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 1345 results
1.

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation.

blue bPAC (BlaC) mIMCD-3 Immediate control of second messengers
EMBO Rep, 13 Jun 2022 DOI: 10.15252/embr.202154315 Link to full text
Abstract: The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.
2.

Precise control of microtubule disassembly in living cells.

blue CRY2/CIB1 Cos-7 Control of cytoskeleton / cell motility / cell shape
EMBO J, 10 Jun 2022 DOI: 10.15252/embj.2021110472 Link to full text
Abstract: Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.
3.

A Single-Component Blue Light-Induced System Based on EL222 in Yarrowia lipolytica.

blue EL222 Y. lipolytica Transgene expression
Int J Mol Sci, 6 Jun 2022 DOI: 10.3390/ijms23116344 Link to full text
Abstract: Optogenetics has the advantages of a fast response time, reversibility, and high spatial and temporal resolution, which make it desirable in the metabolic engineering of chassis cells. In this study, a light-induced expression system of Yarrowia lipolytica was constructed, which successfully achieved the synthesis and functional verification of Bleomycin resistance protein (BleoR). The core of the blue light-induced system, the light-responsive element (TF), is constructed based on the blue photosensitive protein EL222 and the transcription activator VP16. The results show that the light-induced sensor based on TF, upstream activation sequence (C120)5, and minimal promoter CYC102 can respond to blue light and initiate the expression of GFPMut3 report gene. With four copies of the responsive promoter and reporter gene assembled, they can produce a 128.5-fold higher fluorescent signal than that under dark conditions after 8 h of induction. The effects of light dose and periodicity on this system were investigated, which proved that the system has good spatial and temporal controllability. On this basis, the light-controlled system was used for the synthesis of BleoR to realize the expression and verification of functional protein. These results demonstrated that this system has the potential for the transcriptional regulation of target genes, construction of large-scale synthetic networks, and overproduction of the desired product.
4.

Force propagation between epithelial cells depends on active coupling and mechano-structural polarization.

blue CRY2/CIB1 MDCK Control of cell-cell / cell-material interactions
bioRxiv, 3 Jun 2022 DOI: 10.1101/2022.06.01.494332 Link to full text
Abstract: Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern (“cell doublet”). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells shows an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depends on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue is key to maintain signal strength and leads to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.
5.

Spatially Defined Gene Delivery into Native Cells with the Red Light-Controlled OptoAAV Technology.

red PhyB/PIF6 A-431 in vitro
Curr Protoc, Jun 2022 DOI: 10.1002/cpz1.440 Link to full text
Abstract: The OptoAAV technology allows spatially defined delivery of transgenes into native target cells down to single-cell resolution by the illumination with cell-compatible and tissue-penetrating red light. The system is based on an adeno-associated viral (AAV) vector of serotype 2 with an engineered capsid (OptoAAV) and a photoreceptor-containing adapter protein mediating the interaction of the OptoAAV with the surface of the target cell in response to low doses of red and far-red light. In this article, we first provide detailed protocols for the production, purification, and analysis of the OptoAAV and the adapter protein. Afterward, we describe in detail the application of the OptoAAV system for the light-controlled transduction of human cells with global and patterned illumination. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production, purification, and analysis of PhyB-DARPinEGFR adapter protein Basic Protocol 2: Production, purification, and analysis of OptoAAV Basic Protocol 3: Red light-controlled viral transduction with the OptoAAV system Support Protocol: Spatially resolved transduction of two transgenes with the OptoAAV system.
6.

Optogenetic Protein Cleavage in Zebrafish Embryos.

violet PhoCl HEK293T HeLa zebrafish in vivo Transgene expression
bioRxiv, 24 May 2022 DOI: 10.1101/2022.05.23.493165 Link to full text
Abstract: A wide array of optogenetic tools is available that allow for precise spatiotemporal control over many cellular processes. These tools have been especially popular among zebrafish researchers who take advantage of the embryo's transparency. However, photocleavable optogenetic proteins have not been utilized in zebrafish. We demonstrate successful optical control of protein cleavage in embryos using PhoCl, a photocleavable fluorescent protein. This optogenetic tool offers temporal and spatial control over protein cleavage events, which we demonstrate in light-triggered protein translocation and apoptosis.
7.

Spatiotemporal dynamics of membrane surface charge regulates cell polarity and migration.

blue CRY2/CIB1 D. discoideum RAW264.7 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 20 May 2022 DOI: 10.1101/2022.05.19.492577 Link to full text
Abstract: During cell migration and polarization, hundreds of signal transduction and cytoskeletal components self-organize to generate localized protrusions. Although biochemical and genetic analyses have delineated many specific interactions, how the activation and localization of so many different molecules are spatiotemporally orchestrated at the subcellular level has remained unclear. Here we show that the regulation of negative surface charge on the inner leaflet of the plasma membrane plays an integrative role in the molecular interactions. Surface charge, or zeta potential, is transiently lowered at new protrusions and within cortical waves of Ras/PI3K/TORC2/F-actin network activation. Rapid alterations of inner leaflet anionic phospholipids, such as PI(4,5)P2, PI(3,4)P2, phosphatidylserine, and phosphatidic acid, collectively contribute to the surface charge changes. Abruptly reducing the surface charge by recruiting positively charged optogenetic actuators was sufficient to trigger the entire biochemical network, initiate de novo protrusions, and abrogate pre-existing polarity. These effects were blocked by genetic or pharmacological inhibitions of key signaling components such as Akt and PI3K/TORC2. Conversely, increasing the negative surface deactivated the network and locally suppressed chemoattractant-induced protrusions or subverted EGF-induced ERK activation. Computational simulations involving excitable biochemical networks demonstrated that slight changes in feedback loops, induced by recruitment of the actuators, could lead to outsized effects on system activation. We propose that key signaling network components act on, and are in turn acted upon, by surface charge, closing feedback loops which bring about the global-scale molecular self-organization required for spontaneous protrusion formation, cell migration, and polarity establishment.
8.

Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation.

blue bPAC (BlaC) PCCL3 Signaling cascade control Immediate control of second messengers
bioRxiv, 19 May 2022 DOI: 10.1101/2022.05.18.492464 Link to full text
Abstract: cAMP is a key player in many physiological processes. Classically considered to originate solely from the plasma membrane, this view was recently challenged by observations showing that GPCRs can sustain cAMP signaling from intracellular compartments associated with nuclear PKA translocation and activation of transcriptional events. In this report we show that neither PKA translocation nor cAMP diffusion, but rather nuclear sAC activation represents the only source of nuclear cAMP accumulation, PKA activation, and CREB phosphorylation. Both pharmacological and genetic sAC inhibition, that did not affect the cytosolic cAMP levels, completed blunted nuclear cAMP accumulation, PKA activation and proliferation, while an increase in sAC nuclear expression significantly enhanced cell proliferation. Moreover, utilizing novel compartment-specific optogenetic actuators we showed that light-dependent nuclear cAMP synthesis can stimulate PKA, CREB and trigger cell proliferation. Thus, our results show that sAC-mediated nuclear accumulation is not only necessary but sufficient and rate-limiting for cAMP-dependent proliferation.
9.

Killing cells using light (activated) sabers.

blue Cryptochromes Review
J Cell Biol, 16 May 2022 DOI: 10.1083/jcb.202205018 Link to full text
Abstract: Many types of regulated cell death exist, however the non-cell autonomous effects of specific forms of cell death remain poorly understood. Addressing this, Shkarina et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109038) describe an optogenetic method to activate distinct modes of cell death in select cells.
10.

Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine.

blue LOV domains Background
Nat Commun, 12 May 2022 DOI: 10.1038/s41467-022-30252-4 Link to full text
Abstract: In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.
11.

Molecular Research on Oral Diseases and Related Biomaterials: A Journey from Oral Cell Models to Advanced Regenerative Perspectives.

red Phytochromes Review
Int J Mol Sci, 9 May 2022 DOI: 10.3390/ijms23095288 Link to full text
Abstract: Oral diseases such as gingivitis, periodontitis, and oral cancer affect millions of people worldwide. Much research has been conducted to understand the pathogenetic mechanisms of these diseases and translate this knowledge into therapeutics. This review aims to take the reader on a journey from the initial molecular discoveries to complex regenerative issues in oral medicine. For this, a semi-systematic literature search was carried out in Medline and Web of Science databases to retrieve the primary literature describing oral cell models and biomaterial applications in oral regenerative medicine. First, an in vitro cell model of gingival keratinocytes is discussed, which illustrates patho- and physiologic principles in the context of oral epithelial homeostasis and carcinogenesis and represents a cellular tool to understand biomaterial-based approaches for periodontal tissue regeneration. Consequently, a layered gradient nonwoven (LGN) is described, which demonstrates that the key features of biomaterials serve as candidates for oral tissue regeneration. LGN supports proper tissue formation and obeys the important principles for molecular mechanotransduction. Furthermore, current biomaterial-based tissue regeneration trends, including polymer modifications, cell-based treatments, antimicrobial peptides and optogenetics, are introduced to represent the full spectrum of current approaches to oral disease mitigation and prevention. Altogether, this review is a foray through established and new concepts in oral regenerative medicine and illustrates the process of knowledge translation from basic molecular and cell biological research to future clinical applications.
12.

Morphogen Directed Coordination of GPCR Activity Promotes Primary Cilium Function for Downstream Signaling.

blue bPAC (BlaC) mIMCD-3 Signaling cascade control Immediate control of second messengers
bioRxiv, 6 May 2022 DOI: 10.1101/2022.05.06.490951 Link to full text
Abstract: Primary cilium dysfunction triggers catastrophic failure of signal transduction pathways that organize through cilia, thus conferring significant pressure on such signals to ensure ciliary homeostasis. Intraflagellar transport (IFT) of cargo that maintains the primary cilium is powered by high ciliary cAMP. Paradoxically, Sonic Hedgehog (SHH) signaling, for which ciliary function is crucial, triggers a reduction in ciliary cAMP that could blunt downstream signaling by slowing IFT. We investigated this paradox and mapped a novel signal relay driven by SHH-stimulated prostaglandin E2 that stabilizes ciliary cAMP flux through by activating Gαs-coupled EP4 receptor. Chemical or genetic blockade of the SHH-EP4 relay cripples cAMP buffering, which leads to decreased intraciliary cAMP, short cilia, and attenuated SHH pathway induction. Accordingly, EP4-/- mice show pronounced ciliary defects and altered SHH-dependent neural tube patterning. Thus, SHH orchestrates a sophisticated ciliary GPCR-cAMP signaling network that ensures primary cilium fitness for a robust downstream signaling response.
13.

Peeking under the hood of early embryogenesis: Using tools and synthetic biology to understand native control systems and sculpt tissues.

blue red Cryptochromes Phytochromes Review
Semin Cell Dev Biol, 4 May 2022 DOI: 10.1016/j.semcdb.2022.04.016 Link to full text
Abstract: Early embryogenesis requires rapid division of pluripotent blastomeres, regulated genome activation, precise spatiotemporal signaling to pattern cell fate, and morphogenesis to shape primitive tissue architectures. The complexity of this process has inspired researchers to move beyond simple genetic perturbation into engineered devices and synthetic biology tools to permit temporal and spatial manipulation of the control systems guiding development. By precise alteration of embryo organization, it is now possible to advance beyond basic analytical strategies and directly test the sufficiency of models for developmental regulation. Separately, advances in micropatterning and embryoid culture have facilitated the bottom-up construction of complex embryo tissues allowing ex vivo systems to recapitulate even later stages of development. Embryos fertilized and grown ex vivo offer an excellent opportunity to exogenously perturb fundamental pathways governing embryogenesis. Here we review the technologies developed to thermally modulate the embryo cell cycle, and optically regulate morphogen and signaling pathways in space and time, specifically in the blastula embryo. Additionally, we highlight recent advances in cell patterning in two and three dimensions that have helped reveal the self-organizing properties and gene regulatory networks guiding early embryo organization.
14.

Engineered Allosteric Regulation of Protein Function.

blue LOV domains Review
J Mol Biol, 2 May 2022 DOI: 10.1016/j.jmb.2022.167620 Link to full text
Abstract: Allosteric regulation of proteins has been utilized to study various aspects of cell signaling, from unicellular events to organism-wide phenotypes. However, traditional methods of allosteric regulation, such as constitutively active mutants and inhibitors, lack tight spatiotemporal control. This often leads to unintended signaling consequences that interfere with data interpretation. To overcome these obstacles, researchers employed protein engineering approaches that enable tight control of protein function through allosteric mechanisms. These methods provide high specificity as well as spatial and temporal precision in regulation of protein activity in vitro and in vivo. In this review, we focus on the recent advancements in engineered allosteric regulation and discuss the various bioengineered allosteric techniques available now, from chimeric GPCRs to chemogenetic and optogenetic switches. We highlight the benefits and pitfalls of each of these techniques as well as areas in which future improvements can be made. Additionally, we provide a brief discussion on implementation of engineered allosteric regulation approaches, demonstrating that these tools can shed light on elusive biological events and have the potential to be utilized in precision medicine.
15.

Light-dependent modulation of protein localization and function in living bacteria cells.

blue CRY2/CIB1 E. coli Control of cytoskeleton / cell motility / cell shape Cell cycle control
bioRxiv, 1 May 2022 DOI: 10.1101/2022.05.01.490209 Link to full text
Abstract: Most bacteria lack membrane-enclosed organelles to compartmentalize cellular processes. In lieu of physical compartments, bacterial proteins are often recruited to macromolecular scaffolds at specific subcellular locations to carry out their functions. Consequently, the ability to modulate a protein’s subcellular location with high precision and speed bears the potential to manipulate its corresponding cellular functions. Here we demonstrate that the CRY2/CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations inside live E. coli cells including the nucleoid, the cell pole, membrane, and the midcell division plane. We further show that such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. Finally, we demonstrate that the CRY2/CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system.
16.

Engineered Cas9 extracellular vesicles as a novel gene editing tool.

blue red CRY2/CIB1 Magnets PhyB/PIF6 VVD HEK293T Nucleic acid editing
J Extracell Vesicles, May 2022 DOI: 10.1002/jev2.12225 Link to full text
Abstract: Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9-fusions with EV sorting partners. Cas9-loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9-mediated functional gene editing in a Cre-reporter cellular assay. Light-induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation-Palmitoylation-Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock-down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent-sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV-mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing.
17.

Synthetic cells with self-activating optogenetic proteins communicate with natural cells.

blue EL222 iLID in vitro Extracellular optogenetics
Nat Commun, 28 Apr 2022 DOI: 10.1038/s41467-022-29871-8 Link to full text
Abstract: Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.
18.

Light-induced fermenter production of derivatives of the sweet protein monellin is maximized in prestationary Saccharomyces cerevisiae cultures.

blue CRY2/CIB1 S. cerevisiae Transgene expression
Biotechnol J, 28 Apr 2022 DOI: 10.1002/biot.202100676 Link to full text
Abstract: Optogenetics has great potential for biotechnology and metabolic engineering due to the cost-effective control of cellular activities. The usage of optogenetics techniques for the biosynthesis of bioactive molecules ensures reduced costs and enhanced regulatory possibilities. This requires development of efficient methods for light-delivery during a production process in a fermenter. Here, we benchmarked the fermenter production of a low-caloric sweetener in Saccharomyces cerevisiae with optogenetic tools against the production in small scale cell culture flasks. An expression system based on the light-controlled interaction between Cry2 and Cib1 was used for sweet-protein production. Optimization of the fermenter process was achieved by increasing the light-flux during the production phase to circumvent shading by yeast cells at high densities. Maximal amounts of the sweet-protein were produced in a pre-stationary growth phase, whereas at later stages, a decay in protein abundance was observable. Our investigation showcases the upscaling of an optogenetic production process from small flasks to a bioreactor. Optogenetic-controlled production in a fermenter is highly cost-effective due to the cheap inducer and therefore a viable alternative to chemicals for a process that requires an induction step.
19.

Benchmarking of Cph1 Mutants and DrBphP for Light-Responsive Phytochrome-Based Hydrogels with Reversibly Adjustable Mechanical Properties.

red Cph1 DrBphP Benchmarking
Adv Biol (Weinh), 28 Apr 2022 DOI: 10.1002/adbi.202000337 Link to full text
Abstract: In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light-responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome-based hydrogels with an increased dynamic range for the storage modulus but a different light-response for the loss modulus compared to the original Cph1-based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1-R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.
20.

PPARγ phase separates with RXRα at PPREs to regulate target gene expression.

blue CRY2olig HEK293T NIH/3T3 Organelle manipulation
Cell Discov, 26 Apr 2022 DOI: 10.1038/s41421-022-00388-0 Link to full text
Abstract: Peroxisome proliferator-activated receptor (PPAR)-γ is a key transcription activator controlling adipogenesis and lipid metabolism. PPARγ binds PPAR response elements (PPREs) as the obligate heterodimer with retinoid X receptor (RXR) α, but exactly how PPARγ orchestrates the transcriptional response is unknown. This study demonstrates that PPARγ forms phase-separated droplets in vitro and solid-like nuclear condensates in cell, which is intriguingly mediated by its DNA binding domain characterized by the zinc finger motif. Furthermore, PPARγ forms nuclear condensates at PPREs sites through phase separation to compartmentalize its heterodimer partner RXRα to initiate PPARγ-specific transcriptional activation. Finally, using an optogenetic approach, the enforced formation of PPARγ/RXRα condensates leads to preferential enrichment at PPREs sites and significantly promotes the expression of PPARγ target genes. These results define a novel mechanism by which PPARγ engages the phase separation principles for efficient and specific transcriptional activation.
21.

Design and engineering of light-sensitive protein switches.

blue green near-infrared red Cobalamin-binding domains Cryptochromes LOV domains Phytochromes Review
Curr Opin Struct Biol, 20 Apr 2022 DOI: 10.1016/j.sbi.2022.102377 Link to full text
Abstract: Engineered, light-sensitive protein switches are used to interrogate a broad variety of biological processes. These switches are typically constructed by genetically fusing naturally occurring light-responsive protein domains with functional domains from other proteins. Protein activity can be controlled using a variety of mechanisms including light-induced colocalization, caging, and allosteric regulation. Protein design efforts have focused on reducing background signaling, maximizing the change in activity upon light stimulation, and perturbing the kinetics of switching. It is common to combine structure-based modeling with experimental screening to identify ideal fusion points between domains and discover point mutations that optimize switching. Here, we introduce commonly used light-sensitive domains and summarize recent progress in using them to regulate protein activity.
22.

Light-inducible T cell engagers trigger, tune and shape the activation of primary T cells.

red PhyB/PIF6 human T cells Extracellular optogenetics
bioRxiv, 15 Apr 2022 DOI: 10.1101/2022.04.15.488452 Link to full text
Abstract: Cells perceive overtime complex sequences of receptor stimulation that they integrate to mount an appropriate response. Yet, the influence of signal dynamics on cell responses has been poorly characterized due to technical limitations. Here, we present a generalizable approach to control receptor stimulation on unmodified primary cells. Indeed, for applications on primary murine T cells, we have engineered the LiTe system, a new recombinant optogenetics-based Light-inducible T cell engager which allows tunable and reversible spatiotemporal control of the T Cell Receptor (TCR) stimulation. We also provided in vitro evidence that this system enables efficient T cell activation with light, leading to cytokine secretion or tumor cell killing. Using specific time-gated stimulations, we have been able to orient the outcome of the activation of T cells. Overall, the LiTe system constitutes a versatile ON/OFF molecular switch allowing to decipher the cellular response to stimulation dynamics. Its original control over T cell activation opens new avenues for future precision cancer immunotherapy.
23.

Optogenetic activators of apoptosis, necroptosis, and pyroptosis.

blue CRY2olig Caco-2 HaCaT HEK293T HeLa MCF7 RAW264.7 zebrafish in vivo Cell death
J Cell Biol, 14 Apr 2022 DOI: 10.1083/jcb.202109038 Link to full text
Abstract: Targeted and specific induction of cell death in an individual or groups of cells hold the potential for new insights into the response of tissues or organisms to different forms of death. Here, we report the development of optogenetically controlled cell death effectors (optoCDEs), a novel class of optogenetic tools that enables light-mediated induction of three types of programmed cell death (PCD)-apoptosis, pyroptosis, and necroptosis-using Arabidopsis thaliana photosensitive protein Cryptochrome-2. OptoCDEs enable a rapid and highly specific induction of PCD in human, mouse, and zebrafish cells and are suitable for a wide range of applications, such as sub-lethal cell death induction or precise elimination of single cells or cell populations in vitro and in vivo. As the proof-of-concept, we utilize optoCDEs to assess the differences in neighboring cell responses to apoptotic or necrotic PCD, revealing a new role for shingosine-1-phosphate signaling in regulating the efferocytosis of the apoptotic cell by epithelia.
24.

Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies.

blue iLID in vitro Extracellular optogenetics
bioRxiv, 10 Apr 2022 DOI: 10.1101/2021.10.22.465381 Link to full text
Abstract: Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, proces-sivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.
25.

LILAC: Enhanced actin imaging with an optogenetic Lifeact.

blue AsLOV2 Schneider 2
bioRxiv, 8 Apr 2022 DOI: 10.1101/2022.04.07.487416 Link to full text
Abstract: We have designed an improved Lifeact variant that binds to actin under the control of light using the LOV2 protein. Light control enables one to subtract the pre-illumination signal of the unbound label, yielding an enhanced view of F-actin dynamics in cells. Furthermore, the tool eliminates actin network perturbations and cell sickness caused by Lifeact overexpression.
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