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 51 - 75 of 1087 results
51.

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.
52.

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.
53.

Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice.

blue red CRY2/CIB1 CRY2/CRY2 PhyB/PIF6 HEK293 mouse in vivo U-2 OS Transgene expression
Sci Adv, 1 Jan 2021 DOI: 10.1126/sciadv.abd3568 Link to full text
Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.
54.

Photobiologically Directed Assembly of Gold Nanoparticles.

blue PtAU1-LOV VVD in vitro
Adv Biol, 30 Dec 2020 DOI: 10.1002/adbi.202000179 Link to full text
Abstract: In nature, photoreceptor proteins undergo molecular responses to light, that exhibit supreme fidelity in time and space and generally occur under mild reaction conditions. To unlock these traits for material science, the light‐induced homodimerization of light‐oxygen‐voltage (LOV) photoreceptors is leveraged to control the assembly of gold nanoparticles. Conjugated to genetically encodable LOV proteins, the nanoparticles are monodispersed in darkness but rapidly assemble into large aggregates upon blue‐light exposure. The work establishes a new modality for reaction control in macromolecular chemistry and thus augurs enhanced precision in space and time in diverse applications of gold nanoparticles.
55.

Engineering Supramolecular Organizing Centers for Optogenetic Control of Innate Immune Responses.

blue CRY2/CRY2 LOVTRAP HEK293T HeLa RAW264.7 THP-1
Adv Biol, 30 Dec 2020 DOI: 10.1002/adbi.202000147 Link to full text
Abstract: The spatiotemporal organization of oligomeric protein complexes, such as the supramolecular organizing centers (SMOCs) made of MyDDosome and MAVSome, is essential for transcriptional activation of host inflammatory responses and immunometabolism. Light‐inducible assembly of MyDDosome and MAVSome is presented herein to induce activation of nuclear factor‐kB and type‐I interferons. Engineering of SMOCs and the downstream transcription factor permits programmable and customized innate immune operations in a light‐dependent manner. These synthetic molecular tools will likely enable optical and user‐defined modulation of innate immunity at a high spatiotemporal resolution to facilitate mechanistic studies of distinct modes of innate immune activations and potential intervention of immune disorders and cancer.
56.

Engineering an Optogenetic CRISPRi Platform for Improved Chemical Production.

blue EL222 E. coli Transgene expression
ACS Synth Biol, 24 Dec 2020 DOI: 10.1021/acssynbio.0c00488 Link to full text
Abstract: Microbial synthesis of chemicals typically requires the redistribution of metabolic flux toward the synthesis of targeted products. Dynamic control is emerging as an effective approach for solving the hurdles mentioned above. As light could control the cell behavior in a spatial and temporal manner, the optogenetic-CRISPR interference (opto-CRISPRi) technique that allocates the metabolic resources according to different optical signal frequencies will enable bacteria to be controlled between the growth phase and the production stage. In this study, we applied a blue light-sensitive protein EL222 to regulate the expression of the dCpf1-mediated CRISPRi system that turns off the competitive pathways and redirects the metabolic flux toward the heterologous muconic acid synthesis in Escherichia coli. We found that the opto-CRISPRi system dynamically regulating the suppression of the central metabolism and competitive pathways could increase the muconic acid production by 130%. These results demonstrated that the opto-CRISPRi platform is an effective method for enhancing chemical synthesis with broad utilities.
57.

Mechanical frustration of phase separation in the cell nucleus by chromatin.

blue iLID U-2 OS Organelle manipulation
bioRxiv, 24 Dec 2020 DOI: 10.1101/2020.12.24.424222 Link to full text
Abstract: Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically-generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that crosslinked chromatin can mechanically suppress droplets’ coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.
58.

Optogenetic control of gut bacterial metabolism to promote longevity.

green CcaS/CcaR E. coli Transgene expression
Elife, 16 Dec 2020 DOI: 10.7554/elife.56849 Link to full text
Abstract: Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.
59.

Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse.

blue red CRY2/CIB1 Magnets PhyB/PIF3 VVD HEK293T HeLa HEp-2 mouse in vivo SH-SY5Y Nucleic acid editing
Proc Natl Acad Sci U S A, 14 Dec 2020 DOI: 10.1073/pnas.2003991117 Link to full text
Abstract: Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.
60.

Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system.

blue iLID HEK293T HeLa
bioRxiv, 11 Dec 2020 DOI: 10.1101/2020.12.11.421990 Link to full text
Abstract: The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatio-temporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light-induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2–5]). Irradiation yielded a 50-230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.
61.

Improved Photocleavable Proteins with Faster and More Efficient Dissociation.

violet PhoCl HeLa Transgene expression Cell death
bioRxiv, 10 Dec 2020 DOI: 10.1101/2020.12.10.419556 Link to full text
Abstract: The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.
62.

Optical sensors of G protein signaling.

blue red LOV domains Phytochromes Review
FEBS J, 7 Dec 2020 DOI: 10.1111/febs.15655 Link to full text
Abstract: Heterotrimeric G proteins are central mediators of cellular signal transduction. They receive, process, and transduce signals from G protein-coupled receptors to downstream effectors. Since their discovery, a number of optical sensors of G protein localization and function have been developed and applied in living systems. In this minireview, we provide an overview of existing G protein-based sensors and the experimental approaches they utilize, with emphasis on live-cell imaging techniques. We outline recent advances, as well as identify current challenges and likely future directions in the field of G protein sensor development.
63.

Use of Optogenetic Amyloid-β to Monitor Protein Aggregation in Drosophila melanogaster, Danio rerio and Caenorhabditis elegans.

blue CRY2/CRY2 C. elegans in vivo D. melanogaster in vivo zebrafish in vivo
Bio Protoc, 5 Dec 2020 DOI: 10.21769/bioprotoc.3856 Link to full text
Abstract: Alzheimer's Disease (AD) has long been associated with accumulation of extracellular amyloid plaques (Aβ) originating from the Amyloid Precursor Protein. Plaques have, however, been discovered in healthy individuals and not all AD brains show plaques, suggesting that extracellular Aβ aggregates may play a smaller role than anticipated. One limitation to studying Aβ peptide in vivo during disease progression is the inability to induce aggregation in a controlled manner. We developed an optogenetic method to induce Aβ aggregation and tested its biological influence in three model organisms-D. melanogaster, C. elegans and D. rerio. We generated a fluorescently labeled, optogenetic Aβ peptide that oligomerizes rapidly in vivo in the presence of blue light in all organisms. Here, we detail the procedures for expressing this fusion protein in animal models, investigating the effects on the nervous system using time lapse light-sheet microscopy, and performing metabolic assays to measure changes due to intracellular Aβ aggregation. This method, employing optogenetics to study the pathology of AD, allows spatial and temporal control in vivo that cannot be achieved by any other method at present.
64.

Design of smart antibody mimetics with photosensitive switches.

blue AsLOV2 HEK293T HeLa Transgene expression Nucleic acid editing
bioRxiv, 4 Dec 2020 DOI: 10.1101/2020.12.03.410936 Link to full text
Abstract: As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. We introduce herein a generally-applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By paralleled insertion of two LOV2 modules into a single sunbody and the use of bivalent sunbodies, we substantially enhance the range of dynamic changes of photo-switchable sunbodies. Furthermore, we demonstrate the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light.
65.

Regulating enzymatic reactions in Escherichia coli utilizing light-responsive cellular compartments based on liquid-liquid phase separation.

blue CRY2/CIB1 E. coli
bioRxiv, 29 Nov 2020 DOI: 10.1101/2020.11.26.395616 Link to full text
Abstract: Enzymatic reactions in cells are well organized into different compartments, among which protein-based membraneless compartments formed through liquid-liquid phase separation (LLPS) are believed to play important roles1,2. Hijacking them for our own purpose has promising applications in metabolic engineering3. Yet, it is still hard to precisely and dynamically control target enzymatic reactions in those compartments4. To address those problems, we developed Photo-Activated Switch in E. coli (PhASE), based on phase separating scaffold proteins and optogenetic tools. In this system, a protein of interest (POI) can be enriched up to 15-fold by LLPS-based compartments from cytosol within only a few seconds once activated by light, and become fully dispersed again within 15 minutes. Furthermore, we explored the potentiality of the LLPS-based compartment in enriching small organic molecules directly via chemical-scaffold interaction. With enzymes and substrates co-localized under light induction, the overall reaction efficiency could be enhanced. Using luciferin and catechol oxidation as model enzymatic reactions, we found that they could accelerate 2.3-fold and 1.6-fold, respectively, when regulated by PhASE. We anticipate our system to be an extension of the synthetic biology toolkit, facilitating rapid recruitment and release of POIs, and reversible regulation of enzymatic reactions.
66.

A light way for nuclear cell biologists.

blue near-infrared red violet Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
J Biochem, 27 Nov 2020 DOI: 10.1093/jb/mvaa139 Link to full text
Abstract: The nucleus is a very complex organelle present in eukaryotic cells. Having the crucial task to safeguard, organize and manage the genetic information, it must tightly control its molecular constituents, its shape and its internal architecture at any given time. Despite our vast knowledge of nuclear cell biology, much is yet to be unraveled. For instance, only recently we came to appreciate the existence of a dynamic nuclear cytoskeleton made of actin filaments that regulates processes such as gene expression, DNA repair and nuclear expansion. This suggests further exciting discoveries ahead of us. Modern cell biologists embrace a new methodology relying on precise perturbations of cellular processes that require a reversible, highly spatially-confinable, rapid, inexpensive and tunable external stimulus: light. In this review, we discuss how optogenetics, the state-of-the-art technology that uses genetically-encoded light-sensitive proteins to steer biological processes, can be adopted to specifically investigate nuclear cell biology.
67.

Design and Characterization of Rapid Optogenetic Circuits for Dynamic Control in Yeast Metabolic Engineering.

blue EL222 S. cerevisiae Transgene expression Endogenous gene expression
ACS Synth Biol, 24 Nov 2020 DOI: 10.1021/acssynbio.0c00305 Link to full text
Abstract: The use of optogenetics in metabolic engineering for light-controlled microbial chemical production raises the prospect of utilizing control and optimization techniques routinely deployed in traditional chemical manufacturing. However, such mechanisms require well-characterized, customizable tools that respond fast enough to be used as real-time inputs during fermentations. Here, we present OptoINVRT7, a new rapid optogenetic inverter circuit to control gene expression in Saccharomyces cerevisiae. The circuit induces gene expression in only 0.6 h after switching cells from light to darkness, which is at least 6 times faster than previous OptoINVRT optogenetic circuits used for chemical production. In addition, we introduce an engineered inducible GAL1 promoter (PGAL1-S), which is stronger than any constitutive or inducible promoter commonly used in yeast. Combining OptoINVRT7 with PGAL1-S achieves strong and light-tunable levels of gene expression with as much as 132.9 ± 22.6-fold induction in darkness. The high performance of this new optogenetic circuit in controlling metabolic enzymes boosts production of lactic acid and isobutanol by more than 50% and 15%, respectively. The strength and controllability of OptoINVRT7 and PGAL1-S open the door to applying process control tools to engineered metabolisms to improve robustness and yields in microbial fermentations for chemical production.
68.

The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments.

blue green red UV Cryptochromes Cyanobacteriochromes LOV domains Phytochromes PixE/PixD UV receptors Review
Bioengineering (Basel), 24 Nov 2020 DOI: 10.3390/bioengineering7040151 Link to full text
Abstract: Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories.
69.

Spatio-temporal Control of ERK Pulse Frequency Coordinates Fate Decisions during Mammary Acinar Morphogenesis.

blue CRY2/CIB1 CRY2/CRY2 MCF10A Signaling cascade control Cell differentiation Cell death
bioRxiv, 21 Nov 2020 DOI: 10.1101/2020.11.20.387167 Link to full text
Abstract: The signaling events controlling proliferation, survival, and apoptosis during mammary epithelial acinar morphogenesis remain poorly characterized. By imaging single-cell ERK activity dynamics in MCF10A acini, we find that these fates depend on the frequency of ERK pulses. High pulse frequency is observed during initial acinus growth, correlating with rapid cell motility. Subsequent decrease in motility correlates with lower ERK pulse frequency and quiescence. Later, during lumen formation, coordinated ERK waves emerge across multiple cells of an acinus, correlating with high and low ERK pulse frequency in outer surviving and inner dying cells respectively. A PIK3CA H1047R mutation, commonly observed in breast cancer, increases ERK pulse frequency and inner cell survival, causing loss of lumen formation. Optogenetic entrainment of ERK pulses causally connects high ERK pulse frequency with inner cell survival. Thus, fate decisions during acinar morphogenesis are fine-tuned by different spatio-temporal coordination modalities of ERK pulse frequency.
70.

Single-Protein Tracking to Study Protein Interactions During Integrin-Based Migration.

blue CRY2/CIB1 MEF-1 Control of cytoskeleton / cell motility / cell shape
Methods Mol Biol, 20 Nov 2020 DOI: 10.1007/978-1-0716-0962-0_8 Link to full text
Abstract: Cell migration is a complex biophysical process which involves the coordination of molecular assemblies including integrin-dependent adhesions, signaling networks and force-generating cytoskeletal structures incorporating both actin polymerization and myosin activity. During the last decades, proteomic studies have generated impressive protein-protein interaction maps, although the subcellular location, duration, strength, sequence, and nature of these interactions are still concealed. In this chapter we describe how recent developments in superresolution microscopy (SRM) and single-protein tracking (SPT) start to unravel protein interactions and actions in subcellular molecular assemblies driving cell migration.
71.

Spatiotemporal sensitivity of embryonic heart specification to FGFR signaling in Drosophila.

blue CRY2/CRY2 D. melanogaster in vivo Signaling cascade control Developmental processes
bioRxiv, 19 Nov 2020 DOI: 10.1101/2020.11.16.384123 Link to full text
Abstract: Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Htl is involved in shaping the mesoderm at both early and later stages during embryogenesis. How Htl expression levels and timing of signaling affect mesoderm morphogenesis after spreading remains elusive. We have developed an optogenetic tool (Opto-htl) to control the activation of Htl signaling with precise spatiotemporal resolution in vivo. We find that the embryo is most sensitive to Htl over-activation within a developmental window of ~4 hours ranging from late stage 10 until early stage 13, which corresponds to early stages of heart morphogenesis. Opto-htl restores heart cells in htl mutants upon light activation, independent of its role in early mesoderm shaping events. We also successfully generated spatially distinct regions of Htl activity in the mesoderm using light-sheet microscopy. The developing tissue was unable to correct for the ensuing asymmetries in cell fate. Overall, Opto-htl is a powerful tool for studying spatiotemporal regulation of Htl signaling during embryogenesis.
72.

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos.

blue iLID D. melanogaster in vivo Developmental processes
Elife, 17 Nov 2020 DOI: 10.7554/elife.56893 Link to full text
Abstract: Ventral furrow formation, the first step in Drosophila gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements. Here, using an optogenetic probe that rapidly and robustly activates Rho1 in Drosophila tissues, we show that Rho1 activity induces ectopic deformations in the dorsal and ventral epithelia of Drosophila embryos. These perturbations reveal substantial differences in how ventral and dorsal cells, both within and outside the zone of Rho1 activation, respond to spatially and temporally identical patterns of Rho1 activation. Our results demonstrate that an asymmetric zone of Rho1 activity is not sufficient to recapitulate ventral furrow formation and reveal that additional, ventral-specific factors contribute to the cell- and tissue-level behaviors that emerge during ventral furrow formation.
73.

Structural insights into the photoactivation of Arabidopsis CRY2.

blue Cryptochromes Background
Nat Plants, 16 Nov 2020 DOI: 10.1038/s41477-020-00800-1 Link to full text
Abstract: The blue-light receptor cryptochrome (CRY) in plants undergoes oligomerization to transduce blue-light signals after irradiation, but the corresponding molecular mechanism remains poorly understood. Here, we report the cryogenic electron microscopy structure of a blue-light-activated CRY2 tetramer at a resolution of 3.1 Å, which shows how the CRY2 tetramer assembles. Our study provides insights into blue-light-mediated activation of CRY2 and a theoretical basis for developing regulators of CRYs for optogenetic manipulation.
74.

Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.

red DrBphP HEK293T HeLa mouse in vivo S. cerevisiae
ACS Synth Biol, 12 Nov 2020 DOI: 10.1021/acssynbio.0c00397 Link to full text
Abstract: Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.
75.

Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.

blue Magnets Cos-7 HeLa Organelle manipulation
Elife, 11 Nov 2020 DOI: 10.7554/elife.63230 Link to full text
Abstract: Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28oC to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
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