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 164 results
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Optogenetic control of Cdc48 for dynamic metabolic engineering in yeast.

blue AsLOV2 CRY2/CIB1 S. cerevisiae Cell cycle control
Metab Eng, 7 Jul 2023 DOI: 10.1016/j.ymben.2023.06.013 Link to full text
Abstract: Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid β-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Light-switchable transcription factors obtained by direct screening in mammalian cells.

blue AsLOV2 HEK293T Transgene expression
Nat Commun, 2 Jun 2023 DOI: 10.1038/s41467-023-38993-6 Link to full text
Abstract: Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach's utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

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.

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.

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.

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.

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.

Light inducible protein degradation in E. coli with LOVtag.

blue AsLOV2 EL222 E. coli
bioRxiv, 26 Feb 2023 DOI: 10.1101/2023.02.25.530042 Link to full text
Abstract: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVtag, a protein tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVtag by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVtag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVtag system. Finally, we use the LOVtag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVtag system, and introduce a powerful new tool for bacterial optogenetics.

An engineered N-acyltransferase-LOV2 domain fusion protein enables light-inducible allosteric control of enzymatic activity.

blue AsLOV2 in vitro
J Biol Chem, 24 Feb 2023 DOI: 10.1016/j.jbc.2023.103069 Link to full text
Abstract: Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by Light-oxygen-voltage-sensing domain (LOV2). We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/*Dbv8) and identify potential allosteric sites on the protein's surface. We insert LOV2 at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/*Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

blue green red Am1 c0023g2/BAm green Am1 c0023g2/BAm red AsLOV2 TULIP CHO-K1 HEK293T in vitro S. cerevisiae Transgene expression Multichromatic
Nat Methods, 23 Feb 2023 DOI: 10.1038/s41592-023-01764-8 Link to full text
Abstract: Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Crosstalk between Rac and Rho GTPase activity mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

blue AsLOV2 A-431 HeLa Neuro-2a NIH/3T3 U-2 OS Control of cytoskeleton / cell motility / cell shape
bioRxiv, 20 Feb 2023 DOI: 10.1101/2023.02.20.529203 Link to full text
Abstract: Rho GTPase crosstalk is thought to play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigated crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining acute activity perturbation with activity measurements in individual, mammalian cells. As expected for their proposed mutual inhibition, we confirmed that Rho inhibits Rac activity. However, surprisingly, we found that Rac strongly stimulates Rho activity. We hypothesized that this crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. Using new, improved activity sensors for endogenous Rho GTPases, we find that Rac activation is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, we find that a subset of the Rho activating Lbc-type GEFs, in particular Arhgef11 and Arhgef12, are enriched at transient cell protrusions and retractions. Furthermore, via an optogenetic approach, we show that these Lbc GEFs are recruited to the plasma membrane by active Rac, suggesting that they might link cell protrusion and retraction by mediating Rac/Rho activity crosstalk. Indeed, depletion of these GEFs impaired cell protrusion-retraction dynamics, which was accompanied by an increase in migration directionality and reduced migration velocity. Thus, our study shows that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the central cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.

LILAC: enhanced actin imaging with an optogenetic Lifeact.

blue AsLOV2 Schneider 2
Nat Methods, 30 Jan 2023 DOI: 10.1038/s41592-022-01761-3 Link to full text
Abstract: Lifeact is a popular peptide-based label of actin filaments in live cells. We have designed an improved Lifeact variant, LILAC, that binds to actin in light using the LOV2 protein. Light control allows the user to modulate actin labeling, enabling image analysis that leverages modulation for an enhanced view of F-actin dynamics in cells. Furthermore, the tool reduces actin perturbations and cell sickness caused by Lifeact overexpression.

Optogenetic dissection of transcriptional repression in a multicellular organism.

blue AsLOV2 D. melanogaster in vivo Signaling cascade control Transgene expression Developmental processes
bioRxiv, 20 Nov 2022 DOI: 10.1101/2022.11.20.517211 Link to full text
Abstract: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can act as repressors or activators, how these functions are implemented at the molecular level has remained elusive. Here we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 minute) and memoryless. Finally, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Analysis of Slow-Cycling Variants of the Light-Inducible Nuclear Protein Export System LEXY in Mammalian Cells.

blue AsLOV2 HEK293 Transgene expression
ACS Synth Biol, 30 Sep 2022 DOI: 10.1021/acssynbio.2c00232 Link to full text
Abstract: The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.

Optogenetic control of RelA reveals effect of transcription factor dynamics on downstream gene expression.

blue AsLOV2 LOVTRAP HEK293T MCF10A NIH/3T3 Endogenous gene expression
bioRxiv, 5 Aug 2022 DOI: 10.1101/2022.08.03.502739 Link to full text
Abstract: Many transcription factors (TFs) translocate to the nucleus with varied dynamic patterns in response to different inputs. A notable example of such behavior is RelA, a subunit of NF-κB, which translocates to the nucleus with either pulsed or sustained dynamics, depending on the stimulus. Our understanding of how these dynamics are interpreted by downstream genes has remained incomplete, partly because ubiquitously used environmental inputs activate other transcriptional regulators in addition to RelA. Here, we use an optogenetic tool, CLASP (controllable light-activated shuttling and plasma membrane sequestration), to control RelA spatiotemporal dynamics in mouse fibroblasts and quantify their effect on downstream genes using RNA-seq. Using RelA-CLASP, we show for the first time that nuclear translocation of RelA, without post-translational modifications or activation of other transcriptional regulators, is sufficient to activate downstream genes. Furthermore, we find that TNFα, a common endogenous input, regulates many genes independently of RelA, and that this gene regulation is different from that induced by RelA-CLASP. Genes responsive to RelA-CLASP show a wide range of dynamics in response to a constant RelA input. We use a simple promoter model to recapitulate these diverse dynamic responses, as well as data collected in response to a pulsed RelA-CLASP input, and extract features of many RelA-responsive promoters. We also pinpoint many genes for which more complex models, involving feedback or multi-step promoters, may be needed to explain their response to constant and pulsed TF inputs. This study introduces a new robust tool for studying mammalian transcriptional regulation and demonstrates the power of optogenetic tools in dissecting the quantitative features of important cellular pathways.

A genetically encoded photo-proximity labeling approach for mapping protein territories.

blue AsLOV2 miniSOG A549 HEK293T HeLa U-2 OS
bioRxiv, 30 Jul 2022 DOI: 10.1101/2022.07.30.502153 Link to full text
Abstract: Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein interactomes to be captured using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to local generation of reactive radical species within cell-permeable probes. When combined with quantitative proteomics, the system generates ‘snapshots’ of protein interactions with high temporal resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein (MVP). The technology should be broadly useful in the biomedical area.

Optogenetic control of YAP cellular localisation and function.

blue AsLOV2 HEK293T HFF-1 MKN28 zebrafish in vivo Signaling cascade control
EMBO Rep, 25 Jul 2022 DOI: 10.15252/embr.202154401 Link to full text
Abstract: YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Two Rac1 pools integrate the direction and coordination of collective cell migration.

blue AsLOV2 D. melanogaster in vivo Control of cytoskeleton / cell motility / cell shape
bioRxiv, 13 Jun 2022 DOI: 10.1101/2022.06.13.495899 Link to full text
Abstract: Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, here we reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, our studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.

LILAC: Enhanced actin imaging with an optogenetic Lifeact.

blue AsLOV2 Schneider 2
bioRxiv, 8 Apr 2022 DOI: 10.1101/2022.04.07.487416 Link to full text
Abstract: We have designed an improved Lifeact variant that binds to actin under the control of light using the LOV2 protein. Light control enables one to subtract the pre-illumination signal of the unbound label, yielding an enhanced view of F-actin dynamics in cells. Furthermore, the tool eliminates actin network perturbations and cell sickness caused by Lifeact overexpression.

Optical control of protein delivery and partitioning in the nucleolus.

blue AsLOV2 CRY2/CRY2 HeLa Organelle manipulation
Nucleic Acids Res, 23 Mar 2022 DOI: 10.1093/nar/gkac191 Link to full text
Abstract: The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline-arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.

Local temporal Rac1-GTP nadirs and peaks restrict cell protrusions and retractions.

blue AsLOV2 HT-1080 Control of cytoskeleton / cell motility / cell shape
Sci Adv, 23 Mar 2022 DOI: 10.1126/sciadv.abl3667 Link to full text
Abstract: Cells probe their microenvironment using membrane protrusion-retraction cycles. Spatiotemporal coordination of Rac1 and RhoA GTP-binding activities initiates and reinforces protrusions and retractions, but the control of their finite lifetime remains unclear. We examined the relations of Rac1 and RhoA GTP-binding levels to key protrusion and retraction events, as well as to cell-ECM traction forces at physiologically relevant ECM stiffness. High RhoA-GTP preceded retractions and Rac1-GTP elevation before protrusions. Notable temporal Rac1-GTP nadirs and peaks occurred at the maximal edge velocity of local membrane protrusions and retractions, respectively, followed by declined edge velocity. Moreover, altered local Rac1-GTP consistently preceded similarly altered traction force. Local optogenetic Rac1-GTP perturbations defined a function of Rac1 in restricting protrusions and retractions and in promoting local traction force. Together, we show that Rac1 plays a fundamental role in restricting the size and durability of protrusions and retractions, plausibly in part through controlling traction forces.

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation.

blue AsLOV2 D. melanogaster in vivo Signaling cascade control
Cell Rep, 22 Mar 2022 DOI: 10.1016/j.celrep.2022.110543 Link to full text
Abstract: Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor in vivo. We engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirps transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.

MYC amplifies gene expression through global changes in transcription factor dynamics.

blue AsLOV2 HBEC3-KT MCF7 NIH/3T3 U-2 OS Endogenous gene expression
Cell Rep, 25 Jan 2022 DOI: 10.1016/j.celrep.2021.110292 Link to full text
Abstract: The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.

Quantification of nuclear transport inhibition by SARS-CoV-2 ORF6 using a broadly applicable live-cell dose-response pipeline.

blue AsLOV2 U-2 OS
bioRxiv, 14 Dec 2021 DOI: 10.1101/2021.12.10.472151 Link to full text
Abstract: SARS coronavirus ORF6 inhibits the classical nuclear import pathway to antagonize host antiviral responses. Several models were proposed to explain its inhibitory function, but quantitative measurement is needed for model evaluation and refinement. We report a broadly applicable live-cell method for calibrated dose-response characterization of the nuclear transport alteration by a protein of interest. Using this method, we found that SARS-CoV-2 ORF6 is ∼5 times more potent than SARS-CoV-1 ORF6 in inhibiting bidirectional nuclear transport, due to differences in the NUP98-binding C-terminal region that is required for the inhibition. The N-terminal region was also required, but its membrane binding function was dispensable, since loss of the inhibitory function due to N-terminal truncation was rescued by forced oligomerization using a soluble construct. Based on these data, we propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the FG domains of NUP98 at the nuclear pore complex.
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