 Jan 2021 DOI: 10.1007/978-981-15-8763-4_16 Link to full text
Abstract: There are several paths when excited molecules return to the ground state. In the case of fluorescent molecules, the dominant path is fluorescence emission that is greatly contributing to bioimaging. Meanwhile, photosensitizers transfer electron or energy from chromophore to the surrounding molecules, including molecular oxygen. Generated reactive oxygen species has potency to attack other molecules by oxidation. In this chapter, we introduce the chromophore-assisted light inactivation (CALI) method using a photosensitizer to inactivate proteins in a spatiotemporal manner and development of CALI tools, which is useful for investigation of protein functions and dynamics, by inactivation of the target molecules. Moreover, photosensitizers with high efficiency make it possible optogenetic control of cell ablation in living organisms and photodynamic therapy. Further development of photosensitizers with different excitation wavelengths will contribute to the investigation of multiple proteins or cell functions through inactivation in the different positions and timings.
47.

Endosomal cAMP production broadly impacts the cellular phosphoproteome.

blue bPAC (BlaC) HEK293 Signaling cascade control Immediate control of second messengers
bioRxiv, 6 Jan 2021 DOI: 10.1101/2021.01.06.425636 Link to full text
Abstract: Endosomal signaling from G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of activating intracellular GPCRs is incomplete. To address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger cyclic AMP (cAMP) with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that cAMP produced from endosomes led to more robust changes in phosphorylation than cAMP produced from the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56δ as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.
48.

A synthetic gene circuit for imaging-free detection of dynamic cell signaling.

blue iLID NIH/3T3
bioRxiv, 6 Jan 2021 DOI: 10.1101/2021.01.06.425615 Link to full text
Abstract: Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution1. Here, we describe an approach to infer pulsatile signaling dynamics from just a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuit with pulse detecting capability, revealing an incoherent feedforward topology that robustly performs this computation. We then implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our ‘recorder of Erk activity dynamics’ (READer) responds sensitively to both spontaneous and stimulus-driven Erk pulses. READer circuits thus open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.
49.

A Light-Inducible Split-dCas9 System for Inhibiting the Progression of Bladder Cancer Cells by Activating p53 and E-cadherin.

blue CRY2/CIB1 5637 cells HEK293T T24 Nucleic acid editing
Front Mol Biosci, 5 Jan 2021 DOI: 10.3389/fmolb.2020.627848 Link to full text
Abstract: Optogenetic systems have been increasingly investigated in the field of biomedicine. Previous studies had found the inhibitory effect of the light-inducible genetic circuits on cancer cell growth. In our study, we applied an AND logic gates to the light-inducible genetic circuits to inhibit the cancer cells more specifically. The circuit would only be activated in the presence of both the human telomerase reverse transcriptase (hTERT) and the human uroplakin II (hUPII) promoter. The activated logic gate led to the expression of the p53 or E-cadherin protein, which could inhibit the biological function of tumor cells. In addition, we split the dCas9 protein to reduce the size of the synthetic circuit compared to the full-length dCas9. This light-inducible system provides a potential therapeutic strategy for future bladder cancer.
50.

Optogenetics: The Art of Illuminating Complex Signaling Pathways.

blue cyan red Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Physiology (Bethesda), 1 Jan 2021 DOI: 10.1152/physiol.00022.2020 Link to full text
Abstract: Dissection of cell signaling requires tools that can mimic spatiotemporal dynamics of individual pathways in living cells. Optogenetic methods enable manipulation of signaling processes with precise timing and local control. In this review, we describe recent optogenetic approaches for regulation of cell signaling, highlight their advantages and limitations, and discuss examples of their application.
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