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 1283 results

Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control of proximity labeling in living cells.

blue AsLOV2 iLID E. coli HEK293T mouse in vivo rat cortical neurons S. cerevisiae Transgene expression
Nat Methods, 15 May 2023 DOI: 10.1038/s41592-023-01880-5 Link to full text
Abstract: The incorporation of light-responsive domains into engineered proteins has enabled control of protein localization, interactions and function with light. We integrated optogenetic control into proximity labeling, a cornerstone technique for high-resolution proteomic mapping of organelles and interactomes in living cells. Through structure-guided screening and directed evolution, we installed the light-sensitive LOV domain into the proximity labeling enzyme TurboID to rapidly and reversibly control its labeling activity with low-power blue light. 'LOV-Turbo' works in multiple contexts and dramatically reduces background in biotin-rich environments such as neurons. We used LOV-Turbo for pulse-chase labeling to discover proteins that traffic between endoplasmic reticulum, nuclear and mitochondrial compartments under cellular stress. We also showed that instead of external light, LOV-Turbo can be activated by bioluminescence resonance energy transfer from luciferase, enabling interaction-dependent proximity labeling. Overall, LOV-Turbo increases the spatial and temporal precision of proximity labeling, expanding the scope of experimental questions that can be addressed with proximity labeling.

Optogenetic spatial patterning of cooperation in yeast populations.

blue EL222 S. cerevisiae Transgene expression
bioRxiv, 15 May 2023 DOI: 10.1101/2023.05.15.540783 Link to full text
Abstract: Microbial communities are a siege of complex metabolic interactions such as cooperation and competition for resources. Methods to control such interactions could lead to major advances in our ability to engineer microbial consortia for bioproduction and synthetic biology applications. Here, we used optogenetics to control invertase production in yeast, thereby creating landscapes of cooperator and cheater cells. Yeast cells behave as cooperators (i.e., transform sucrose into glucose, a public “good”) upon blue light illumination or cheaters (i.e., consume glucose produced by cooperators to grow) in the dark. We show that cooperators benefit best from the hexoses they produce when their domain size is constrained between two cut-off length-scales. From an engineering point of view, the system behaves as a band pass filter. The lower limit is the trace of cheaters’ competition for hexoses, while the upper limit is defined by cooperators’ competition for sucrose. Hence, cooperation mostly occurs at the frontiers with cheater cells, which not only compete for hexoses but also cooperate passively by letting sucrose reach cooperators. We anticipate that this optogenetic method could be applied to shape metabolic interactions in a variety of microbial ecosystems.

Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.

blue CRY2/CIB1 zebrafish in vivo Control of cytoskeleton / cell motility / cell shape Developmental processes
PLoS Genet, 15 May 2023 DOI: 10.1371/journal.pgen.1010765 Link to full text
Abstract: An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

OptIC Notch reveals mechanism that regulates receptor interactions with CSL.

blue AsLOV2 CRY2/CIB1 D. melanogaster in vivo Signaling cascade control
Development, 12 May 2023 DOI: 10.1242/dev.201785 Link to full text
Abstract: Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.

Directed differentiation of human iPSCs into mesenchymal lineages by optogenetic control of TGF-β signaling.

blue CRY2/CIB1 human IPSCs Signaling cascade control Cell differentiation
Cell Rep, 12 May 2023 DOI: 10.1016/j.celrep.2023.112509 Link to full text
Abstract: In tissue development and homeostasis, transforming growth factor (TGF)-β signaling is finely coordinated by latent forms and matrix sequestration. Optogenetics can offer precise and dynamic control of cell signaling. We report the development of an optogenetic human induced pluripotent stem cell system for TGF-β signaling and demonstrate its utility in directing differentiation into the smooth muscle, tenogenic, and chondrogenic lineages. Light-activated TGF-β signaling resulted in expression of differentiation markers at levels close to those in soluble factor-treated cultures, with minimal phototoxicity. In a cartilage-bone model, light-patterned TGF-β gradients allowed the establishment of hyaline-like layer of cartilage tissue at the articular surface while attenuating with depth to enable hypertrophic induction at the osteochondral interface. By selectively activating TGF-β signaling in co-cultures of light-responsive and non-responsive cells, undifferentiated and differentiated cells were simultaneously maintained in a single culture with shared medium. This platform can enable patient-specific and spatiotemporally precise studies of cellular decision making.

Optogenetic control of YAP can enhance the rate of wound healing.

blue AsLOV2 HEK293T MKN28 rat cardiomyocytes Signaling cascade control
Cell Mol Biol Lett, 11 May 2023 DOI: 10.1186/s11658-023-00446-9 Link to full text
Abstract: Tissues need to regenerate to restore function after injury. Yet, this regenerative capacity varies significantly between organs and between species. For example, in the heart, some species retain full regenerative capacity throughout their lifespan but human cardiac cells display a limited ability to repair the injury. After a myocardial infarction, the function of cardiomyocytes is impaired and reduces the ability of the heart to pump, causing heart failure. Therefore, there is a need to restore the function of an injured heart post myocardial infarction. We investigate in cell culture the role of the Yes-associated protein (YAP), a transcriptional co-regulator with a pivotal role in growth, in driving repair after injury.

Optogenetic inhibition of Gα signalling alters and regulates circuit functionality and early circuit formation

blue CRY2/CIB1 C. elegans in vivo D. melanogaster in vivo HEK293A rat dorsal root ganglion NSCs zebrafish in vivo Signaling cascade control Developmental processes
bioRxiv, 8 May 2023 DOI: 10.1101/2023.05.06.539674 Link to full text
Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαq signaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach, Photo-induced Modulation of Gα protein – Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. We alter the behavior of C. elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαi signaling.

Self-Regulated and Bidirectional Communication in Synthetic Cell Communities.

blue iLID in vitro Extracellular optogenetics
ACS Nano, 8 May 2023 DOI: 10.1021/acsnano.2c09908 Link to full text
Abstract: Cell-to-cell communication is not limited to a sender releasing a signaling molecule and a receiver perceiving it but is often self-regulated and bidirectional. Yet, in communities of synthetic cells, such features that render communication efficient and adaptive are missing. Here, we report the design and implementation of adaptive two-way signaling with lipid-vesicle-based synthetic cells. The first layer of self-regulation derives from coupling the temporal dynamics of the signal, H2O2, production in the sender to adhesions between sender and receiver cells. This way the receiver stays within the signaling range for the duration sender produces the signal and detaches once the signal fades. Specifically, H2O2 acts as both a forward signal and a regulator of the adhesions by activating photoswitchable proteins at the surface for the duration of the chemiluminescence. The second layer of self-regulation arises when the adhesions render the receiver permeable and trigger the release of a backward signal, resulting in bidirectional exchange. These design rules provide a concept for engineering multicellular systems with adaptive communication.

Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions.

bioRxiv, 6 May 2023 DOI: 10.1101/2022.10.22.513249 Link to full text
Abstract: Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the resolution and accuracy of proximity labeling methods are limited by a lack of control over the enzymatic labeling process. Here, we present a high spatial and temporal resolution technology that can be activated on demand using light, for high accuracy proximity labeling. Our system, called Light Activated BioID (LAB), is generated by fusing the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. Using live cell imaging, immunofluorescence, western blotting, and mass spectrometry, we show that upon exposure to blue light, CRY2 and CIB1 dimerize, reconstitute the split-TurboID enzyme, and biotinylate proximate proteins. Turning off the light halts the biotinylation reaction. We validate LAB in different cell types and demonstrate that it can identify known binding partners of proteins while reducing background labeling and false positives.

Light-Regulated Pro-Angiogenic Engineered Living Materials

blue YtvA E. coli Transgene expression
Adv Funct Mater, 5 May 2023 DOI: 10.1002/adfm.202212695 Link to full text
Abstract: Regenerative medicine aims to restore damaged cells, tissues, and organs, for which growth factors are vital to stimulate regenerative cellular transformations. Major advances have been made in growth factor engineering and delivery like the development of robust peptidomimetics and controlled release matrices. However, their clinical applicability remains limited due to their poor stability in the body and need for careful regulation of their local concentration to avoid unwanted side-effects. In this study, a strategy to overcome these limitations is explored using engineered living materials (ELMs), which contain live microorganisms that can be programmed with stimuli-responsive functionalities. Specifically, the development of an ELM that releases a pro-angiogenic protein in a light-regulated manner is described. This is achieved by optogenetically engineering bacteria to synthesize and secrete a vascular endothelial growth factor peptidomimetic (QK) linked to a collagen-binding domain. The bacteria are securely encapsulated in bilayer hydrogel constructs that support bacterial functionality but prevent their escape from the ELM. In situ control over the release profiles of the pro-angiogenic protein using light is demonstrated. Finally, it is shown that the released protein is able to bind collagen and promote angiogenic network formation among vascular endothelial cells, indicating the regenerative potential of these ELMs.

The clinical potential of optogenetic interrogation of pathogenesis.

blue cyan green red UV Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Clin Transl Med, May 2023 DOI: 10.1002/ctm2.1243 Link to full text
Abstract: Opsin-based optogenetics has emerged as a powerful biomedical tool using light to control protein conformation. Such capacity has been initially demonstrated to control ion flow across the cell membrane, enabling precise control of action potential in excitable cells such as neurons or muscle cells. Further advancement in optogenetics incorporates a greater variety of photoactivatable proteins and results in flexible control of biological processes, such as gene expression and signal transduction, with commonly employed light sources such as LEDs or lasers in optical microscopy. Blessed by the precise genetic targeting specificity and superior spatiotemporal resolution, optogenetics offers new biological insights into physiological and pathological mechanisms underlying health and diseases. Recently, its clinical potential has started to be capitalized, particularly for blindness treatment, due to the convenient light delivery into the eye.

Optogenetic control of kinesins -1, -2, -3 and dynein reveals their specific roles in vesicular transport.

blue LOVTRAP Cos-7 U-2 OS Control of vesicular transport
bioRxiv, 29 Apr 2023 DOI: 10.1101/2023.04.18.537380 Link to full text
Abstract: Each cargo in a cell employs a unique set of motor proteins for its transport. Often multiple types of kinesins are bound to the same cargo. It is puzzling why several types of motors are required for robust transport. To dissect the roles of each type of motor, we developed optogenetic inhibitors of kinesin-1, -2, -3 and dynein. This system allows us to control the activity of the endogenous set of motor proteins that are bound to intracellular cargoes. We examined the effect of optogenetic inhibition of kinesins-1, -2, and -3 and dynein on the transport of early endosomes, late endosomes, and lysosomes. While kinesin-1, kinesin-3, and dynein transport vesicles at all stages of endocytosis, kinesin-2 primarily drives late endosomes and lysosomes. In agreement with previous studies, sustained inhibition of either kinesins or dynein results in reduced motility in both directions. However, transient, optogenetic inhibition of kinesin-1 or dynein causes both early and late endosomes to move more processively by relieving competition with opposing motors. In contrast, optogenetic inhibition of kinesin-2 reduces the motility of late endosomes and lysosomes, and inhibition of kinesin-3 reduces the motility of endosomes and lysosomes. These results suggest that the directionality of transport is likely controlled through regulating kinesin-1 and dynein activity. On vesicles transported by several kinesin and dynein motors, motility can be directed by modulating the activity of a single type of motor on the cargo.

Transcription factor localization dynamics and DNA binding drive distinct promoter interpretations.

blue LOVTRAP S. cerevisiae Transgene expression
Cell Rep, 21 Apr 2023 DOI: 10.1016/j.celrep.2023.112426 Link to full text
Abstract: Environmental information may be encoded in the temporal dynamics of transcription factor (TF) activation and subsequently decoded by gene promoters to enact stimulus-specific gene expression programs. Previous studies of this behavior focused on the encoding and decoding of information in TF nuclear localization dynamics, yet cells control the activity of TFs in myriad ways, including by regulating their ability to bind DNA. Here, we use light-controlled mutants of the yeast TF Msn2 as a model system to investigate how promoter decoding of TF localization dynamics is affected by changes in the ability of the TF to bind DNA. We find that yeast promoters directly decode the light-controlled localization dynamics of Msn2 and that the effects of changing Msn2 affinity on that decoding behavior are highly promoter dependent, illustrating how cells could regulate TF localization dynamics and DNA binding in concert for improved control of gene expression.

Engineering of NEMO as calcium indicators with large dynamics and high sensitivity.

blue AsLOV2 HeLa Immediate control of second messengers
Nat Methods, 20 Apr 2023 DOI: 10.1038/s41592-023-01852-9 Link to full text
Abstract: Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal peak signal-to-baseline ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.

Requirements for mammalian promoters to decode transcription factor dynamics.

blue AsLOV2 HEK293 HeLa Transgene expression Endogenous gene expression
Nucleic Acids Res, 18 Apr 2023 DOI: 10.1093/nar/gkad273 Link to full text
Abstract: In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

Focal adhesions are controlled by microtubules through local contractility regulation.

blue iLID FAK-/- HT-1080 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 17 Apr 2023 DOI: 10.1101/2023.04.17.535593 Link to full text
Abstract: Microtubules regulate cell polarity and migration by local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with the major component of focal adhesions, talin. Local optogenetic activation of KANK1-mediated links which promoted microtubule targeting to individual focal adhesion resulting in its centripetal sliding and rapid disassembly. The sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesion upon KANK activation. Other players participating in microtubule-driven KANK-dependent focal adhesion disassembly include kinases ROCK and PAK, as well as microtubules/focal adhesions associated proteins Kinesin-1, APC and αTAT. Finally, we propose a physical model of a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK dependent activation of contractility which is consistent with experimental data.

Controlling protein stability with SULI, a highly sensitive tag for stabilization upon light induction.

blue PtAU1-LOV VVD S. cerevisiae zebrafish in vivo Cell cycle control Developmental processes
Nat Commun, 15 Apr 2023 DOI: 10.1038/s41467-023-37830-0 Link to full text
Abstract: Optogenetics tools for precise temporal and spatial control of protein abundance are valuable in studying diverse complex biological processes. In the present study, we engineer a monomeric tag of stabilization upon light induction (SULI) for yeast and zebrafish based on a single light-oxygen-voltage domain from Neurospora crassa. Proteins of interest fused with SULI are stable upon light illumination but are readily degraded after transfer to dark conditions. SULI shows a high dynamic range and a high tolerance to fusion at different positions of the target protein. Further studies reveal that SULI-mediated degradation occurs through a lysine ubiquitination-independent proteasome pathway. We demonstrate the usefulness of SULI in controlling the cell cycle in yeast and regulating protein stability in zebrafish, respectively. Overall, our data indicate that SULI is a simple and robust tool to quantitatively and spatiotemporally modulate protein levels for biotechnological or biomedical applications.

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos.

blue CRY2/CIB1 D. melanogaster in vivo
J Vis Exp, 14 Apr 2023 DOI: 10.3791/65314 Link to full text
Abstract: Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at multiple length scales, such as cell division, cell migration, epithelial folding, and branching morphogenesis. An in-depth understanding of the role of actomyosin contractility in morphogenesis requires approaches that allow the rapid inactivation of actomyosin, which is difficult to achieve using conventional genetic or pharmacological approaches. The presented protocol demonstrates the use of a CRY2-CIBN based optogenetic dimerization system, Opto-Rho1DN, to inhibit actomyosin contractility in Drosophila embryos with precise temporal and spatial controls. In this system, CRY2 is fused to the dominant negative form of Rho1 (Rho1DN), whereas CIBN is anchored to the plasma membrane. Blue light-mediated dimerization of CRY2 and CIBN results in rapid translocation of Rho1DN from the cytoplasm to the plasma membrane, where it inactivates actomyosin by inhibiting endogenous Rho1. In addition, this article presents a detailed protocol for coupling Opto-Rho1DN-mediated inactivation of actomyosin with laser ablation to investigate the role of actomyosin in generating epithelial tension during Drosophila ventral furrow formation. This protocol can be applied to many other morphological processes that involve actomyosin contractility in Drosophila embryos with minimal modifications. Overall, this optogenetic tool is a powerful approach to dissect the function of actomyosin contractility in controlling tissue mechanics during dynamic tissue remodeling.

Bioelectricity in Developmental Patterning and Size Control: Evidence and Genetically Encoded Tools in the Zebrafish Model.

blue AsLOV BLUF domains Cryptochromes LOV domains Review
Cells, 13 Apr 2023 DOI: 10.3390/cells12081148 Link to full text
Abstract: Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Genetically encoded imaging tools for investigating cell dynamics at a glance.

blue red UV Cryptochromes LOV domains Phytochromes UV receptors Review
J Cell Sci, 11 Apr 2023 DOI: 10.1242/jcs.260783 Link to full text
Abstract: The biology of a cell is the sum of many highly dynamic processes, each orchestrated by a plethora of proteins and other molecules. Microscopy is an invaluable approach to spatially and temporally dissect the molecular details of these processes. Hundreds of genetically encoded imaging tools have been developed that allow cell scientists to determine the function of a protein of interest in the context of these dynamic processes. Broadly, these tools fall into three strategies: observation, inhibition and activation. Using examples for each strategy, in this Cell Science at a Glance and the accompanying poster, we provide a guide to using these tools to dissect protein function in a given cellular process. Our focus here is on tools that allow rapid modification of proteins of interest and how observing the resulting changes in cell states is key to unlocking dynamic cell processes. The aim is to inspire the reader's next set of imaging experiments.

Lustro: High-throughput optogenetic experiments enabled by automation and a yeast optogenetic toolkit.

blue CRY2/CIB1 Magnets S. cerevisiae Transgene expression
bioRxiv, 10 Apr 2023 DOI: 10.1101/2023.04.07.536078 Link to full text
Abstract: Optogenetic systems use genetically-encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light, however these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and Enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized Enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to high-throughput characterization of optogenetic systems across a range of biological systems and applications.

A disordered tether to iLID improves photoswitchable protein patterning on model membranes.

blue iLID in vitro
Chem Commun (Camb), 6 Apr 2023 DOI: 10.1039/d3cc00709j Link to full text
Abstract: Reversible protein patterning on model membranes is important to reproduce spatiotemporal protein dynamics in vitro. An engineered version of iLID, disiLID, with a disordered domain as a membrane tether improves the recruitment of Nano under blue light and the reversibility in the dark, which enables protein patterning on membranes with higher spatiotemporal precision.

Light-activated macromolecular phase separation modulates transcription by reconfiguring chromatin interactions.

blue CRY2clust HeLa Transgene expression Endogenous gene expression
Sci Adv, 31 Mar 2023 DOI: 10.1126/sciadv.adg1123 Link to full text
Abstract: Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.

Allosteric inactivation of an engineered optogenetic GTPase.

blue AsLOV2 in vitro
Proc Natl Acad Sci U S A, 27 Mar 2023 DOI: 10.1073/pnas.2219254120 Link to full text
Abstract: Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

Development and Application of an Optogenetic Manipulation System to Suppress Actomyosin Activity in Ciona Epidermis.

blue BcLOV4 HeLa Control of cytoskeleton / cell motility / cell shape
Int J Mol Sci, 16 Mar 2023 DOI: 10.3390/ijms24065707 Link to full text
Abstract: Studying the generation of biomechanical force and how this force drives cell and tissue morphogenesis is challenging for understanding the mechanical mechanisms underlying embryogenesis. Actomyosin has been demonstrated to be the main source of intracellular force generation that drives membrane and cell contractility, thus playing a vital role in multi-organ formation in ascidian Ciona embryogenesis. However, manipulation of actomyosin at the subcellular level is impossible in Ciona because of the lack of technical tools and approaches. In this study, we designed and developed a myosin light chain phosphatase fused with a light-oxygen-voltage flavoprotein from Botrytis cinerea (MLCP-BcLOV4) as an optogenetics tool to control actomyosin contractility activity in the Ciona larva epidermis. We first validated the light-dependent membrane localization and regulatory efficiency on mechanical forces of the MLCP-BcLOV4 system as well as the optimum light intensity that activated the system in HeLa cells. Then, we applied the optimized MLCP-BcLOV4 system in Ciona larval epidermal cells to realize the regulation of membrane elongation at the subcellular level. Moreover, we successfully applied this system on the process of apical contraction during atrial siphon invagination in Ciona larvae. Our results showed that the activity of phosphorylated myosin on the apical surface of atrial siphon primordium cells was suppressed and apical contractility was disrupted, resulting in the failure of the invagination process. Thus, we established an effective technique and system that provide a powerful approach in the study of the biomechanical mechanisms driving morphogenesis in marine organisms.
Submit a new publication to our database