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 25 results
1.

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.
2.

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.
3.

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.
4.

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

blue red CRY2/CIB1 CRY2/CRY2 PhyB/PIF6 HEK293 mouse in vivo U-2 OS Transgene expression
Sci Adv, 1 Jan 2021 DOI: 10.1126/sciadv.abd3568 Link to full text
Abstract: Light-inducible gene switches represent a key strategy for the precise manipulation of cellular events in fundamental and applied research. However, the performance of widely used gene switches is limited due to low tissue penetrance and possible phototoxicity of the light stimulus. To overcome these limitations, we engineer optogenetic synthetic transcription factors to undergo liquid-liquid phase separation in close spatial proximity to promoters. Phase separation of constitutive and optogenetic synthetic transcription factors was achieved by incorporation of intrinsically disordered regions. Supported by a quantitative mathematical model, we demonstrate that engineered transcription factor droplets form at target promoters and increase gene expression up to fivefold. This increase in performance was observed in multiple mammalian cells lines as well as in mice following in situ transfection. The results of this work suggest that the introduction of intrinsically disordered domains is a simple yet effective means to boost synthetic transcription factor activity.
5.

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

blue iLID U-2 OS Organelle manipulation
bioRxiv, 24 Dec 2020 DOI: 10.1101/2020.12.24.424222 Link to full text
Abstract: Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically-generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that crosslinked chromatin can mechanically suppress droplets’ coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.
6.

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.
7.

Livecell reporters reveal bidirectional acceleration of nucleocytoplasmic transport by O-GlcNAc modification of the nuclear pore complex.

blue AsLOV2 U-2 OS
bioRxiv, 10 Oct 2020 DOI: 10.1101/2020.10.09.334029 Link to full text
Abstract: Macromolecular transportacross the nuclear envelope is fundamental to eukaryotic cells and depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains apermeability barriermade of phenylalanine-glycine (FG) repeat domainsthat selectively facilitatesthe permeation ofcargoes bound to nuclear transport receptors (NTRs)1,2.The NPC is enriched in O-linked N-acetylglucosamine (O-GlcNAc) modification3-8, but itsfunctional rolein nucleocytoplasmic transport isunclear. We developed high-throughput assaysbased on optogenetic probes to quantify the kinetics of nuclear import and exportin living human cells and showedthat the O-GlcNAc modification of the NPC accelerates the nucleocytoplasmic transport in both directions.Super-resolution imaging of O-GlcNAc revealed strong enrichmentat the FG barrier ofthe NPC channel. O-GlcNAcmodificationalso promoted the passive permeation of a small,inert protein through NPCs.Our results suggest that O-GlcNAc modification acceleratesnucleocytoplasmic transport by enhancingthe non-specific permeabilitythe FG-repeat barrier.
8.

Light-Inducible CRISPR Labeling.

blue AsLOV2 U-2 OS
Methods Mol Biol, 11 Jul 2020 DOI: 10.1007/978-1-0716-0755-8_9 Link to full text
Abstract: CRISPR labeling is a powerful technique to study the chromatin architecture in live cells. In CRISPR labeling, a catalytically dead CRISPR-Cas9 mutant is employed as programmable DNA-binding domain to recruit fluorescent proteins to selected genomic loci. The fluorescently labeled loci can then be identified as fluorescent spots and tracked over time by microscopy. A limitation of this approach is the lack of temporal control of the labeling process itself: Cas9 binds to the g(uide)RNA-complementary target loci as soon as it is expressed. The decoration of the genome with Cas9 molecules will, however, interfere with gene regulation and-possibly-affect the genome architecture itself. The ability to switch on and off Cas9 DNA binding in CRISPR labeling experiments would thus be important to enable more precise interrogations of the chromatin spatial organization and dynamics and could further be used to study Cas9 DNA binding kinetics directly in living human cells.Here, we describe a detailed protocol for light-inducible CRISPR labeling. Our method employs CASANOVA, an engineered, optogenetic anti-CRISPR protein, which efficiently traps the Streptococcus pyogenes (Spy)Cas9 in the dark, but permits Cas9 DNA targeting upon illumination with blue light. Using telomeres as exemplary target loci, we detail the experimental steps required for inducible CRISPR labeling with CASANOVA. We also provide instructions on how to analyze the resulting microscopy data in a fully automated fashion.
9.

Nuclear actin regulates inducible transcription by enhancing RNA polymerase II clustering.

blue CRY2olig U-2 OS Organelle manipulation
Sci Adv, 15 Apr 2020 DOI: 10.1126/sciadv.aay6515 Link to full text
Abstract: Gene expression in response to external stimuli underlies a variety of fundamental cellular processes. However, how the transcription machinery is regulated under these scenarios is largely unknown. Here, we discover a novel role of nuclear actin in inducible transcriptional regulation using next-generation transcriptome sequencing and super-resolution microscopy. The RNA-seq data reveal that nuclear actin is required for the establishment of the serum-induced transcriptional program. Using super-resolution imaging, we found a remarkable enhancement of RNA polymerase II (Pol II) clustering upon serum stimulation and this enhancement requires the presence of nuclear actin. To study the molecular mechanisms, we firstly observed that Pol II clusters co-localized with the serum-response genes and nuclear actin polymerized in adjacent to Pol II clusters upon serum stimulation. Furthermore, N-WASP and Arp2/3 are reported to interact with Pol II, and we demonstrated N-WASP is required for serum-enhanced Pol II clustering. Importantly, using an optogenetic tool, we revealed that N-WASP phase-separated with the carboxy-terminal domain of Pol II and nuclear actin. In addition to serum stimulation, we found nuclear actin also essential in enhancing Pol II clustering upon interferon-γ treatment. Taken together, our work unveils nuclear actin promotes the formation of transcription factory on inducible genes, acting as a general mechanism underlying the rapid response to environmental cues.
10.

An optimized toolbox for the optogenetic control of intracellular transport.

blue iLID VVD Cos-7 HeLa U-2 OS Control of cytoskeleton / cell motility / cell shape
J Cell Biol, 6 Apr 2020 DOI: 10.1083/jcb.201907149 Link to full text
Abstract: Cellular functioning relies on active transport of organelles by molecular motors. To explore how intracellular organelle distributions affect cellular functions, several optogenetic approaches enable organelle repositioning through light-inducible recruitment of motors to specific organelles. Nonetheless, robust application of these methods in cellular populations without side effects has remained challenging. Here, we introduce an improved toolbox for optogenetic control of intracellular transport that optimizes cellular responsiveness and limits adverse effects. To improve dynamic range, we employed improved optogenetic heterodimerization modules and engineered a photosensitive kinesin-3, which is activated upon blue light-sensitive homodimerization. This opto-kinesin prevented motor activation before experimental onset, limited dark-state activation, and improved responsiveness. In addition, we adopted moss kinesin-14 for efficient retrograde transport with minimal adverse effects on endogenous transport. Using this optimized toolbox, we demonstrate robust reversible repositioning of (endogenously tagged) organelles within cellular populations. More robust control over organelle motility will aid in dissecting spatial cell biology and transport-related diseases.
11.

ActuAtor, a molecular tool for generating force in living cells: Controlled deformation of intracellular structures.

blue iLID U-2 OS Control of cytoskeleton / cell motility / cell shape
bioRxiv, 31 Mar 2020 DOI: 10.1101/2020.03.30.016360 Link to full text
Abstract: Mechanical force underlies fundamental cell functions such as division, migration and differentiation. While physical probes and devices revealed cellular mechano-responses, how force is translated inside cells to exert output functions remains largely unknown, due to the limited techniques to manipulate force intracellularly. By engineering an ActA protein, an actin nucleation promoting factor derived from Listeria monocytogenes, and implementing this in protein dimerization paradigms, we developed a molecular tool termed ActuAtor, with which actin polymerization can be triggered at intended subcellular locations to generate constrictive force in a rapidly inducible manner. The ActuAtor operation led to striking deformation of target intracellular structures including mitochondria, Golgi apparatus, nucleus, and non-membrane-bound RNA granules. Based on functional analysis before and after organelle deformation, we found the form-function relationship of mitochondria to be generally marginal. The modular design and genetically-encoded nature enable wide applications of ActuAtor for studies of intracellular mechanobiology processes.
12.

Light-inducible generation of membrane curvature in live cells with engineered BAR domain proteins.

blue cyan iLID pdDronpa1 Cos-7 U-2 OS Organelle manipulation
ACS Synth Biol, 26 Mar 2020 DOI: 10.1021/acssynbio.9b00516 Link to full text
Abstract: Nanoscale membrane curvature is now understood to play an active role in essential cellular processes such as endocytosis, exocytosis and actin dynamics. Previous studies have shown that membrane curvature can directly affect protein function and intracellular signaling. However, few methods are able to precisely manipulate membrane curvature in live cells. Here, we report the development of a new method of generating nanoscale membrane curvature in live cells that is controllable, reversible, and capable of precise spatial and temporal manipulation. For this purpose, we make use of BAR domain proteins, a family of well-studied membrane-remodeling and membrane-sculpting proteins. Specifically, we engineered two optogenetic systems, opto-FBAR and opto-IBAR, that allow light-inducible formation of positive and negative membrane curvature, respectively. Using opto-FBAR, blue light activation results in the formation of tubular membrane invaginations (positive curvature), controllable down to the subcellular level. Using opto-IBAR, blue light illumination results in the formation of membrane protrusions or filopodia (negative curvature). These systems present a novel approach for light-inducible manipulation of nanoscale membrane curvature in live cells.
13.

Optogenetic regulation of endogenous proteins.

blue near-infrared AsLOV2 BphP1/Q-PAS1 HeLa U-2 OS Signaling cascade control Control of cytoskeleton / cell motility / cell shape Multichromatic
Nat Commun, 30 Jan 2020 DOI: 10.1038/s41467-020-14460-4 Link to full text
Abstract: Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.
14.

Light-Induced Transcription Activation for Time-Lapse Microscopy Experiments in Living Cells.

blue CRY2/CIB1 U-2 OS
Methods Mol Biol, 13 Aug 2019 DOI: 10.1007/978-1-4939-9674-2_17 Link to full text
Abstract: Gene expression can be monitored in living cells via the binding of fluorescently tagged proteins to RNA repeats engineered into a reporter transcript. This approach makes it possible to trace temporal changes of RNA production in real time in living cells to dissect transcription regulation. For a mechanistic analysis of the underlying activation process, it is essential to induce gene expression with high accuracy. Here, we describe how this can be accomplished with an optogenetic approach termed blue light-induced chromatin recruitment (BLInCR). It employs the recruitment of an activator protein to a target promoter via the interaction between the PHR and CIBN plant protein domains. This process occurs within seconds after setting the light trigger and is reversible. Protocols for continuous activation as well as pulsed activation and reactivation with imaging either by laser scanning confocal microscopy or automated widefield microscopy are provided. For the semiautomated quantification of the resulting image series, an approach has been implemented in a set of scripts in the R programming language. Thus, the complete workflow of the BLInCR method is described for mechanistic studies of the transcription activation process as well as the persistence and memory of the activated state.
15.

m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins.

blue CRY2olig U-2 OS
bioRxiv, 7 Jul 2019 DOI: 10.1101/694455 Link to full text
Abstract: Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.
16.

Phase separation of 53BP1 determines liquid-like behavior of DNA repair compartments.

blue CRY2olig U-2 OS Organelle manipulation
EMBO J, 1 Jul 2019 DOI: 10.15252/embj.2018101379 Link to full text
Abstract: The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9-mediated endogenous protein tagging, that 53BP1-marked repair compartments are dynamic, show droplet-like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light-induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1-dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53-dependent gene activation and cell fate decisions.
17.

Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology.

blue CRY2/CRY2 human IPSCs U-2 OS Organelle manipulation
Elife, 20 Mar 2019 DOI: 10.7554/elife.39578 Link to full text
Abstract: Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.
18.

Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.

blue CRY2/CRY2 iLID HEK293 HEK293T NIH/3T3 U-2 OS Organelle manipulation
Cell, 29 Nov 2018 DOI: 10.1016/j.cell.2018.10.057 Link to full text
Abstract: Phase transitions involving biomolecular liquids are a fundamental mechanism underlying intracellular organization. In the cell nucleus, liquid-liquid phase separation of intrinsically disordered proteins (IDPs) is implicated in assembly of the nucleolus, as well as transcriptional clusters, and other nuclear bodies. However, it remains unclear whether and how physical forces associated with nucleation, growth, and wetting of liquid condensates can directly restructure chromatin. Here, we use CasDrop, a novel CRISPR-Cas9-based optogenetic technology, to show that various IDPs phase separate into liquid condensates that mechanically exclude chromatin as they grow and preferentially form in low-density, largely euchromatic regions. A minimal physical model explains how this stiffness sensitivity arises from lower mechanical energy associated with deforming softer genomic regions. Targeted genomic loci can nonetheless be mechanically pulled together through surface tension-driven coalescence. Nuclear condensates may thus function as mechanoactive chromatin filters, physically pulling in targeted genomic loci while pushing out non-targeted regions of the neighboring genome.
19.

Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

blue iLID C. elegans in vivo HEK293 HeLa NIH/3T3 S. cerevisiae U-2 OS Organelle manipulation
Cell, 29 Nov 2018 DOI: 10.1016/j.cell.2018.10.048 Link to full text
Abstract: Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, ‘‘Corelets,’’ and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.
20.

Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.

blue AsLOV2 HEK293T U-2 OS Epigenetic modification Endogenous gene expression Nucleic acid editing
Nat Methods, 30 Oct 2018 DOI: 10.1038/s41592-018-0178-9 Link to full text
Abstract: Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.
21.

Optogenetic reversible knocksideways, laser ablation, and photoactivation on the mitotic spindle in human cells.

blue iLID U-2 OS
Methods Cell Biol, 26 Apr 2018 DOI: 10.1016/bs.mcb.2018.03.024 Link to full text
Abstract: At the onset of mitosis, cells assemble the mitotic spindle, a dynamic micromachine made of microtubules and associated proteins. Although most of these proteins have been identified, it is still unknown how their collective behavior drives spindle formation and function. Over the last decade, RNA interference has been the main tool for revealing the role of spindle proteins. However, the effects of this method are evident only after a longer time period, leading to difficulties in the interpretation of phenotypes. Optogenetics is a novel technology that enables fast, reversible, and precise control of protein activity by utilization of light. In this chapter, we present an optogenetic knocksideways method for rapid and reversible translocation of proteins from the mitotic spindle to mitochondria using blue light. Furthermore, we discuss other optical approaches, such as laser ablation of microtubule bundles in the spindle and creation of reference marks on the bundles by photoactivation of photoactivatable GFP. Finally, we show how different optical perturbations can be combined in order to acquire deeper understanding of the mechanics of mitosis.
22.

Near-infrared light-controlled gene expression and protein targeting in neurons and non-neuronal cells.

blue near-infrared AsLOV2 BphP1/Q-PAS1 Cos-7 HEK293 HeLa Neuro-2a rat cortical neurons SH-SY5Y U-2 OS Multichromatic
Chembiochem, 21 Feb 2018 DOI: 10.1002/cbic.201700642 Link to full text
Abstract: Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner QPAS1 is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested functionality of two BphP1-QPAS1-based optogenetic tools, such as an NIR and blue light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA), in several cell types including cortical neurons. We found that the performance of these optogenetic tools often rely on physiological properties of a specific cell type, such as nuclear transport, which may limit applicability of blue light-sensitive component of iRIS. In contrast, the NIR-light-sensing part of iRIS performed well in all tested cell types. The TA system showed the best performance in HeLa, U-2 OS and HEK-293 cells. Small size of the QPAS1 component allows designing AAV viral particles, which were applied to deliver the TA system to neurons.
23.

Real-time observation of light-controlled transcription in living cells.

blue CRY2/CIB1 U-2 OS
J Cell Sci, 9 Nov 2017 DOI: 10.1242/jcs.205534 Link to full text
Abstract: Gene expression is tightly regulated in space and time. To dissect this process with high temporal resolution, we introduce an optogenetic tool termed blue light-induced chromatin recruitment (BLInCR) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a cluster of reporter genes in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable ∼2 min after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models of transcription activation suggests that the gene cluster undergoes a maturation process when activated. We anticipate that BLInCR will facilitate the study of transcription dynamics in living cells.This article has an associated First Person interview with the first author of the paper.
24.

A Phytochrome-Derived Photoswitch for Intracellular Transport.

blue red PhyB/PIF6 TULIP Cos-7 U-2 OS Organelle manipulation Multichromatic
ACS Synth Biol, 30 Mar 2017 DOI: 10.1021/acssynbio.6b00333 Link to full text
Abstract: Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.
25.

Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells.

UV UVR8/COP1 S. cerevisiae U-2 OS
Nat Commun, 2013 DOI: 10.1038/ncomms2800 Link to full text
Abstract: Light-sensitive proteins are useful tools to control protein localization, activation and gene expression, but are currently limited to excitation with red or blue light. Here we report a novel optogenetic system based on the ultraviolet-B-dependent interaction of the Arabidopsis ultraviolet-B photoreceptor UVR8 with COP1 that can be performed in visible light background. We use this system to induce nuclear accumulation of cytoplasmic green fluorescent protein fused to UVR8 in cells expressing nuclear COP1, and to recruit a nucleoplasmic red fluorescent protein fused to COP1 to chromatin in cells expressing UVR8-H2B. We also show that ultraviolet-B-dependent interactions between DNA-binding and transcription activation domains result in a linear induction of gene expression. The UVR8-COP1 interactions in mammalian cells can be induced using subsecond pulses of ultraviolet-B light and last several hours. As UVR8 photoperception is based on intrinsic tryptophan residues, these interactions do not depend on the addition of an exogenous chromophore.
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