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 55 results
1.

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

blue CRY2/CIB1 LOVTRAP VVD A549 Cos-7 HEK293 HEK293T HeLa mouse in vivo NCI-H1299 PC-3 U-87 MG Transgene expression
Nat Commun, 27 Jan 2021 DOI: 10.1038/s41467-021-20913-1 Link to full text
Abstract: Pulsing cellular dynamics in genetic circuits have been shown to provide critical capabilities to cells in stress response, signaling and development. Despite the fascinating discoveries made in the past few years, the mechanisms and functional capabilities of most pulsing systems remain unclear, and one of the critical challenges is the lack of a technology that allows pulsatile regulation of transgene expression both in vitro and in vivo. Here, we describe the development of a synthetic BRET-based transgene expression (LuminON) system based on a luminescent transcription factor, termed luminGAVPO, by fusing NanoLuc luciferase to the light-switchable transcription factor GAVPO. luminGAVPO allows pulsatile and quantitative activation of transgene expression via both chemogenetic and optogenetic approaches in mammalian cells and mice. Both the pulse amplitude and duration of transgene expression are highly tunable via adjustment of the amount of furimazine. We further demonstrated LuminON-mediated blood-glucose homeostasis in type 1 diabetic mice. We believe that the BRET-based LuminON system with the pulsatile dynamics of transgene expression provides a highly sensitive tool for precise manipulation in biological systems that has strong potential for application in diverse basic biological studies and gene- and cell-based precision therapies in the future.
2.

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

blue violet Cryptochromes Fluorescent proteins LOV domains Review
Adv Biol, 14 Jan 2021 DOI: 10.1002/adbi.202000234 Link to full text
Abstract: To reveal the underpinnings of complex biological systems, a variety of approaches have been developed that allow switchable control of protein function. One powerful approach for switchable control is the use of inducible dimerization systems, which can be configured to control activity of a target protein upon induced dimerization triggered by chemicals or light. Individually, many inducible dimerization systems suffer from pre‐defined dynamic ranges and overwhelming sensitivity to expression level and cellular context. Such systems often require extensive engineering efforts to overcome issues of background leakiness and restricted dynamic range. To address these limitations, recent tool development efforts have explored overlaying dimerizer systems with a second layer of regulation. Albeit more complex, the resulting layered systems have enhanced functionality, such as tighter control that can improve portability of these tools across platforms.
3.

Steering Molecular Activity with Optogenetics: Recent Advances and Perspectives.

blue cyan green near-infrared red UV violet BLUF domains Cobalamin-binding domains Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Adv Biol, 14 Jan 2021 DOI: 10.1002/adbi.202000180 Link to full text
Abstract: Optogenetics utilizes photosensitive proteins to manipulate the localization and interaction of molecules in living cells. Because light can be rapidly switched and conveniently confined to the sub‐micrometer scale, optogenetics allows for controlling cellular events with an unprecedented resolution in time and space. The past decade has witnessed an enormous progress in the field of optogenetics within the biological sciences. The ever‐increasing amount of optogenetic tools, however, can overwhelm the selection of appropriate optogenetic strategies. Considering that each optogenetic tool may have a distinct mode of action, a comparative analysis of the current optogenetic toolbox can promote the further use of optogenetics, especially by researchers new to this field. This review provides such a compilation that highlights the spatiotemporal accuracy of current optogenetic systems. Recent advances of optogenetics in live cells and animal models are summarized, the emerging work that interlinks optogenetics with other research fields is presented, and exciting clinical and industrial efforts to employ optogenetic strategy toward disease intervention are reported.
4.

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.
5.

Synthetic protein condensates that recruit and release protein activity in living cells.

blue LOVTRAP HeLa
bioRxiv, 10 Nov 2020 DOI: 10.1101/2020.11.09.375766 Link to full text
Abstract: Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. We first present chemogenetic protein-recruiting and -releasing condensates, which rapidly inhibited and activated signaling proteins, respectively. An optogenetic condensate system was successfully constructed that enables reversible release and sequestration of protein activity using light. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step towards tailor-made engineering of synthetic protein condensates with various functionalities.
6.

Quantifying signal persistence in the T cell signaling network using an optically controllable antigen receptor.

blue LOVTRAP HEK293T Jurkat Signaling cascade control
bioRxiv, 30 Oct 2020 DOI: 10.1101/2020.10.30.362194 Link to full text
Abstract: T cells discriminate between healthy and infected cells with remarkable sensitivity when mounting an immune response. It has been hypothesized that this efficient detection requires combining signals from discrete antigen-presenting cell interactions into a more potent response, requiring T cells to maintain a ‘memory’ of previous encounters. To quantify the magnitude of this phenomenon, we have developed an antigen receptor that is both optically and chemically tunable, providing control over the initiation, duration, and intensity of intracellular T-cell signaling within physiological cell conjugates. We observe very limited persistence within the T cell intracellular network on disruption of receptor input, with signals dissipating entirely in ~15 minutes, and directly confirm that sustained proximal receptor signaling is required to maintain active gene transcription. Our data suggests that T cells are largely incapable of integrating discrete antigen receptor signals but instead simply accumulate the output of gene expression. By engineering optical control in a clinically relevant chimeric antigen receptor, we show that this limited signal persistence can be exploited to increase the activation of primary T cells by ~3-fold by using pulsatile stimulation. Our results are likely to apply more generally to the signaling dynamics of other cellular networks.
7.

The rise and shine of yeast optogenetics.

blue green near-infrared red UV BLUF domains Cryptochromes Cyanobacteriochromes LOV domains Phytochromes UV receptors Review
Yeast, 29 Oct 2020 DOI: 10.1002/yea.3529 Link to full text
Abstract: Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared to traditional chemically-inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, or protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.
8.

Optogenetic interrogation and control of cell signaling.

blue cyan green near-infrared red Cryptochromes Cyanobacteriochromes Fluorescent proteins LOV domains Phytochromes Review
Curr Opin Biotechnol, 11 Oct 2020 DOI: 10.1016/j.copbio.2020.07.007 Link to full text
Abstract: Signaling networks control the flow of information through biological systems and coordinate the chemical processes that constitute cellular life. Optogenetic actuators - genetically encoded proteins that undergo light-induced changes in activity or conformation - are useful tools for probing signaling networks over time and space. They have permitted detailed dissections of cellular proliferation, differentiation, motility, and death, and enabled the assembly of synthetic systems with applications in areas as diverse as photography, chemical synthesis, and medicine. In this review, we provide a brief introduction to optogenetic systems and describe their application to molecular-level analyses of cell signaling. Our discussion highlights important research achievements and speculates on future opportunities to exploit optogenetic systems in the study and assembly of complex biochemical networks.
9.

Open-Closed Structure of Light Responsive Protein LOV2 Regulates its Molecular Interaction with Binding Partner.

blue LOV domains Background
J Phys Chem Lett, 18 Sep 2020 DOI: 10.1021/acs.jpclett.0c02252 Link to full text
Abstract: Optogenetic approaches have broad applications including regulating cell signalling and gene expression. Photo-responsive protein LOV2 and its binding partner ZDK represent an important protein caging/uncaging optogenetic system. Herein, we combine time-resolved small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) to reveal different structural states of LOV2 and the light-controlled mechanism of interaction between LOV2 and ZDK. In response to blue light within a time frame of ca. 70 s, LOV2 has a significantly higher value of radius of gyration Rg (29.6± 0.3 Å vs 26.4± 0.4 Å) than its dark state, suggesting unwinding of the C-terminal Jα-helix into an open structure. Atomic force microscopy was used to characterise molecular interactions of LOV2 in open and closed states with ZDK at a single molecule level. The closed state of LOV2 enables strong binding with ZDK, characterised by 60-fold lower dissociation rate and ~1.5 times higher activation energy barrier than its open state. In combination, these data support a light-switching mechanism that is modulated by the proximity of multiple binding sites of LOV2 for ZDK.
10.

Optogenetic Control Reveals Differential Promoter Interpretation of Transcription Factor Nuclear Translocation Dynamics.

blue AsLOV2 LOVTRAP S. cerevisiae
Cell Syst, 7 Sep 2020 DOI: 10.1016/j.cels.2020.08.009 Link to full text
Abstract: Gene expression is thought to be affected not only by the concentration of transcription factors (TFs) but also the dynamics of their nuclear translocation. Testing this hypothesis requires direct control of TF dynamics. Here, we engineer CLASP, an optogenetic tool for rapid and tunable translocation of a TF of interest. Using CLASP fused to Crz1, we observe that, for the same integrated concentration of nuclear TF over time, changing input dynamics changes target gene expression: pulsatile inputs yield higher expression than continuous inputs, or vice versa, depending on the target gene. Computational modeling reveals that a dose-response saturating at low TF input can yield higher gene expression for pulsatile versus continuous input, and that multi-state promoter activation can yield the opposite behavior. Our integrated tool development and modeling approach characterize promoter responses to Crz1 nuclear translocation dynamics, extracting quantitative features that may help explain the differential expression of target genes.
11.

Optogenetic Tuning of Protein-protein Binding in Bilayers Using LOVTRAP.

blue LOVTRAP in vitro
Bio Protoc, 5 Sep 2020 DOI: 10.21769/bioprotoc.3745 Link to full text
Abstract: Modern microscopy methods are powerful tools for studying live cell signaling and biochemical reactions, enabling us to observe when and where these reactions take place from the level of a cell down to single molecules. With microscopy, each cell or molecule can be observed both before and after a given perturbation, facilitating better inference of cause and effect than is possible with destructive modes of signaling quantitation. As many inputs to cell signaling and biochemical systems originate as protein-protein interactions near the cell membrane, an outstanding challenge lies in controlling the timing, location and the magnitude of protein-protein interactions in these unique environments. Here, we detail our procedure for manipulating such spatial and temporal protein-protein interactions in a closed microscopy system using a LOVTRAP-based light-responsive protein-protein interaction system on a supported lipid bilayer. The system responds in seconds and can pattern details down to the one micron level. We used this technique to unlock fundamental aspects of T cell signaling, and this approach is generalizable to many other cell signaling and biochemical contexts.
12.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.

blue LOV domains Background
ACS Chem Biol, 3 Sep 2020 DOI: 10.1021/acschembio.0c00543 Link to full text
Abstract: Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light, oxygen, voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output partner. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in loss of the interaction between the side chain of N414 and the backbone C=O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.
13.

Lights up on organelles: Optogenetic tools to control subcellular structure and organization.

blue cyan near-infrared red UV BLUF domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Wiley Interdiscip Rev Syst Biol Med, 26 Jul 2020 DOI: 10.1002/wsbm.1500 Link to full text
Abstract: Since the neurobiological inception of optogenetics, light-controlled molecular perturbations have been applied in many scientific disciplines to both manipulate and observe cellular function. Proteins exhibiting light-sensitive conformational changes provide researchers with avenues for spatiotemporal control over the cellular environment and serve as valuable alternatives to chemically inducible systems. Optogenetic approaches have been developed to target proteins to specific subcellular compartments, allowing for the manipulation of nuclear translocation and plasma membrane morphology. Additionally, these tools have been harnessed for molecular interrogation of organelle function, location, and dynamics. Optogenetic approaches offer novel ways to answer fundamental biological questions and to improve the efficiency of bioengineered cell factories by controlling the assembly of synthetic organelles. This review first provides a summary of available optogenetic systems with an emphasis on their organelle-specific utility. It then explores the strategies employed for organelle targeting and concludes by discussing our perspective on the future of optogenetics to control subcellular structure and organization. This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Physiology > Physiology of Model Organisms Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Cellular Models.
14.

Engineering Optogenetic Protein Analogs.

blue LOV domains Review
Methods Mol Biol, 11 Jul 2020 DOI: 10.1007/978-1-0716-0755-8_7 Link to full text
Abstract: This chapter provides an overview of the technologies we have developed to control proteins with light. First, we focus on the LOV domain, a versatile building block with reversible photo-response, kinetics tunable through mutagenesis, and ready expression in a broad range of cells and animals. Incorporation of LOV into proteins produced a variety of approaches: simple steric block of the active site released when irradiation lengthened a linker (PA-GTPases), reversible release from sequestration at mitochondria (LOVTRAP), and Z-lock, a method in which a light-cleavable bridge is placed where it occludes the active site. The latter two methods make use of Zdk, small engineered proteins that bind selectively to the dark state of LOV. In order to control endogenous proteins, inhibitory peptides are embedded in the LOV domain where they are exposed only upon irradiation (PKA and MLCK inhibition). Similarly, controlled exposure of a nuclear localization sequence and nuclear export sequence is used to reversibly send proteins into the nucleus. Another avenue of engineering makes use of the heterodimerization of FKBP and FRB proteins, induced by the small molecule rapamycin. We control rapamycin with light or simply add it to target cells. Incorporation of fused FKBP-FRB into kinases, guanine exchange factors, or GTPases leads to rapamycin-induced protein activation. Kinases are engineered so that they can interact with only a specific substrate upon activation. Recombination of split proteins using rapamycin-induced conformational changes minimizes spontaneous reassembly. Finally, we explore the insertion of LOV or rapamycin-responsive domains into proteins such that light-induced conformational changes exert allosteric control of the active site. We hope these design ideas will inspire new applications and broaden our reach towards dynamic biological processes that unfold when studied in vivo.
15.

An optogenetic method for interrogating YAP1 and TAZ nuclear-cytoplasmic shuttling.

blue LOVTRAP HaCaT
bioRxiv, 9 Jun 2020 DOI: 10.1101/2020.06.08.140228 Link to full text
Abstract: The shuttling of transcription factors and transcriptional regulators in and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive AsLOV (Avena sativa Light Oxygen Voltage) domain is used to sequester the transcriptional regulators, YAP1 and TAZ/WWTR1, on the surface of mitochondria. Illumination with blue light is used to release fluorophore-tagged YAP1 and TAZ from mitochondria and their entry into the nucleus can be observed. Cessation of blue light illumination leads to re-sequestration on the surface of mitochondria. This method generates three distinct curves that are then used to fit ordinary differential equations for the rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on sites of canonical regulation by LATS1/2 enhances its rate of nuclear export. Moreover, we provide evidences that YAP1 import and export rates, despite high intercellular variability, are correlated within the same cell. Interestingly, the ratio of import and export rates correlated with nuclear-cytoplasmic (NC) distribution of YAP1 and TAZ proteins at steady state.
16.

LITESEC-T3SS - Light-controlled protein delivery into eukaryotic cells with high spatial and temporal resolution.

blue iLID LOVTRAP HEp-2 Y. enterocolitica Cell death
Nat Commun, 13 May 2020 DOI: 10.1038/s41467-020-16169-w Link to full text
Abstract: Many bacteria employ a type III secretion system (T3SS) injectisome to translocate proteins into eukaryotic host cells. Although the T3SS can efficiently export heterologous cargo proteins, a lack of target cell specificity currently limits its application in biotechnology and healthcare. In this study, we exploit the dynamic nature of the T3SS to govern its activity. Using optogenetic interaction switches to control the availability of the dynamic cytosolic T3SS component SctQ, T3SS-dependent effector secretion can be regulated by light. The resulting system, LITESEC-T3SS (Light-induced translocation of effectors through sequestration of endogenous components of the T3SS), allows rapid, specific, and reversible activation or deactivation of the T3SS upon illumination. We demonstrate the light-regulated translocation of heterologous reporter proteins, and induction of apoptosis in cultured eukaryotic cells. LITESEC-T3SS constitutes a new method to control protein secretion and translocation into eukaryotic host cells with unparalleled spatial and temporal resolution.
17.

The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics.

blue LOVTRAP hTERT RPE-1
bioRxiv, 3 May 2020 DOI: 10.1101/2020.05.02.070680 Link to full text
Abstract: Eukaryotic cells typically form a single, round nucleus after mitosis, and failures to do so can compromise genomic integrity. How mammalian cells form such a nucleus remains incompletely understood. NuMA is a spindle protein whose disruption results in nuclear fragmentation. What role NuMA plays in nuclear integrity, or whether its perceived role stems from its spindle function, is unclear. Here, we use live imaging to demonstrate that NuMA plays a spindle-independent role in forming a single, round nucleus. NuMA keeps the decondensing chromosome mass compact at mitotic exit, and promotes a mechanically robust nucleus. NuMA’s C-terminus binds DNA in vitro and chromosomes in interphase, while its coiled-coil acts as a regulatory and structural hub: it prevents NuMA from binding chromosomes at mitosis, regulates its nuclear mobility and is essential for nuclear formation. Thus, NuMA plays a long-range structural role in building and maintaining an intact nucleus, as it does for the spindle, playing a protective role over the cell cycle.
18.

Lights, cytoskeleton, action: Optogenetic control of cell dynamics.

blue cyan red Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Curr Opin Cell Biol, 1 May 2020 DOI: 10.1016/j.ceb.2020.03.003 Link to full text
Abstract: Cell biology is moving from observing molecules to controlling them in real time, a critical step towards a mechanistic understanding of how cells work. Initially developed from light-gated ion channels to control neuron activity, optogenetics now describes any genetically encoded protein system designed to accomplish specific light-mediated tasks. Recent photosensitive switches use many ingenious designs that bring spatial and temporal control within reach for almost any protein or pathway of interest. This next generation optogenetics includes light-controlled protein-protein interactions and shape-shifting photosensors, which in combination with live microscopy enable acute modulation and analysis of dynamic protein functions in living cells. We provide a brief overview of various types of optogenetic switches. We then discuss how diverse approaches have been used to control cytoskeleton dynamics with light through Rho GTPase signaling, microtubule and actin assembly, mitotic spindle positioning and intracellular transport and highlight advantages and limitations of different experimental strategies.
19.

Actin waves transport RanGTP to the neurite tip to regulate non-centrosomal microtubules in neurons.

blue LOVTRAP HeLa primary mouse cortical neurons primary mouse hippocampal neurons Control of cytoskeleton / cell motility / cell shape
J Cell Sci, 6 Apr 2020 DOI: 10.1242/jcs.241992 Link to full text
Abstract: Microtubule (MT) is the most abundant cytoskeleton in neurons and controls multiple facets of their development. While the MT-organizing center (MTOC) in mitotic cells is typically located at the centrosome, MTOC in neurons switches to non-centrosomal sites. A handful of cellular components have been shown to promote non-centrosomal MT (ncMT) formation in neurons, yet the regulation mechanism remains unknown. Here we demonstrate that the small GTPase Ran is a key regulator of ncMTs in neurons. Using an optogenetic tool that enables light-induced local production of RanGTP, we demonstrate that RanGTP promotes ncMT plus-end growth along the neurite. Additionally, we discovered that actin waves drive the anterograde transport of RanGTP. Pharmacological disruption of actin waves abolishes the enrichment of RanGTP and reduces growing ncMT plus-ends at the neurite tip. These observations identify a novel regulation mechanism of ncMTs and pinpoint an indirect connection between the actin and MT cytoskeletons in neurons.
20.

Optogenetics: Rho GTPases Activated by Light in Living Macrophages.

blue LOVTRAP MEF-1 RAW264.7
Methods Mol Biol, 15 Jan 2020 DOI: 10.1007/978-1-0716-0247-8_24 Link to full text
Abstract: Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.
21.

Functional Modulation of Receptor Proteins on Cellular Interface with Optogenetic System.

blue green red UV violet Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Adv Exp Med Biol, 6 Jan 2020 DOI: 10.1007/978-981-15-8763-4_15 Link to full text
Abstract: In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.
22.

Photoreaction Mechanisms of Flavoprotein Photoreceptors and Their Applications.

blue red BLUF domains Cryptochromes LOV domains Phytochromes Review
Adv Exp Med Biol, 6 Jan 2020 DOI: 10.1007/978-981-15-8763-4_11 Link to full text
Abstract: Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.
23.

A Computational Protocol for Regulating Protein Binding Reactions with a Light-Sensitive Protein Dimer.

blue LOVTRAP HEK293
J Mol Biol, 27 Dec 2019 DOI: 10.1016/j.jmb.2019.12.033 Link to full text
Abstract: Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. On activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 on stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.
24.

Optogenetic Control of Microtubule Dynamics.

blue LOVTRAP NCI-H1299
Methods Mol Biol, 27 Dec 2019 DOI: 10.1007/978-1-0716-0219-5_14 Link to full text
Abstract: Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.
25.

Strategies for Engineering and Rewiring Kinase Regulation.

blue cyan red Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Trends Biochem Sci, 19 Dec 2019 DOI: 10.1016/j.tibs.2019.11.005 Link to full text
Abstract: Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.
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