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 138 results
1.

Biophysical and biochemical properties of Deup1 self-assemblies: a potential driver for deuterosome formation during multiciliogenesis.

blue CRY2clust HeLa Organelle manipulation
Biol Open, 3 Mar 2021 DOI: 10.1242/bio.056432 Link to full text
Abstract: The deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.
2.

Optogenetic Tools for Manipulating Protein Subcellular Localization and Intracellular Signaling at Organelle Contact Sites.

blue Magnets Cos-7 HeLa U-2 OS
Curr Protoc, 3 Mar 2021 DOI: 10.1002/cpz1.71 Link to full text
Abstract: Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein-protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light-activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light-tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Genetic engineering strategy for the generation of modular light-activated protein dimerization units Support Protocol 1: Molecular cloning Basic Protocol 2: Cell culture and transfection Support Protocol 2: Production of dark containers for optogenetic samples Basic Protocol 3: Confocal microscopy and light-dependent activation of the dimerization system Alternate Protocol 1: Protein recruitment to intracellular compartments Alternate Protocol 2: Induction of organelles' membrane tethering Alternate Protocol 3: Optogenetic reconstitution of protein function Basic Protocol 4: Image analysis Support Protocol 3: Analysis of apparent on- and off-kinetics Support Protocol 4: Analysis of changes in organelle overlap over time.
3.

Optogenetic Control of Myocardin‐Related Transcription Factor A Subcellular Localization and Transcriptional Activity Steers Membrane Blebbing and Invasive Cancer Cell Motility.

blue AsLOV2 HEK293 HeLa NIH/3T3 Endogenous gene expression
Adv Biol, 8 Feb 2021 DOI: 10.1002/adbi.202000208 Link to full text
Abstract: The myocardin‐related transcription factor A (MRTF‐A) controls the transcriptional activity of the serum response factor (SRF) in a tightly controlled actin‐dependent manner. In turn, MRTF‐A is crucial for many actin‐dependent processes including adhesion, migration, and contractility and has emerged as novel targets for anti‐tumor strategies. MRTF‐A rapidly shuttles between cytoplasmic and nuclear compartment via dynamic actin interactions within its N‐terminal RPEL domain. Here, optogenetics is used to spatiotemporally control MRTF‐A nuclear localization by blue light using the light‐oxygen‐voltage‐sensing domain 2‐domain based system LEXY (light‐inducible nuclear export system). It is found that light‐regulated nuclear export of MRTF‐A occurs within 10–20 min. Importantly, MRTF‐A‐LEXY shuttling is independent of perturbations of actin dynamics. Furthermore, light‐regulation of MRTF‐A‐LEXY is reversible and repeatable for several cycles of illumination and its subcellular localization correlates with SRF transcriptional activity. As a consequence, optogenetic control of MRTF‐A subcellular localization determines subsequent cytoskeletal dynamics such as non‐apoptotic plasma membrane blebbing as well as invasive tumor‐cell migration through 3D collagen matrix. This data demonstrate robust optogenetic regulation of MRTF as a powerful tool to control SRF‐dependent transcription as well as cell motile behavior.
4.

Transcription activation is enhanced by multivalent interactions independent of liquid-liquid phase separation.

blue CRY2/CIB1 HeLa U-2 OS Transgene expression
bioRxiv, 28 Jan 2021 DOI: 10.1101/2021.01.27.428421 Link to full text
Abstract: Transcription factors (TFs) consist of a DNA binding and an activation domain (AD) that are considered to be independent and exchangeable modules. However, recent studies conclude that also the physico-chemical properties of the AD can control TF assembly at chromatin via driving a phase separation into “transcriptional condensates”. Here, we dissected the mechanism of transcription activation at a reporter gene array with real-time single-cell fluorescence microscopy readouts. Our comparison of different synthetic TFs reveals that the phase separation propensity of the AD correlates with high transcription activation capacity by increasing binding site occupancy, residence time and the recruitment of co-activators. However, we find that the actual formation of phase separated TF liquid-like droplets has a neutral or inhibitory effect on transcription induction. Thus, our study suggests that the ability of a TF to phase separate reflects the functionally important property of the AD to establish multivalent interactions but does not by itself enhance transcription.
5.

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.
6.

Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.

blue iLID HeLa U-2 OS Control of cytoskeleton / cell motility / cell shape
Elife, 22 Jan 2021 DOI: 10.7554/elife.61170 Link to full text
Abstract: During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.
7.

Optogenetic control of small GTPases reveals RhoA mediates intracellular calcium signaling.

blue CRY2/CIB1 iLID HEK293T HeLa hTERT RPE-1 MDCK Signaling cascade control
J Biol Chem, 13 Jan 2021 DOI: 10.1016/j.jbc.2021.100290 Link to full text
Abstract: Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.
8.

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.
9.

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

blue red CRY2/CIB1 Magnets PhyB/PIF3 VVD HEK293T HeLa HEp-2 mouse in vivo SH-SY5Y Nucleic acid editing
Proc Natl Acad Sci U S A, 14 Dec 2020 DOI: 10.1073/pnas.2003991117 Link to full text
Abstract: Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.
10.

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

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

Improved Photocleavable Proteins with Faster and More Efficient Dissociation.

violet PhoCl HeLa Transgene expression Cell death
bioRxiv, 10 Dec 2020 DOI: 10.1101/2020.12.10.419556 Link to full text
Abstract: The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain cleavage followed by spontaneous dissociation of the resulting fragments. The first generation PhoCl (PhoCl1) exhibited a relative slow rate of dissociation, potentially limiting its utilities for optogenetic control of cell physiology. In this work, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and expressed in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein-protein interactions in living cells.
12.

Design of smart antibody mimetics with photosensitive switches.

blue AsLOV2 HEK293T HeLa Transgene expression Nucleic acid editing
bioRxiv, 4 Dec 2020 DOI: 10.1101/2020.12.03.410936 Link to full text
Abstract: As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. We introduce herein a generally-applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By paralleled insertion of two LOV2 modules into a single sunbody and the use of bivalent sunbodies, we substantially enhance the range of dynamic changes of photo-switchable sunbodies. Furthermore, we demonstrate the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light.
13.

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

red DrBphP HEK293T HeLa mouse in vivo S. cerevisiae
ACS Synth Biol, 12 Nov 2020 DOI: 10.1021/acssynbio.0c00397 Link to full text
Abstract: Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.
14.

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

blue Magnets Cos-7 HeLa Organelle manipulation
Elife, 11 Nov 2020 DOI: 10.7554/elife.63230 Link to full text
Abstract: Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28oC to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.
15.

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.
16.

Improvement of Phycocyanobilin Synthesis for Genetically Encoded Phytochrome-Based Optogenetics.

red PhyB/PIF3 HeLa mESCs Xenopus in vivo
ACS Chem Biol, 9 Nov 2020 DOI: 10.1021/acschembio.0c00477 Link to full text
Abstract: Optogenetics is a powerful technique using photoresponsive proteins, and the light-inducible dimerization (LID) system, an optogenetic tool, allows to manipulate intracellular signaling pathways. One of the red/far-red responsive LID systems, phytochrome B (PhyB)-phytochrome interacting factor (PIF), has a unique property of controlling both association and dissociation by light on the second time scale, but PhyB requires a linear tetrapyrrole chromophore such as phycocyanobilin (PCB), and such chromophores are present only in higher plants and cyanobacteria. Here, we report that we further improved our previously developed PCB synthesis system (SynPCB) and successfully established a stable cell line containing a genetically encoded PhyB-PIF LID system. First, four genes responsible for PCB synthesis, namely, PcyA, HO1, Fd, and Fnr, were replaced with their counterparts derived from thermophilic cyanobacteria. Second, Fnr was truncated, followed by fusion with Fd to generate a chimeric protein, tFnr-Fd. Third, these genes were concatenated with P2A peptide cDNAs for polycistronic expression, resulting in an approximately 4-fold increase in PCB synthesis compared with the previous version. Finally, we incorporated the PhyB, PIF, and SynPCB system into drug inducible lentiviral and transposon vectors, which enabled us to induce PCB synthesis and the PhyB-PIF LID system by doxycycline treatment. These tools provide a new opportunity to advance our understanding of the causal relationship between intracellular signaling and cellular functions.
17.

Light-inducible Deformation of Mitochondria in Live Cells.

blue CRY2/CIB1 iLID 3T3-L1 Cos-7 HeLa U-2 OS Organelle manipulation
bioRxiv, 2 Nov 2020 DOI: 10.1101/2020.11.01.363663 Link to full text
Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the correlation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with other optical dimerizers and molecular motors. This method presents a novel mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.
18.

Light-Regulated allosteric switch enables temporal and subcellular control of enzyme activity.

blue VVD HEK293T HeLa Signaling cascade control
Elife, 23 Sep 2020 DOI: 10.7554/elife.60647 Link to full text
Abstract: Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.
19.

Control of Cell Migration Using Optogenetics.

blue CRY2/CIB1 HeLa
Methods Mol Biol, 17 Sep 2020 DOI: 10.1007/978-1-0716-0779-4_29 Link to full text
Abstract: Optogenetics uses light to manipulate protein localization or activity from subcellular to supra-cellular level with unprecedented spatiotemporal resolution. We used it to control the activity of the Cdc42 Rho GTPase, a major regulator of actin polymerization and cell polarity. In this chapter, we describe how to trigger and guide cell migration using optogenetics as a way to mimic EMT in an artificial yet highly controllable fashion.
20.

Spatiotemporal regulation of ubiquitin-mediated protein degradation via upconversion optogenetic nanosystem.

blue VVD HEK293T HeLa MARC145 mouse in vivo
Nano Res, 14 Aug 2020 DOI: 10.1007/s12274-020-2998-z Link to full text
Abstract: Protein degradation technology, which is one of the most direct and effective ways to regulate the life activities of cells, is expected to be applied to the treatment of various diseases. However, current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation, making them difficult to transform into clinical applications. In this article, an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation. This system worked via two interconnected parts: 1) the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure; 2) the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation. Experimental results based on model protein (Green Fluorescent Protein, GFP) validated that this system could fulfill protein degradation both in vitro (both Hela and 293T cells) and in vivo (by upconversion optogenetic nanosystem), and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time (0–25 h) and the illumination frequency (the illuminating frequency of 0–30 s every 1 min). We further took another functional protein (The Nonstructural Protein 9, NSP9) into experiment. Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV) was inhibited by degrading the NSP9 in this light-induced system, and PRRSV proliferation was affected by different light conditions (illumination time varies from 0–24 h). We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.
21.

Biliverdin reductase-A deficiency brighten and sensitize biliverdin-binding chromoproteins.

near-infrared BphP1/PpsR2 HeLa
Cell Struct Funct, 25 Jun 2020 DOI: 10.1247/csf.20010 Link to full text
Abstract: Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools. Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.
22.

Photoactivatable RNA N6 -Methyladenosine Editing with CRISPR-Cas13.

blue CRY2/CIB1 HEK293T HeLa primary mouse BMSCs Epigenetic modification
Small, 25 Jun 2020 DOI: 10.1002/smll.201907301 Link to full text
Abstract: RNA has important and diverse biological roles, but the molecular methods to manipulate it spatiotemporally are limited. Here, an engineered photoactivatable RNA N6 -methyladenosine (m6 A) editing system with CRISPR-Cas13 is designed to direct specific m6 A editing. Light-inducible heterodimerizing proteins CIBN and CRY2PHR are fused to catalytically inactive PguCas13 (dCas13) and m6 A effectors, respectively. This system, referred to as PAMEC, enables the spatiotemporal control of m6 A editing in response to blue light. Further optimization of this system to create a highly efficient version, known as PAMECR , allows the manipulation of multiple genes robustly and simultaneously. When coupled with an upconversion nanoparticle film, the optogenetic operation window is extended from the visible range to tissue-penetrable near-infrared wavelengths, which offers an appealing avenue to remotely control RNA editing. These results show that PAMEC is a promising optogenetic platform for flexible and efficient targeting of RNA, with broad applicability for epitranscriptome engineering, imaging, and future therapeutic development.
23.

CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells.

blue CRY2/CIB1 HeLa Control of cytoskeleton / cell motility / cell shape
J Biol Chem, 18 May 2020 DOI: 10.1074/jbc.ra119.012427 Link to full text
Abstract: The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species (ROS), and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as amyloid β (Aβ) fibrils and Tau tangles in Alzheimer's disease (AD) are accessible only via invasive cerebrospinal fluid assays, and ROS can be fleeting and challenging to monitor in vivo. Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early-stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the AD brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the "CofActor," we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied, depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early-stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.
24.

CLIC4 is a cytokinetic cleavage furrow protein that regulates cortical cytoskeleton stability during cell division.

blue CRY2/CIB1 CRY2/CRY2 HeLa Control of cytoskeleton / cell motility / cell shape
J Cell Sci, 14 May 2020 DOI: 10.1242/jcs.241117 Link to full text
Abstract: During mitotic cell division, the actomyosin cytoskeleton undergoes several dynamic changes that play key roles in progression through mitosis. Although the regulators of cytokinetic ring formation and contraction are well established, proteins that regulate cortical stability during anaphase and telophase have been understudied. Here, we describe a role for CLIC4 in regulating actin and actin regulators at the cortex and cytokinetic cleavage furrow during cytokinesis. We first describe CLIC4 as a new component of the cytokinetic cleavage furrow that is required for successful completion of mitotic cell division. We also demonstrate that CLIC4 regulates the remodeling of the sub-plasma-membrane actomyosin network within the furrow by recruiting MST4 kinase (also known as STK26) and regulating ezrin phosphorylation. This work identifies and characterizes new molecular players involved in regulating cortex stiffness and blebbing during the late stages of cytokinetic furrowing.
25.

Dynamic Fas signaling network regulates neural stem cell proliferation and memory enhancement.

blue CRY2/CRY2 HeLa mouse in vivo primary rat hippocampal neurons rat astrocytes rat oligodendrocyte Signaling cascade control
Sci Adv, 22 Apr 2020 DOI: 10.1126/sciadv.aaz9691 Link to full text
Abstract: Activation of Fas (CD95) is observed in various neurological disorders and can lead to both apoptosis and prosurvival outputs, yet how Fas signaling operates dynamically in the hippocampus is poorly understood. The optogenetic dissection of a signaling network can yield molecular-level explanations for cellular responses or fates, including the signaling dysfunctions seen in numerous diseases. Here, we developed an optogenetically activatable Fas that works in a physiologically plausible manner. Fas activation in immature neurons of the dentate gyrus triggered mammalian target of rapamycin (mTOR) activation and subsequent brain-derived neurotrophic factor secretion. Phosphorylation of extracellular signal-regulated kinase (Erk) in neural stem cells was induced under prolonged Fas activation. Repetitive activation of this signaling network yielded proliferation of neural stem cells and a transient increase in spatial working memory in mice. Our results demonstrate a novel Fas signaling network in the dentate gyrus and illuminate its consequences for adult neurogenesis and memory enhancement.
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