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 933 results
1.

Loss of Nrf1 rather than Nrf2 leads to inflammatory accumulation of lipids and reactive oxygen species in human hepatoma cells, which is alleviated by 2-bromopalmitate.

blue AsLOV2 cpLOV2 LOVTRAP HEK293T HeLa Hep G2 Neuro-2a U-2 OS Transgene expression Endogenous gene expression
Biochim Biophys Acta Mol Cell Res, 21 Nov 2023 DOI: 10.1101/2023.11.06.565805 Link to full text
Abstract: Since Nrf1 and Nrf2 are essential for regulating the lipid metabolism pathways, their dysregulation has thus been shown to be critically involved in the non-controllable inflammatory transformation into cancer. Herein, we have explored the molecular mechanisms underlying their distinct regulation of lipid metabolism, by comparatively analyzing the changes in those lipid metabolism-related genes in Nrf1α-/- and/or Nrf2-/- cell lines relative to wild-type controls. The results revealed that loss of Nrf1α leads to lipid metabolism disorders. That is, its lipid synthesis pathway was up-regulated by the JNK-Nrf2-AP1 signaling, while its lipid decomposition pathway was down-regulated by the nuclear receptor PPAR-PGC1 signaling, thereby resulting in severe accumulation of lipids as deposited in lipid droplets. By contrast, knockout of Nrf2 gave rise to decreases in lipid synthesis and uptake capacity. These demonstrate that Nrf1 and Nrf2 contribute to significant differences in the cellular lipid metabolism profiles and relevant pathological responses. Further experimental evidence unraveled that lipid deposition in Nrf1α-/- cells resulted from CD36 up-regulation by activating the PI3K-AKT-mTOR pathway, leading to abnormal activation of the inflammatory response. This was also accompanied by a series of adverse consequences, e.g., accumulation of reactive oxygen species (ROS) in Nrf1α-/- cells. Interestingly, treatment of Nrf1α-/- cells with 2-bromopalmitate (2BP) enabled the yield of lipid droplets to be strikingly alleviated, as accompanied by substantial abolishment of CD36 and critical inflammatory cytokines. Such Nrf1α-/- led inflammatory accumulation of lipids, as well as ROS, was significantly ameliorated by 2BP. Overall, this study provides a potential strategy for cancer prevention and treatment by precision targeting of Nrf1, Nrf2 alone or both.
2.

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime.

blue LOV domains Background
JAmChemSoc, 21 Nov 2023 DOI: 10.1021/jacsau.3c00440 Link to full text
Abstract: Naturally occurring and engineered flavin-binding, blue-light-sensing, light, oxygen, voltage (LOV) photoreceptor domains have been used widely to design fluorescent reporters, optogenetic tools, and photosensitizers for the visualization and control of biological processes. In addition, natural LOV photoreceptors with engineered properties were recently employed for optimizing plant biomass production in the framework of a plant-based bioeconomy. Here, the understanding and fine-tuning of LOV photoreceptor (kinetic) properties is instrumental for application. In response to blue-light illumination, LOV domains undergo a cascade of photophysical and photochemical events that yield a transient covalent FMN-cysteine adduct, allowing for signaling. The rate-limiting step of the LOV photocycle is the dark-recovery process, which involves adduct scission and can take between seconds and days. Rational engineering of LOV domains with fine-tuned dark recovery has been challenging due to the lack of a mechanistic model, the long time scale of the process, which hampers atomistic simulations, and a gigantic protein sequence space covering known mutations (combinatorial challenge). To address these issues, we used machine learning (ML) trained on scarce literature data and iteratively generated and implemented experimental data to design LOV variants with faster and slower dark recovery. Over the three prediction–validation cycles, LOV domain variants were successfully predicted, whose adduct-state lifetimes spanned 7 orders of magnitude, yielding optimized tools for synthetic (opto)biology. In summary, our results demonstrate ML as a viable method to guide the design of proteins even with limited experimental data and when no mechanistic model of the underlying physical principles is available.
3.

A single-component, light-assisted uncaging switch for endoproteolytic release.

blue violet CRY2/CIB1 iLID PhoCl HEK293T primary rat hippocampal neurons Signaling cascade control Transgene expression
Nat Chem Biol, 16 Nov 2023 DOI: 10.1038/s41589-023-01480-6 Link to full text
Abstract: Proteases function as pivotal molecular switches, initiating numerous biological events. Notably, potyviral protease, derived from plant viruses, has emerged as a trusted proteolytic switch in synthetic biological circuits. To harness their capabilities, we have developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), by employing a circularly permutated tobacco etch virus protease and a blue-light-gated substrate, which are connected by fine-tuned intermodular linkers. As a single-component system, LAUNCHER exhibits a superior signal-to-noise ratio compared with multi-component systems, enabling precise and user-controllable release of payloads. This characteristic renders LAUNCHER highly suitable for diverse cellular applications, including transgene expression, tailored subcellular translocation and optochemogenetics. Additionally, the plug-and-play integration of LAUNCHER into existing synthetic circuits facilitates the enhancement of circuit performance. The demonstrated efficacy of LAUNCHER in improving existing circuitry underscores its significant potential for expanding its utilization in various applications.
4.

Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo.

blue EL222 HEK293FT HEK293T mouse in vivo mouse T cells Transgene expression Endogenous gene expression Nucleic acid editing
bioRxiv, 10 Nov 2023 DOI: 10.1101/2023.11.09.566272 Link to full text
Abstract: There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. CRISPR’s ease of use and flexibility, coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool, BLU-VIPR, that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. This simplifies spatiotemporal gene perturbation and works in vivo with cells previously intractable to optogenetic gene editing. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript, allowing for optogenetic gene editing in T lymphocytes in vivo.
5.

Light-Oxygen-Voltage (LOV)-sensing domains: activation mechanism and optogenetic stimulation.

blue UV LOV domains UV receptors Review
J Mol Biol, 7 Nov 2023 DOI: 10.1016/j.jmb.2023.168356 Link to full text
Abstract: The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.
6.

Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.

blue LOV domains Background
bioRxiv, 6 Nov 2023 DOI: 10.1101/2023.11.06.565770 Link to full text
Abstract: Light-Oxygen-Voltage (LOV) domains are small photosensory flavoprotein modules that allow converting external stimuli (sunlight) into intracellular signals responsible for various cell behavior (e.g., phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that results in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the observation of the activation cascade of the LOV domain in real-time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to the actual data collection process, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the obtained crystals preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.
7.

Critical capillary waves of biomolecular condensates.

blue iLID U-2 OS Organelle manipulation
bioRxiv, 5 Nov 2023 DOI: 10.1101/2023.10.29.564316 Link to full text
Abstract: Membraneless compartments known as biomolecular condensates are thought to form through liquid-liquid phase separation (LLPS). When forces are applied to the fluid interfaces of these condensates, surface fluctuation are generated, a phenomenon known as capillary waves. The spatiotemporal dynamics of these fluctuations, characterized by the amplitude and velocity, reflect the physical properties of condensates. Moreover, unraveling the nature of fluctuations near the critical point is crucial for understanding the universal physical underpinnings of phase transitions. Although fluid condensate interfaces are ubiquitous within living cells, little is known about their surface fluctuations. Here, we quantify the interface fluctuations of light-induced synthetic and endogenous nuclear condensates, including nucleoli and nuclear speckles, in real and Fourier space. Measured fluctuations align with a theory assuming thermal driving, which enables measurement of surface tension and effective viscosity. The surface tensions fall within the range of 10−6 to 10−5 N/m for all tested condensates; in contrast, we find significant difference of fluctuation velocities, highlighting much higher viscosity of nucleoli ∼ 104 Pa·s, compared to synthetic condensates and nuclear speckles. We further find that the interface fluctuations become enhanced and slower as the system nears the critical point. These findings elucidate key aspects of intracellular condensate properties, and suggest that the critical trend of surface tension is more consistent with theoretical predictions by the mean-field model than those by the 3D Ising model.
8.

RudLOV—a new optically synchronized cargo transport method reveals unexpected effect of dynasore.

blue LOVTRAP HeLa Control of vesicular transport
bioRxiv, 5 Nov 2023 DOI: 10.1101/2023.11.04.565648 Link to full text
Abstract: Live imaging of secretory cargoes is a powerful method for understanding the mechanisms of membrane trafficking. Inducing the synchronous release of cargoes from an organelle is a key for enhancing microscopic observation. We developed an optical cargo-releasing method named as retention using dark state of LOV2 (RudLOV), which enables exceptional spatial, temporal, and quantity control during cargo release. A limited amount of cargo-release using RudLOV successfully visualized cargo cisternal-movement and cargo-specific exit sites on the Golgi/trans-Golgi network. Moreover, by controlling the timing of cargo-release using RudLOV, we revealed the canonical and non-canonical effects of the well-known dynamin inhibitor dynasore, which inhibits early-Golgi but not late-Golgi transport and exit from the trans-Golgi network where dynamin-2 is active. Accumulation of COPI vesicles at the cis-side of the Golgi stacks in dynasore-treated cells suggests that dynasore targets COPI-uncoating/tethering/fusion machinery in the early-Golgi cisternae or endoplasmic reticulum but not in the late-Golgi cisternae. These results provide insight into the cisternal maturation of Golgi stacks.
9.

Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid interference system.

blue AsLOV2 E. coli HEK293T Nucleic acid editing
Nucleic Acids Res, 1 Nov 2023 DOI: 10.1093/nar/gkad995 Link to full text
Abstract: Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.
10.

Emerging optogenetics technologies in biomedical applications.

blue green near-infrared red UV violet Cryptochromes Cyanobacteriochromes LOV domains Phytochromes UV receptors Review
Smart Med, 1 Nov 2023 DOI: 10.1002/smmd.20230026 Link to full text
Abstract: Optogenetics is a cutting-edge technology that merges light control and genetics to achieve targeted control of tissue cells. Compared to traditional methods, optogenetics offers several advantages in terms of time and space precision, accuracy, and reduced damage to the research object. Currently, optogenetics is primarily used in pathway research, drug screening, gene expression regulation, and the stimulation of molecule release to treat various diseases. The selection of light-sensitive proteins is the most crucial aspect of optogenetic technology; structural changes occur or downstream channels are activated to achieve signal transmission or factor release, allowing efficient and controllable disease treatment. In this review, we examine the extensive research conducted in the field of biomedicine concerning optogenetics, including the selection of light-sensitive proteins, the study of carriers and delivery devices, and the application of disease treatment. Additionally, we offer critical insights and future implications of optogenetics in the realm of clinical medicine.
11.

Full-field exposure of larval zebrafish to narrow waveband LED light sources at defined power and energy for optogenetic applications.

blue VVD zebrafish in vivo Transgene expression
J Neurosci Methods, 31 Oct 2023 DOI: 10.1016/j.jneumeth.2023.110001 Link to full text
Abstract: Optogenetic approaches in transparent zebrafish models have provided numerous insights into vertebrate neurobiology. The purpose of this study was to develop methods to activate light-sensitive transgene products simultaneously throughout an entire larval zebrafish.
12.

Optogenetic control of YAP reveals a dynamic communication code for stem cell fate and proliferation.

blue AsLOV2 mESCs Signaling cascade control Cell differentiation
Nat Commun, 30 Oct 2023 DOI: 10.1038/s41467-023-42643-2 Link to full text
Abstract: YAP is a transcriptional regulator that controls pluripotency, cell fate, and proliferation. How cells ensure the selective activation of YAP effector genes is unknown. This knowledge is essential to rationally control cellular decision-making. Here we leverage optogenetics, live-imaging of transcription, and cell fate analysis to understand and control gene activation and cell behavior. We reveal that cells decode the steady-state concentrations and timing of YAP activation to control proliferation, cell fate, and expression of the pluripotency regulators Oct4 and Nanog. While oscillatory YAP inputs induce Oct4 expression and proliferation optimally at frequencies that mimic native dynamics, cellular differentiation requires persistently low YAP levels. We identify the molecular logic of the Oct4 dynamic decoder, which acts through an adaptive change sensor. Our work reveals how YAP levels and dynamics enable multiplexing of information transmission for the regulation of developmental decision-making and establishes a platform for the rational control of these behaviors.
13.

Optogenetics for sensors: On-demand fluorescent labeling of histone epigenetics.

blue AsLOV2 HeLa Epigenetic modification
Biochem Biophys Res Commun, 29 Oct 2023 DOI: 10.1016/j.bbrc.2023.149174 Link to full text
Abstract: Post-translational modifications of histones to a large extent determine the functional state of chromatin loci. Dynamic visualization of histone modifications with genetically encoded fluorescent sensors makes it possible to monitor changes in the epigenetic state of a single living cell. At the same time, the sensors can potentially compete with endogenous factors recognizing these modifications. Thus, prolonged binding of the sensors to chromatin can affect normal epigenetic regulation. Here, we report an optogenetic sensor for live-cell visualization of histone H3 methylated at lysine-9 (H3K9me3) named MPP8-LAMS (MPP8-based light-activated modification sensor). MPP8-LAMS consists of several fusion protein parts (from N- to C-terminus): i) nuclear export signal (NES), ii) far-red fluorescent protein Katushka, iii) H3K9me3-binding reader domain of the human M phase phosphoprotein 8 (MPP8), iv) the light-responsive AsLOV2 domain, which exposes a nuclear localization signal (NLS) upon blue light stimulation. In the dark, due to the NES, MPP8-LAMS is localized in the cytosol. Under blue light illumination, MPP8-LAMS underwent an efficient translocation from cytosol to nucleus, enabling visualization of H3K9me3-enriched loci. Such an on-demand visualization minimizes potential impact on cell physiology as most of the time the sensor is separated from its target. In general, the present work extends the application of optogenetics to the area of advanced use of genetically encoded sensors.
14.

Optogenetic Signaling Activation in Zebrafish Embryos.

blue VfAU1-LOV zebrafish in vivo
J Vis Exp, 27 Oct 2023 DOI: 10.3791/65733 Link to full text
Abstract: Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.
15.

Turn-On Protein Switches for Controlling Actin Binding in Cells.

blue AsLOV2 HEK293T HeLa MDCK Control of cytoskeleton / cell motility / cell shape
bioRxiv, 26 Oct 2023 DOI: 10.1101/2023.10.26.561921 Link to full text
Abstract: Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP’s influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.
16.

A programmable protease-based protein secretion platform for therapeutic applications.

blue red BphS CRY2/CIB1 Magnets PhyA/FHY1 Hana3A HEK293T hMSCs mouse IPSCs Control of vesicular transport
Nat Chem Biol, 23 Oct 2023 DOI: 10.1038/s41589-023-01433-z Link to full text
Abstract: Cell-based therapies represent potent enabling technologies in biomedical science. However, current genetic control systems for engineered-cell therapies are predominantly based on the transcription or translation of therapeutic outputs. Here we report a protease-based rapid protein secretion system (PASS) that regulates the secretion of pretranslated proteins retained in the endoplasmic reticulum (ER) owing to an ER-retrieval signal. Upon cleavage by inducible proteases, these proteins are secreted. Three PASS variants (chemPASS, antigenPASS and optoPASS) are developed. With chemPASS, we demonstrate the reversal of hyperglycemia in diabetic mice within minutes via drug-induced insulin secretion. AntigenPASS-equipped cells recognize the tumor antigen and secrete granzyme B and perforin, inducing targeted cell apoptosis. Finally, results from mouse models of diabetes, hypertension and inflammatory pain demonstrate light-induced, optoPASS-mediated therapeutic peptide secretion within minutes, conferring anticipated therapeutic benefits. PASS is a flexible platform for rapid delivery of therapeutic proteins that can facilitate the development and adoption of cell-based precision therapies.
17.

Direct investigation of cell contraction signal networks by light-based perturbation methods.

blue cyan red Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Pflugers Arch, 18 Oct 2023 DOI: 10.1007/s00424-023-02864-2 Link to full text
Abstract: Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.
18.

Optogenetics in Alzheimer's Disease: Focus on Astrocytes.

blue red violet Cryptochromes Fluorescent proteins LOV domains Phytochromes Review
Antioxidants (Basel), 13 Oct 2023 DOI: 10.3390/antiox12101856 Link to full text
Abstract: Alzheimer's disease (AD) is the most common form of dementia, resulting in disability and mortality. The global incidence of AD is consistently surging. Although numerous therapeutic agents with promising potential have been developed, none have successfully treated AD to date. Consequently, the pursuit of novel methodologies to address neurodegenerative processes in AD remains a paramount endeavor. A particularly promising avenue in this search is optogenetics, enabling the manipulation of neuronal activity. In recent years, research attention has pivoted from neurons to glial cells. This review aims to consider the potential of the optogenetic correction of astrocyte metabolism as a promising strategy for correcting AD-related disorders. The initial segment of the review centers on the role of astrocytes in the genesis of neurodegeneration. Astrocytes have been implicated in several pathological processes associated with AD, encompassing the clearance of β-amyloid, neuroinflammation, excitotoxicity, oxidative stress, and lipid metabolism (along with a critical role in apolipoprotein E function). The effect of astrocyte-neuronal interactions will also be scrutinized. Furthermore, the review delves into a number of studies indicating that changes in cellular calcium (Ca2+) signaling are one of the causes of neurodegeneration. The review's latter section presents insights into the application of various optogenetic tools to manipulate astrocytic function as a means to counteract neurodegenerative changes.
19.

Reversible photoregulation of cell-cell adhesions with opto-E-cadherin.

blue AsLOV2 A-431 HeLa MDA-MB-231 NCTC clone 929 Control of cell-cell / cell-material interactions
Nat Commun, 9 Oct 2023 DOI: 10.1038/s41467-023-41932-0 Link to full text
Abstract: E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.
20.

Comprehensive Screening of a Light-Inducible Split Cre Recombinase with Domain Insertion Profiling.

blue Magnets E. coli Transgene expression
ACS Synth Biol, 3 Oct 2023 DOI: 10.1021/acssynbio.3c00328 Link to full text
Abstract: Splitting proteins with light- or chemically inducible dimers provides a mechanism for post-translational control of protein function. However, current methods for engineering stimulus-responsive split proteins often require significant protein engineering expertise and the laborious screening of individual constructs. To address this challenge, we use a pooled library approach that enables rapid generation and screening of nearly all possible split protein constructs in parallel, where results can be read out by using sequencing. We perform our method on Cre recombinase with optogenetic dimers as a proof of concept, resulting in comprehensive data on the split sites throughout the protein. To improve the accuracy in predicting split protein behavior, we develop a Bayesian computational approach to contextualize errors inherent to experimental procedures. Overall, our method provides a streamlined approach for achieving inducible post-translational control of a protein of interest.
21.

Light-induced trapping of endogenous proteins reveals spatiotemporal roles of microtubule and kinesin-1 in dendrite patterning of Drosophila sensory neurons.

blue CRY2/CIB1 CRY2/CRY2 CRY2olig Magnets D. melanogaster in vivo Control of cytoskeleton / cell motility / cell shape
bioRxiv, 2 Oct 2023 DOI: 10.1101/2023.09.30.560303 Link to full text
Abstract: Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. We generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed us to instantly disrupt microtubules and inhibit the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, we obtained direct evidence that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages.
22.

Current Trends of Bacterial and Fungal Optoproteins for Novel Optical Applications.

blue cyan near-infrared red UV BLUF domains Cryptochromes Fluorescent proteins LOV domains Phytochromes UV receptors Review
Int J Mol Sci, 29 Sep 2023 DOI: 10.3390/ijms241914741 Link to full text
Abstract: Photoproteins, luminescent proteins or optoproteins are a kind of light-response protein responsible for the conversion of light into biochemical energy that is used by some bacteria or fungi to regulate specific biological processes. Within these specific proteins, there are groups such as the photoreceptors that respond to a given light wavelength and generate reactions susceptible to being used for the development of high-novel applications, such as the optocontrol of metabolic pathways. Photoswitchable proteins play important roles during the development of new materials due to their capacity to change their conformational structure by providing/eliminating a specific light stimulus. Additionally, there are bioluminescent proteins that produce light during a heatless chemical reaction and are useful to be employed as biomarkers in several fields such as imaging, cell biology, disease tracking and pollutant detection. The classification of these optoproteins from bacteria and fungi as photoreceptors or photoresponse elements according to the excitation-emission spectrum (UV-Vis-IR), as well as their potential use in novel applications, is addressed in this article by providing a structured scheme for this broad area of knowledge.
23.

Local negative feedback of Rac activity at the leading edge underlies a pilot pseudopod-like program for amoeboid cell guidance.

blue iLID HL-60 Signaling cascade control Control of cytoskeleton / cell motility / cell shape
PLoS Biol, 25 Sep 2023 DOI: 10.1371/journal.pbio.3002307 Link to full text
Abstract: To migrate efficiently, neutrophils must polarize their cytoskeletal regulators along a single axis of motion. This polarization process is thought to be mediated through local positive feedback that amplifies leading edge signals and global negative feedback that enables sites of positive feedback to compete for dominance. Though this two-component model efficiently establishes cell polarity, it has potential limitations, including a tendency to "lock" onto a particular direction, limiting the ability of cells to reorient. We use spatially defined optogenetic control of a leading edge organizer (PI3K) to probe how neutrophil-like HL-60 cells balance "decisiveness" needed to polarize in a single direction with the flexibility needed to respond to new cues. Underlying this balancing act is a local Rac inhibition process that destabilizes the leading edge to promote exploration. We show that this local inhibition enables cells to process input signal dynamics, linking front stability and orientation to local temporal increases in input signals.
24.

Allosteric regulation of kinase activity in living cells.

blue cyan red Fluorescent proteins LOV domains Phytochromes Review
bioRxiv, 25 Sep 2023 DOI: 10.1101/2023.07.19.549709 Link to full text
Abstract: The dysregulation of protein kinases is associated with multiple diseases due to the kinases’ involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities making it difficult to specifically target one kinase, allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or “sensors” are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation.
25.

Spatiotemporal, optogenetic control of gene expression in organoids.

blue CRY2/CIB1 Magnets HEK293T human IPSCs Endogenous gene expression Nucleic acid editing
Nat Methods, 21 Sep 2023 DOI: 10.1038/s41592-023-01986-w Link to full text
Abstract: Organoids derived from stem cells have become an increasingly important tool for studying human development and modeling disease. However, methods are still needed to control and study spatiotemporal patterns of gene expression in organoids. Here we combined optogenetics and gene perturbation technologies to activate or knock-down RNA of target genes in programmable spatiotemporal patterns. To illustrate the usefulness of our approach, we locally activated Sonic Hedgehog (SHH) signaling in an organoid model for human neurodevelopment. Spatial and single-cell transcriptomic analyses showed that this local induction was sufficient to generate stereotypically patterned organoids and revealed new insights into SHH's contribution to gene regulation in neurodevelopment. With this study, we propose optogenetic perturbations in combination with spatial transcriptomics as a powerful technology to reprogram and study cell fates and tissue patterning in organoids.
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