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 78 results

Assembly Domain-Based Optogenetic System for the Efficient Control of Cellular Signaling.

blue Magnets Cos-7 HEK293T Control of cytoskeleton / cell motility / cell shape
ACS Synth Biol, 3 Mar 2017 DOI: 10.1021/acssynbio.7b00022 Link to full text
Abstract: We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca(2+)/calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation.

A Photoactivatable Innate Immune Receptor for Optogenetic Inflammation.

blue CRY2/CIB1 HEK293 RAW264.7 Signaling cascade control
ACS Chem Biol, 29 Dec 2016 DOI: 10.1021/acschembio.6b01012 Link to full text
Abstract: Although spatial and temporal elements of immune activation mediate the intensity of the immune response, few tools exist to directly examine these effects. To elucidate the spatiotemporal aspects of innate immune responses, we designed an optogenetic pattern recognition receptor that activates in response to blue light. We demonstrate direct receptor activation, leading to spatial and temporal control of downstream signaling pathways in a variety of relevant cell types. We combined our platform with Bi-molecular Fluorescence Complementation (BiFC), resulting in selective fluorescent labeling of cells in which receptor activation has occurred.

Engineered Photoactivatable Genetic Switches Based on the Bacterium Phage T7 RNA Polymerase.

blue Magnets VVD E. coli
ACS Synth Biol, 15 Nov 2016 DOI: 10.1021/acssynbio.6b00248 Link to full text
Abstract: Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects. The split fragments show high modularity when they are combined with different regulatory domains such as those with chemically inducible interaction, enabling chemically controlled switches. To summarize, the T7 RNA polymerase-based switches are powerful tools to implement light-activated gene expression in different contexts. Moreover, results about the studied split positions and domain organizations may facilitate future engineering studies on this and on related proteins.

Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli.

violet UirS/UirR E. coli
ACS Synth Biol, 11 May 2016 DOI: 10.1021/acssynbio.6b00068 Link to full text
Abstract: We have previously engineered green/red and red/far red photoreversible E. coli phytochrome and cyanobacteriochrome (CBCR) two-component systems (TCSs) and utilized them to program tailor-made gene expression signals for gene circuit characterization. Here, we transport the UV-violet/green photoreversible CBCR TCS UirS-UirR from Synechocystis PCC6803 to E. coli. We demonstrate that the promoter of the small RNA csiR1, previously shown to be activated by inorganic carbon stress, is a UirS-UirR output. Additionally, in contrast to a recently proposed sequestration model, we show that the sensor histidine kinase UirS phosphorylates the response regulator UirR to activate PcsiR1 transcription in response to UV-violet light. Finally, we measure changes in UirS-UirR output minutes after a change in light input and exploit these rapid dynamics to program a challenging gene expression signal with high predictability. UirS-UirR is the first engineered transcriptional regulatory tool activated exclusively by UV-violet light, and the most blue shifted photoreversible transcriptional regulatory tool.

Illuminating Cell Signaling with Near-Infrared Light-Responsive Nanomaterials.

blue Cryptochromes Review
ACS Nano, 14 Apr 2016 DOI: 10.1021/acsnano.6b02284 Link to full text
Abstract: The regulation of cellular signaling in vivo has been a challenging task owing to the lack of effective methods for tunable control of the amplitude, location, and duration of cell-signaling events at a deep-tissue level. In this issue of ACS Nano, an intriguing paper by Ambrosone et al. demonstrates that deep-tissue-penetrating near-infrared (NIR) light can be used to control the Wnt/β-catenin-signaling pathway in a single-cell organism (Hydra) by utilizing microcapsules that contain plasmonic gold nanoparticles. In parallel, in recent work, we proposed upconversion nanoparticles (UCNPs) as NIR-light-activatable "wireless" optogenetic tools, and we showed their ability to modulate cell signaling pathways in both mammalian cells and mice. We believe that these interesting NIR-light-responsive nanotechnologies will open new avenues for both basic research and clinical applications.

Rewiring Multidomain Protein Switches: Transforming a Fluorescent Zn(2+) Sensor into a Light-Responsive Zn(2+) Binding Protein.

blue VVD HeLa in vitro
ACS Synth Biol, 12 Apr 2016 DOI: 10.1021/acssynbio.6b00027 Link to full text
Abstract: Protein-based sensors and switches provide attractive tools for the real-time monitoring and control of molecular processes in complex biological environments. Fluorescent sensor proteins have been developed for a wide variety of small molecules, but the construction of genetically encoded light-responsive ligand binding proteins remains mostly unexplored. Here we present a generic approach to reengineer a previously developed FRET-based Zn(2+) sensor into a light-activatable Zn(2+) binding protein using a design strategy based on mutually exclusive domain interactions. These so-called VividZn proteins consist of two light-responsive Vivid domains that homodimerize upon illumination with blue light, thus preventing the binding of Zn(2+) between two Zn(2+) binding domains, Atox1 and WD4. Following optimization of the linker between WD4 and the N-terminus of one of the Vivid domains, VividZn variants were obtained that show a 9- to 55-fold decrease in Zn(2+) affinity upon illumination, which is fully reversible following dark adaptation. The Zn(2+) affinities of the switch could be rationally tuned between 1 pM and 2 nM by systematic variation of linker length and mutation of one of the Zn(2+) binding residues. Similarly, introduction of mutations in the Vivid domains allowed tuning of the switching kinetics between 10 min and 7 h. Low expression levels in mammalian cells precluded the demonstration of light-induced perturbation of cytosolic Zn(2+) levels. Nonetheless, our results firmly establish the use of intramolecular Vivid dimerization as an attractive light-sensitive input module to rationally engineer light-responsive protein switches based on mutually exclusive domain interactions.

Library-Aided Probing of Linker Determinants in Hybrid Photoreceptors.

blue LOV domains Background
ACS Synth Biol, 21 Mar 2016 DOI: 10.1021/acssynbio.6b00028 Link to full text
Abstract: Signaling proteins comprise interaction and effector modules connected by linkers. Throughout evolution, these recurring modules have multiply been recombined to produce the present-day plethora of signaling proteins. Likewise, modular recombination lends itself to the engineering of hybrid signal receptors, whose functionality hinges on linker topology, sequence, and length. Often, numerous linkers must be assessed to obtain functional receptors. To expedite linker optimization, we devised the PATCHY strategy (primer-aided truncation for the creation of hybrid proteins) for the facile construction of hybrid gene libraries with defined linker distributions. Empowered by PATCHY, we engineered photoreceptors whose signal response was governed by linker length: whereas blue-light-repressed variants possessed linkers of 7n or 7n+5 residues, variants with 7n+1 residues were blue-light-activated. Related natural receptors predominantly displayed linker lengths of 7n and 7n+5 residues but rarely of 7n+1 residues. PATCHY efficiently explores linker sequence space to yield functional hybrid proteins including variants transcending the natural repertoire of signaling proteins.

Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

blue Cryptochromes LOV domains Review
ACS Chem Biol, 9 Feb 2016 DOI: 10.1021/acschembio.5b01019 Link to full text
Abstract: The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

red PhyB/PIF6 HEK293T HeLa hMSCs HUVEC in vitro NIH/3T3
ACS Nano, 30 Nov 2015 DOI: 10.1021/acsnano.5b05558 Link to full text
Abstract: Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.

Optical Control of Peroxisomal Trafficking.

blue AsLOV2 Cos-7 HEK293T HeLa S. cerevisiae
ACS Synth Biol, 2 Nov 2015 DOI: 10.1021/acssynbio.5b00144 Link to full text
Abstract: The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times.

Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.

blue CRY2/CIB1 iLID TULIP in vitro mouse IA32 fibroblasts S. cerevisiae Control of cytoskeleton / cell motility / cell shape Benchmarking
ACS Synth Biol, 30 Oct 2015 DOI: 10.1021/acssynbio.5b00119 Link to full text
Abstract: Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.

The Dual Characteristics of Light-Induced Cryptochrome 2, Homo-oligomerization and Heterodimerization, for Optogenetic Manipulation in Mammalian Cells.

blue CRY2/CIB1 CRY2/CRY2 Cos-7 HEK293T NIH/3T3
ACS Synth Biol, 8 Jun 2015 DOI: 10.1021/acssynbio.5b00048 Link to full text
Abstract: The photoreceptor cryptochrome 2 (CRY2) has become a powerful optogenetic tool that allows light-inducible manipulation of various signaling pathways and cellular processes in mammalian cells with high spatiotemporal precision and ease of application. However, it has also been shown that the behavior of CRY2 under blue light is complex, as the photoexcited CRY2 can both undergo homo-oligomerization and heterodimerization by binding to its dimerization partner CIB1. To better understand the light-induced CRY2 activities in mammalian cells, this article systematically characterizes CRY2 homo-oligomerization in different cellular compartments, as well as how CRY2 homo-oligomerization and heterodimerization activities affect each other. Quantitative analysis reveals that membrane-bound CRY2 has drastically enhanced oligomerization activity compared to that of its cytoplasmic form. While CRY2 homo-oligomerization and CRY2-CIB1 heterodimerization could happen concomitantly, the presence of certain CIB1 fusion proteins can suppress CRY2 homo-oligomerization. However, the homo-oligomerization of cytoplasmic CRY2 can be significantly intensified by its recruitment to the membrane via interaction with the membrane-bound CIB1. These results contribute to the understanding of the light-inducible CRY2-CRY2 and CRY2-CIB1 interaction systems and can be used as a guide to establish new strategies utilizing the dual optogenetic characteristics of CRY2 to probe cellular processes.

Probing Yeast Polarity with Acute, Reversible, Optogenetic Inhibition of Protein Function.

red PhyB/PIF6 S. cerevisiae Control of cytoskeleton / cell motility / cell shape Cell cycle control
ACS Synth Biol, 2 Jun 2015 DOI: 10.1021/acssynbio.5b00053 Link to full text
Abstract: We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inhibition of Bem1 produces profound defects in cell polarization and cell viability that are not observed in bem1Δ. By disrupting Bem1 activity at specific points in the cell cycle, we demonstrate that Bem1 is essential for the establishment of polarity and bud emergence but is dispensable for the growth of an emerged bud. By taking advantage of the reversibility of Bem1 inactivation, we show that pole size scales with cell size, and that this scaling is dependent on the actin cytoskeleton. Our experiments reveal how rapid reversible inactivation of protein function complements traditional genetic approaches. This strategy should be widely applicable to other biological contexts.

Red Light-Regulated Reversible Nuclear Localization of Proteins in Mammalian Cells and Zebrafish.

red PhyB/PIF3 CHO-K1 Cos-7 HEK293T HeLa NIH/3T3 zebrafish in vivo
ACS Synth Biol, 30 Mar 2015 DOI: 10.1021/acssynbio.5b00004 Link to full text
Abstract: Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices.

Structural details of light activation of the LOV2-based photoswitch PA-Rac1.

blue AsLOV2 in vitro
ACS Chem Biol, 17 Nov 2014 DOI: 10.1021/cb500744m Link to full text
Abstract: Optical control of cellular processes is an emerging approach for studying biological systems, affording control with high spatial and temporal resolution. Specifically designed artificial photoswitches add an interesting extension to naturally occurring light-regulated functionalities. However, despite a great deal of structural information, the generation of new tools cannot be based fully on rational design yet; in many cases design is limited by our understanding of molecular details of light activation and signal transduction. Our biochemical and biophysical studies on the established optogenetic tool PA-Rac1, the photoactivatable small GTPase Rac1, reveal how unexpected details of the sensor-effector interface, such as metal coordination, significantly affect functionally important structural elements of this photoswitch. Together with solution scattering experiments, our results favor differences in the population of pre-existing conformations as the underlying allosteric activation mechanism of PA-Rac1, rather than the assumed release of the Rac1 domain from the caging photoreceptor domain. These results have implications for the design of new optogenetic tools and highlight the importance of including molecular details of the sensor-effector interface, which is however difficult to assess during the initial design of novel artificial photoswitches.

Benchmarking of optical dimerizer systems.

blue red CRY2/CIB1 PhyB/PIF3 PhyB/PIF6 TULIP S. cerevisiae Signaling cascade control Benchmarking
ACS Synth Biol, 5 Nov 2014 DOI: 10.1021/sb500291r Link to full text
Abstract: Optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions. Such tools have been useful for regulating cellular pathways and processes with high spatiotemporal resolution in live cells, and a growing number of dimerizer systems are available. As these systems have been characterized by different groups using different methods, it has been difficult for users to compare their properties. Here, we set about to systematically benchmark the properties of four optical dimerizer systems, CRY2/CIB1, TULIPs, phyB/PIF3, and phyB/PIF6. Using a yeast transcriptional assay, we find significant differences in light sensitivity and fold-activation levels between the red light regulated systems but similar responses between the CRY2/CIB and TULIP systems. Further comparison of the ability of the CRY2/CIB1 and TULIP systems to regulate a yeast MAPK signaling pathway also showed similar responses, with slightly less background activity in the dark observed with CRY2/CIB. In the process of developing this work, we also generated an improved blue-light-regulated transcriptional system using CRY2/CIB in yeast. In addition, we demonstrate successful application of the CRY2/CIB dimerizers using a membrane-tethered CRY2, which may allow for better local control of protein interactions. Taken together, this work allows for a better understanding of the capacities of these different dimerization systems and demonstrates new uses of these dimerizers to control signaling and transcription in yeast.

Orthogonal optogenetic triple-gene control in Mammalian cells.

blue red UV PhyB/PIF6 TULIP UVR8/COP1 VVD CHO-K1 Multichromatic
ACS Synth Biol, 28 Oct 2014 DOI: 10.1021/sb500305v Link to full text
Abstract: Optogenetic gene switches allow gene expression control at an unprecedented spatiotemporal resolution. Recently, light-responsive transgene expression systems that are activated by UV-B, blue, or red light have been developed. These systems perform well on their own, but their integration into genetic networks has been hampered by the overlapping absorbance spectra of the photoreceptors. We identified a lack of orthogonality between UV-B and blue light-controlled gene expression as the bottleneck and employed a model-based approach that identified the need for a blue light-responsive gene switch that is insensitive to low-intensity light. Based on this prediction, we developed a blue light-responsive and rapidly reversible expression system. Finally, we employed this expression system to demonstrate orthogonality between UV-B, blue, and red/far-red light-responsive gene switches in a single mammalian cell culture. We expect this approach to enable the spatiotemporal control of gene networks and to expand the applications of optogenetics in synthetic biology.

Refactoring and optimization of light-switchable Escherichia coli two-component systems.

green red CcaS/CcaR Cph1 E. coli
ACS Synth Biol, 11 Oct 2014 DOI: 10.1021/sb500273n Link to full text
Abstract: Light-switchable proteins enable unparalleled control of molecular biological processes in live organisms. Previously, we have engineered red/far-red and green/red photoreversible two-component signal transduction systems (TCSs) with transcriptional outputs in E. coli and used them to characterize and control synthetic gene circuits with exceptional quantitative, temporal, and spatial precision. However, the broad utility of these light sensors is limited by bulky DNA encoding, incompatibility with commonly used ligand-responsive transcription factors, leaky output in deactivating light, and less than 10-fold dynamic range. Here, we compress the four genes required for each TCS onto two streamlined plasmids and replace all chemically inducible and evolved promoters with constitutive, engineered versions. Additionally, we systematically optimize the expression of each sensor histidine kinase and response regulator, and redesign both pathway output promoters, resulting in low leakiness and 72- and 117-fold dynamic range, respectively. These second-generation light sensors can be used to program the expression of more genes over a wider range and can be more easily combined with additional plasmids or moved to different host strains. This work demonstrates that bacterial TCSs can be optimized to function as high-performance sensors for scientific and engineering applications.

Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides.

blue AsLOV2 Cos-7 HEK293 primary mouse cortical neurons Signaling cascade control Control of cytoskeleton / cell motility / cell shape
ACS Synth Biol, 13 Jun 2014 DOI: 10.1021/sb5001356 Link to full text
Abstract: Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently inhibited kinases in the light but were sterically blocked from kinase interaction in the dark. Photoactivatable inhibitors for cyclic-AMP dependent kinase (PKA) and myosin light chain kinase (MLCK) are described, together with studies that shed light on proper positioning of the peptides in the LOV domain. These inhibitors altered endogenous signaling in living cells and produced light-dependent changes in cell morphodynamics.

Optical control of protein function through unnatural amino acid mutagenesis and other optogenetic approaches.

blue red Cryptochromes LOV domains Phytochromes Review
ACS Chem Biol, 21 May 2014 DOI: 10.1021/cb500176x Link to full text
Abstract: Biological processes are naturally regulated with high spatial and temporal resolution at the molecular, cellular, and systems level. To control and study processes with the same resolution, light-sensitive groups and domains have been employed to optically activate and deactivate protein function. Optical control is a noninvasive technique in which the amplitude, wavelength, spatial location, and timing of the light illumination can be easily controlled. This review focuses on applications of genetically encoded unnatural amino acids containing light-removable protecting groups to optically trigger protein function, while also discussing select optogenetic approaches using natural light-sensitive domains to engineer optical control of biological processes.

Optical control of the Ca2+ concentration in a live specimen with a genetically encoded Ca2+-releasing molecular tool.

blue AsLOV2 C. elegans in vivo HeLa in vitro Immediate control of second messengers Neuronal activity control
ACS Chem Biol, 24 Mar 2014 DOI: 10.1021/cb400849n Link to full text
Abstract: Calcium ion (Ca2+) is an important second messenger implicated in the control of many different cellular processes in living organisms. Ca2+ is typically studied by direct visualization using chemically or genetically encoded indicators. A complementary, and perhaps more useful, approach involves direct manipulation of Ca2+ concentration; tools for this exist but are rather poorly developed compared to the indicators at least. Here, we report a photoactivatable Ca2+-releasing protein, photoactivatable Ca2+ releaser (PACR), made by the insertion of a photosensitive protein domain (LOV2) into a Ca2+ binding protein (calmodulin fused with the M13 peptide). As the PACR is genetically encoded, and unlike conventional optical control tools (e.g., channel rhodopsin) not membrane bound, we are able to restrict expression within the cell, to allow subcellular perturbation of Ca2+ levels. In whole animals, we are able to control the behavior of Caenorhabditis elegans with light by expressing the PACR only in the touch neuron.

Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors.

blue LOV domains Background
ACS Synth Biol, 5 Mar 2014 DOI: 10.1021/sb400205x Link to full text
Abstract: As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutations near the flavin chromophore modulate response kinetics and the effective light responsiveness. To probe for potential, inadvertent effects on receptor activity, we introduced these mutations into the engineered LOV photoreceptor YF1 and determined their impact on light regulation. While several mutations severely impaired the dynamic range of the receptor (e.g., I39V, R63K, and N94A), residue substitutions in a second group were benign with little effect on regulation (e.g., V28T, N37C, and L82I). Electron paramagnetic resonance and absorption spectroscopy identified correlated effects for certain of the latter mutations on chromophore environment and response kinetics in YF1 and the LOV2 domain from Avena sativa phototropin 1. Carefully chosen mutations provide a powerful means to adjust the light-response function of photoreceptors as demanded for diverse applications.

Near-infrared light responsive synthetic c-di-GMP module for optogenetic applications.

red BphG BphS E. coli in vitro Immediate control of second messengers
ACS Synth Biol, 28 Jan 2014 DOI: 10.1021/sb400182x Link to full text
Abstract: Enormous potential of cell-based therapeutics is hindered by the lack of effective means to control genetically engineered cells in mammalian tissues. Here, we describe a synthetic module for remote photocontrol of engineered cells that can be adapted for such applications. The module involves photoactivated synthesis of cyclic dimeric GMP (c-di-GMP), a stable small molecule that is not produced by higher eukaryotes and therefore is suitable for orthogonal regulation. The key component of the photocontrol module is an engineered bacteriophytochrome diguanylate cyclase, which synthesizes c-di-GMP from GTP in a light-dependent manner. Bacteriophytochromes are particularly attractive photoreceptors because they respond to light in the near-infrared window of the spectrum, where absorption by mammalian tissues is minimal, and also because their chromophore, biliverdin IXα, is naturally available in mammalian cells. The second component of the photocontrol module, a c-di-GMP phosphodiesterase, maintains near-zero background levels of c-di-GMP in the absence of light, which enhances the photodynamic range of c-di-GMP concentrations. In the E. coli model used in this study, the intracellular c-di-GMP levels could be upregulated by light by >50-fold. Various c-di-GMP-responsive proteins and riboswitches identified in bacteria can be linked downstream of the c-di-GMP-mediated photocontrol module for orthogonal regulation of biological activities in mammals as well as in other organisms lacking c-di-GMP signaling. Here, we linked the photocontrol module to a gene expression output via a c-di-GMP-responsive transcription factor and achieved a 40-fold photoactivation of gene expression.

Genetically engineered photoinducible homodimerization system with improved dimer-forming efficiency.

blue VVD Cos-7 HEK293 Cell death
ACS Chem Biol, 17 Jan 2014 DOI: 10.1021/cb400836k Link to full text
Abstract: Vivid (VVD) is a photoreceptor derived from Neurospora Crassa that rapidly forms a homodimer in response to blue light. Although VVD has several advantages over other photoreceptors as photoinducible homodimerization system, VVD has a critical limitation in its low dimer-forming efficiency. To overcome this limitation of wild-type VVD, here we conduct site-directed saturation mutagenesis in the homodimer interface of VVD. We have found that the Ile52Cys mutation of VVD (VVD-52C) substantially improves its homodimer-forming efficiency up to 180%. We have demonstrated the utility of VVD-52C for making a light-inducible gene expression system more robust. In addition, using VVD-52C, we have developed photoactivatable caspase-9, which enables optical control of apoptosis of mammalian cells. The present genetically engineered photoinducible homodimerization system can provide a powerful tool to optically control a broad range of molecular processes in the cell.

General method for regulating protein stability with light.

blue AsLOV2 NIH/3T3 zebrafish in vivo
ACS Chem Biol, 8 Nov 2013 DOI: 10.1021/cb400755b Link to full text
Abstract: Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.
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