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 26 - 50 of 60 results
26.

Light-based control of metabolic flux through assembly of synthetic organelles.

blue CRY2/CRY2 CRY2olig PixD/PixE S. cerevisiae Organelle manipulation
Nat Chem Biol, 13 May 2019 DOI: 10.1038/s41589-019-0284-8 Link to full text
Abstract: To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
27.

NF-κB signaling dynamics is controlled by a dose-sensing autoregulatory loop.

blue CRY2olig NIH/3T3 Signaling cascade control
Sci Signal, 30 Apr 2019 DOI: 10.1126/scisignal.aau3568 Link to full text
Abstract: Over the last decade, multiple studies have shown that signaling proteins activated in different temporal patterns, such as oscillatory, transient, and sustained, can result in distinct gene expression patterns or cell fates. However, the molecular events that ensure appropriate stimulus- and dose-dependent dynamics are not often understood and are difficult to investigate. Here, we used single-cell analysis to dissect the mechanisms underlying the stimulus- and dose-encoding patterns in the innate immune signaling network. We found that Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling dynamics relied on a dose-dependent, autoinhibitory loop that rendered cells refractory to further stimulation. Using inducible gene expression and optogenetics to perturb the network at different levels, we identified IL-1R-associated kinase 1 (IRAK1) as the dose-sensing node responsible for limiting signal flow during the innate immune response. Although the kinase activity of IRAK1 was not required for signal propagation, it played a critical role in inhibiting the nucleocytoplasmic oscillations of the transcription factor NF-κB. Thus, protein activities that may be "dispensable" from a topological perspective can nevertheless be essential in shaping the dynamic response to the external environment.
28.

RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43.

blue CRY2/CRY2 CRY2olig HEK293 ReNcell VM Organelle manipulation
Neuron, 27 Feb 2019 DOI: 10.1016/j.neuron.2019.01.048 Link to full text
Abstract: TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.
29.

Photodimerization systems for regulating protein-protein interactions with light.

blue cyan near-infrared red UV Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Curr Opin Struct Biol, 25 Feb 2019 DOI: 10.1016/j.sbi.2019.01.021 Link to full text
Abstract: Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.
30.

Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells.

blue CRY2/CIB1 CRY2olig Schneider 2
Biomolecules, 12 Feb 2019 DOI: 10.3390/biom9020061 Link to full text
Abstract: Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells.
31.

A bright future: optogenetics to dissect the spatiotemporal control of cell behavior.

blue cyan BLUF domains Cryptochromes Fluorescent proteins LOV domains Review
Curr Opin Chem Biol, 4 Dec 2018 DOI: 10.1016/j.cbpa.2018.11.010 Link to full text
Abstract: Cells sense, process, and respond to extracellular information using signaling networks: collections of proteins that act as precise biochemical sensors. These protein networks are characterized by both complex temporal organization, such as pulses of signaling activity, and by complex spatial organization, where proteins assemble structures at particular locations and times within the cell. Yet despite their ubiquity, studying these spatial and temporal properties has remained challenging because they emerge from the entire protein network rather than a single node, and cannot be easily tuned by drugs or mutations. These challenges are being met by a new generation of optogenetic tools capable of directly controlling the activity of individual signaling nodes over time and the assembly of protein complexes in space. Here, we outline how these recent innovations are being used in conjunction with engineering-influenced experimental design to address longstanding questions in signaling biology.
32.

Mechanobiology of Protein Droplets: Force Arises from Disorder.

blue Cryptochromes LOV domains Review
Cell, 29 Nov 2018 DOI: 10.1016/j.cell.2018.11.020 Link to full text
Abstract: The use of optogenetic approaches has revealed new roles for intracellular protein condensates described in two papers in this issue of Cell (Bracha et. al., 2018; Shin et al., 2018). These results show that growing condensates are able to exert mechanical forces resulting in chromatin rearrangement, establishing a new role for liquid-liquid phase separation in the mechanobiology of the cell.
33.

Light‐Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.

blue cyan green near-infrared red UV BLUF domains Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Adv Sci, 30 Sep 2018 DOI: 10.1002/advs.201800952 Link to full text
Abstract: The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology‐inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light‐controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non‐neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light‐sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light‐controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.
34.

Increasing spatial resolution of photoregulated GTPases through immobilized peripheral membrane proteins.

blue CRY2olig iLID HEK293T HeLa
Small GTPases, 5 Sep 2018 DOI: 10.1080/21541248.2018.1507411 Link to full text
Abstract: Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible.
35.

Activation of EphB2 Forward Signaling Enhances Memory Consolidation.

blue CRY2olig HEK293 mouse in vivo NIH/3T3 Signaling cascade control
Cell Rep, 15 May 2018 DOI: 10.1016/j.celrep.2018.04.042 Link to full text
Abstract: EphB2 is involved in enhancing synaptic transmission and gene expression. To explore the roles of EphB2 in memory formation and enhancement, we used a photoactivatable EphB2 (optoEphB2) to activate EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning, but not minutes afterward, enhanced long-term, but not short-term, auditory fear conditioning. Photoactivation of optoEphB2 during fear conditioning led to activation of the cAMP/Ca2+ responsive element binding (CREB) protein. Application of light to a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long-term, but not short-term, auditory fear conditioning memory was impaired in mice lacking EphB2 forward signaling (EphB2lacZ/lacZ). Activation of optoEphB2 in LA of EphB2lacZ/lacZ mice enhanced long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.
36.

Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.

blue CRY2/CRY2 CRY2olig HEK293T primary mouse cortical neurons rat cortical neurons Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Neuron, 30 Apr 2018 DOI: 10.1016/j.neuron.2018.04.011 Link to full text
Abstract: Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle.
37.

New approaches for solving old problems in neuronal protein trafficking.

blue red UV BLUF domains Cryptochromes LOV domains Phytochromes UV receptors Review
Mol Cell Neurosci, 10 Apr 2018 DOI: 10.1016/j.mcn.2018.04.004 Link to full text
Abstract: Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.
38.

Analysis of the CaMKIIα and β splice-variant distribution among brain regions reveals isoform-specific differences in holoenzyme formation.

blue CRY2/CIB1 CRY2olig HEK293
Sci Rep, 3 Apr 2018 DOI: 10.1038/s41598-018-23779-4 Link to full text
Abstract: Four CaMKII isoforms are encoded by distinct genes, and alternative splicing within the variable linker-region generates additional diversity. The α and β isoforms are largely brain-specific, where they mediate synaptic functions underlying learning, memory and cognition. Here, we determined the α and β splice-variant distribution among different mouse brain regions. Surprisingly, the nuclear variant αB was detected in all regions, and even dominated in hypothalamus and brain stem. For CaMKIIβ, the full-length variant dominated in most regions (with higher amounts of minor variants again seen in hypothalamus and brain stem). The mammalian but not fish CaMKIIβ gene lacks exon v3Nthat encodes the nuclear localization signal in αB, but contains three exons not found in the CaMKIIα gene (exons v1, v4, v5). While skipping of exons v1 and/or v5 generated the minor splice-variants β', βe and βe', essentially all transcripts contained exon v4. However, we instead detected another minor splice-variant (now termed βH), which lacks part of the hub domain that mediates formation of CaMKII holoenzymes. Surprisingly, in an optogenetic cellular assay of protein interactions, CaMKIIβH was impaired for binding to the β hub domain, but still bound CaMKIIα. This provides the first indication for isoform-specific differences in holoenzyme formation.
39.

Induction of signal transduction using non-channelrhodopsin-type optogenetic tools.

blue cyan near-infrared red UV Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Chembiochem, 25 Mar 2018 DOI: 10.1002/cbic.201700635 Link to full text
Abstract: Signal transductions are the basis for all cellular functions. Previous studies investigating signal transductions mainly relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies do not allow the modulation of protein activity in cells, tissues, and organs in animals with high spatial and temporal precision. Recently, non-channelrhodopsin-type optogenetic tools for regulating signal transduction have emerged. These photoswitches address several disadvantages of previous techniques, and allow us to control a variety of signal transductions such as cell membrane dynamics, calcium signaling, lipid signaling, and apoptosis. In this review, we summarize recent advances in the development of such photoswitches and how these optotools are applied to signaling processes.
40.

Illuminating developmental biology with cellular optogenetics.

blue Cryptochromes LOV domains Review
Curr Opin Biotechnol, 2 Mar 2018 DOI: 10.1016/j.copbio.2018.02.003 Link to full text
Abstract: In developmental biology, localization is everything. The same stimulus-cell signaling event or expression of a gene-can have dramatically different effects depending on the time, spatial position, and cell types in which it is applied. Yet the field has long lacked the ability to deliver localized perturbations with high specificity in vivo. The advent of optogenetic tools, capable of delivering highly localized stimuli, is thus poised to profoundly expand our understanding of development. We describe the current state-of-the-art in cellular optogenetic tools, review the first wave of major studies showcasing their application in vivo, and discuss major obstacles that must be overcome if the promise of developmental optogenetics is to be fully realized.
41.

Optogenetic Reconstitution for Determining the Form and Function of Membraneless Organelles.

blue Cryptochromes LOV domains Review
Biochemistry, 26 Jan 2018 DOI: 10.1021/acs.biochem.7b01173 Link to full text
Abstract: It has recently become clear that large-scale macromolecular self-assembly is a rule, rather than an exception, of intracellular organization. A growing number of proteins and RNAs have been shown to self-assemble into micrometer-scale clusters that exhibit either liquid-like or gel-like properties. Given their proposed roles in intracellular regulation, embryo development, and human disease, it is becoming increasingly important to understand how these membraneless organelles form and to map their functional consequences for the cell. Recently developed optogenetic systems make it possible to acutely control cluster assembly and disassembly in live cells, driving the separation of proteins of interest into liquid droplets, hydrogels, or solid aggregates. Here we propose that these approaches, as well as their evolution into the next generation of optogenetic biophysical tools, will allow biologists to determine how the self-assembly of membraneless organelles modulates diverse biochemical processes.
42.

Optogenetic activation of EphB2 receptor in dendrites induced actin polymerization by activating Arg kinase.

blue CRY2olig 3T3MEF Cos-7 HEK293 rat hippocampal neurons Signaling cascade control Control of cytoskeleton / cell motility / cell shape
Biol Open, 20 Nov 2017 DOI: 10.1242/bio.029900 Link to full text
Abstract: Erythropoietin-producing hepatocellular (Eph) receptors regulate a wide array of developmental processes by responding to cell-cell contacts. EphB2 is well-expressed in brain and known to be important for dendritic spine development, as well as for the maintenance of the synapses, although the mechanisms of these functions have not been fully understood. Here we studied EphB2's functions in hippocampal neurons with an optogenetic approach, which allows us to specify spatial regions of signal activation and monitor in real-time the consequences of signal activation. We designed and constructed OptoEphB2, a genetically encoded photoactivatable EphB2. Photoactivation of OptoEphB2 in fibroblast cells induced receptor phosphorylation and resulted in cell rounding - a well-known cellular response to EphB2 activation. In contrast, local activation of OptoEphb2 in dendrites of hippocampal neurons induces rapid actin polymerization, resulting dynamic dendritic filopodial growth. Inhibition of Rac1 and CDC42 did not abolish OptoEphB2-induced actin polymerization. Instead, we identified Abelson Tyrosine-Protein Kinase 2 (Abl2/Arg) as a necessary effector in OptoEphB2-induced filopodia growth in dendrites. These findings provided new mechanistic insight into EphB2's role in neural development and demonstrated the advantage of OptoEphB as a new tool for studying EphB signaling.
43.

Cell membrane dynamics induction using optogenetic tools.

blue near-infrared red Cryptochromes LOV domains Phytochromes Review
Biochem Biophys Res Commun, 16 Nov 2017 DOI: 10.1016/j.bbrc.2017.11.091 Link to full text
Abstract: Structures arising from actin-based cell membrane movements, including ruffles, lamellipodia, and filopodia, play important roles in a broad spectrum of cellular functions, such as cell motility, axon guidance in neurons, wound healing, and micropinocytosis. Previous studies investigating these cell membrane dynamics often relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies did not allow the modulation of protein activity at specific regions of cells, tissues, and organs in animals with high spatial and temporal precision. Recently, optogenetic tools for inducing cell membrane dynamics have been developed which address several of the disadvantages of previous techniques. In a recent study, we developed a powerful optogenetic tool, called the Magnet system, to change cell membrane dynamics through Tiam1 and PIP3 signal transductions with high spatial and temporal resolution. In this review, we summarize recent advances in optogenetic tools that allow us to induce actin-regulated cell membrane dynamics and unique membrane ruffles that we discovered using our Magnet system.
44.

Understanding CRY2 interactions for optical control of intracellular signaling.

blue CRY2/CIB1 CRY2/CRY2 CRY2olig Cos-7 HEK293T Signaling cascade control
Nat Commun, 15 Sep 2017 DOI: 10.1038/s41467-017-00648-8 Link to full text
Abstract: Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2-CRY2 homo-oligomerization and CRY2-CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2-CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2-CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2-CIB1 and CRY2-CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2-CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.
45.

Applications of optobiology in intact cells and multi-cellular organisms.

blue cyan green near-infrared red Cobalamin-binding domains Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
J Mol Biol, 4 Sep 2017 DOI: 10.1016/j.jmb.2017.08.015 Link to full text
Abstract: Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.
46.

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells.

blue cyan red UV Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Theranostics, 18 Aug 2017 DOI: 10.7150/thno.20593 Link to full text
Abstract: Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells.
47.

Optogenetic protein clustering through fluorescent protein tagging and extension of CRY2.

blue CRY2/CRY2 CRY2clust CRY2olig HeLa Signaling cascade control Immediate control of second messengers
Nat Commun, 23 Jun 2017 DOI: 10.1038/s41467-017-00060-2 Link to full text
Abstract: Protein homo-oligomerization is an important molecular mechanism in many biological processes. Therefore, the ability to control protein homo-oligomerization allows the manipulation and interrogation of numerous cellular events. To achieve this, cryptochrome 2 (CRY2) from Arabidopsis thaliana has been recently utilized for blue light-dependent spatiotemporal control of protein homo-oligomerization. However, limited knowledge on molecular characteristics of CRY2 obscures its widespread applications. Here, we identify important determinants for efficient cryptochrome 2 clustering and introduce a new CRY2 module, named ''CRY2clust'', to induce rapid and efficient homo-oligomerization of target proteins by employing diverse fluorescent proteins and an extremely short peptide. Furthermore, we demonstrate advancement and versatility of CRY2clust by comparing against previously reported optogenetic tools. Our work not only expands the optogenetic clustering toolbox but also provides a guideline for designing CRY2-based new optogenetic modules.Cryptochrome 2 (CRY2) from A. thaliana can be used to control light-dependent protein homo-oligomerization, but the molecular mechanism of CRY2 clustering is not known, limiting its application. Here the authors identify determinants of CRY2 clustering and engineer fusion partners to modulate clustering efficiency.
48.

At Light Speed: Advances in Optogenetic Systems for Regulating Cell Signaling and Behavior.

blue near-infrared red UV Cryptochromes LOV domains Phytochromes UV receptors Review
Annu Rev Chem Biomol Eng, 7 Jun 2017 DOI: 10.1146/annurev-chembioeng-060816-101254 Link to full text
Abstract: Cells are bombarded by extrinsic signals that dynamically change in time and space. Such dynamic variations can exert profound effects on behaviors, including cellular signaling, organismal development, stem cell differentiation, normal tissue function, and disease processes such as cancer. Although classical genetic tools are well suited to introduce binary perturbations, new approaches have been necessary to investigate how dynamic signal variation may regulate cell behavior. This fundamental question is increasingly being addressed with optogenetics, a field focused on engineering and harnessing light-sensitive proteins to interface with cellular signaling pathways. Channelrhodopsins initially defined optogenetics; however, through recent use of light-responsive proteins with myriad spectral and functional properties, practical applications of optogenetics currently encompass cell signaling, subcellular localization, and gene regulation. Now, important questions regarding signal integration within branch points of signaling networks, asymmetric cell responses to spatially restricted signals, and effects of signal dosage versus duration can be addressed. This review summarizes emerging technologies and applications within the expanding field of optogenetics.
49.

Engineering genetically-encoded tools for optogenetic control of protein activity.

blue near-infrared red Cryptochromes LOV domains Phytochromes Review
Curr Opin Chem Biol, 17 May 2017 DOI: 10.1016/j.cbpa.2017.05.001 Link to full text
Abstract: Optogenetic tools offer fast and reversible control of protein activity with subcellular spatial precision. In the past few years, remarkable progress has been made in engineering photoactivatable systems regulating the activity of cellular proteins. In this review, we discuss general strategies in designing and optimizing such optogenetic tools and highlight recent advances in the field, with specific focus on applications regulating protein catalytic activity.
50.

The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology.

blue cyan red Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
F1000Res, 11 Apr 2017 DOI: 10.12688/f1000research.10617.1 Link to full text
Abstract: The zebrafish ( Danio rerio) is a powerful vertebrate model to study cellular and developmental processes in vivo. The optical clarity and their amenability to genetic manipulation make zebrafish a model of choice when it comes to applying optical techniques involving genetically encoded photoresponsive protein technologies. In recent years, a number of fluorescent protein and optogenetic technologies have emerged that allow new ways to visualize, quantify, and perturb developmental dynamics. Here, we explain the principles of these new tools and describe some of their representative applications in zebrafish.
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