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Mononegaviruses are promising tools as oncolytic vectors and transgene delivery vectors for gene therapy and regenerative medicine. By using the Magnet proteins, which reversibly heterodimerize upon blue light illumination, photocontrollable mononegaviruses (measles and rabies viruses) were generated. The Magnet proteins were inserted into the flexible domain of viral polymerase, and viruses showed strong replication and oncolytic activities only when the viral polymerases were activated by blue light illumination.
A molecular toolbox for interrogation of membrane contact sites.
Membrane contact sites (MCSs) are specialized subcellular compartments formed by closely apposed membranes from two organelles. The intermembrane gap is separated by a distance ranging from 10 nm to 35 nm. MCSs are typically maintained through dynamic protein-protein and protein-lipid interactions. These inter-membrane contact sites constitute important intracellular signaling hotspots to mediate a plethora of cellular processes, including calcium homeostasis, lipid metabolism, membrane biogenesis and organelle remodeling. In recent years, a series of genetically-encoded probes and chemogenetic or optogenetic actuators have been invented to aid the visualization and interrogation of MCSs in both fixed and living cells. These molecular tools have greatly accelerated the pace of mechanistic dissection of membrane contact sites at the molecular level. In this review, we present an overview on the latest progress in this endeavor, and provide a general guide to the selection of methods and molecular tools for probing interorganellar membrane contact sites. This article is protected by copyright. All rights reserved.
Independent Blue and Red Light Triggered Narcissistic Self-Sorting Self-Assembly of Colloidal Particles.
The ability of living systems to self-sort different cells into separate assemblies and the ability to independently regulate different structures are one ingredient that gives rise to their spatiotemporal complexity. Here, this self-sorting behavior is replicated in a synthetic system with two types of colloidal particles; where each particle type independently self-assembles either under blue or red light into distinct clusters, known as narcissistic self-sorting. For this purpose, each particle type is functionalized either with the light-switchable protein VVDHigh or Cph1, which homodimerize under blue and red light, respectively. The response to different wavelengths of light and the high specificity of the protein interactions allows for the independent self-assembly of each particle type with blue or red light and narcissistic self-sorting. Moreover, as both of the photoswitchable protein interactions are reversible in the dark; also, the self-sorting is reversible and dynamic. Overall, the independent blue and red light controlled self-sorting in a synthetic system opens new possibilities to assemble adaptable, smart, and advanced materials similar to the complexity observed in tissues.
Engineering proteins for allosteric control by light or ligands.
Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.
RhoA mediates epithelial cell shape changes via mechanosensitive endocytosis.
Morphogenetic movements require tight spatiotemporal control over cell-cell junction lengths. Contractile forces, acting at adherens junctions, alter cell-cell contact lengths in a cyclic fashion as a mechanical ratchet. Pulsatile RhoA activity is thought to drive ratcheting through acute periods of junction contraction followed by stabilization. Currently, we lack a mechanistic under-standing of if and how RhoA activity governs junction length and subsequent cell shape within epi-thelia. In this study we use optogenetics to exogenously control RhoA activity in model Caco-2 epi-thelium. We find that at short timescales, RhoA activation drives reversible junction contraction. Sus-tained RhoA activity drives irreversible junction shortening but the amount of shortening saturates for a single pulse. To capture these data, we develop a vertex model modified to include strain-dependent junction length and tension remodeling. We find that, to account for experimental data, tension remodeling requires a strain-dependent threshold. Our model predicts that temporal structur-ing of RhoA activity allows for subsequent tension remodeling events to overcome the limited short-ening within a single pulse and this is confirmed by our experimental data. We find that RhoA-mediated junction remodeling requires activities of formin and dynamin, indicating the closely inter-connected activities of contractility, E-cadherin clustering, and endocytosis. Junction length is there-fore regulated by the coordinated action of RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling. Altogether these data provide insights into the underlying molecular and biophysical mechanisms of RhoA-mediated regulation of epithelial cell shape.
Optogenetic downregulation of protein levels with an ultrasensitive switch.
Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, e.g. for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycycline-dependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low light-fluxes as well as fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.
Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells.
Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noisein cells. Optogenetic systems have recently been employed for controlling gene expression levelsin mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback. Here, we engineer optogenetic negative-feedback gene circuits inmammalian cellsto achievenoise-reduction forprecisegene expression control. We builda toolset of these noise-reducingLight-Inducible Tuner (LITer)gene circuits usingtheTetR repressor fused witha Tet-Inhibitory peptide (TIP)oradegradation tagthrough the light-sensitive LOV2 protein domain. These LITers provide nearly a range of 4-fold gene expression control and up to five-fold noise reductionfrom existing optogenetic systems. Moreover, we usethe LITergene circuit architecture to controlgene expression of the cancer oncogene KRAS(G12V)and studyits downstream effectsthrough phospho-ERK levels and cellular proliferation.Overall, these novel LITer optogenetic platformsshould enable precisespatiotemporal perturbations for studyingmulticellular phenotypes in developmental biology, oncology,and other biomedical fields of research.
Light-based tuning of ligand half-life supports kinetic proofreading model of T cell signaling.
T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.
Optical induction of autophagy via Transcription factor EB (TFEB) reduces pathological tau in neurons.
Aggregation and accumulation of microtubule associated protein tau in neurons is major neuropathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Attempts have been made to promote clearance of pathological tau (p-Tau) from neurons via autophagy. Over expression of transcription factor EB (TFEB), has shown to clear pTau from neurons via autophagy. However, sustained TFEB activation and/or autophagy can create burden on cellular bioenergetics and can be deleterious. Thus, we engineered a minimally invasive optical system that could transiently alter autophagic flux. We optimized and testedan optogenetic gene expression system derived from apreviouslyengineered bacterial transcription factor, EL222. For the first time, our group utilized this system not only to spatial-temporally control nuclear TFEB expression, we also show light-induced TFEB has the capacity to reduce p-Tau burden in AD patient-derived human iPSC-neurons. Together, these results suggest that optically-regulatable gene expression of TFEB unlocks opto-therapeutics to treat AD and other dementias.
Reversible induction of mitophagy by an optogenetic bimodular system.
Autophagy-mediated degradation of mitochondria (mitophagy) is a key process in cellular quality control. Although mitophagy impairment is involved in several patho-physiological conditions, valuable methods to induce mitophagy with low toxicity in vivo are still lacking. Herein, we describe a new optogenetic tool to stimulate mitophagy, based on light-dependent recruitment of pro-autophagy protein AMBRA1 to mitochondrial surface. Upon illumination, AMBRA1-RFP-sspB is efficiently relocated from the cytosol to mitochondria, where it reversibly mediates mito-aggresome formation and reduction of mitochondrial mass. Finally, as a proof of concept of the biomedical relevance of this method, we induced mitophagy in an in vitro model of neurotoxicity, fully preventing cell death, as well as in human T lymphocytes and in zebrafish in vivo. Given the unique features of this tool, we think it may turn out to be very useful for a wide range of both therapeutic and research applications.
Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions.
Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested 'AND' gate design of SPARK2-in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest-yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.
Use of Exogenous and Endogenous Photomediators as Efficient ROS Modulation Tools: Results and Perspectives for Therapeutic Purposes.
Reactive Oxygen Species (ROS) play an essential dual role in living systems. Healthy levels of ROS modulate several signaling pathways, but at the same time, when they exceed normal physiological amounts, they work in the opposite direction, playing pivotal functions in the pathophysiology of multiple severe medical conditions (i.e., cancer, diabetes, neurodegenerative and cardiovascular diseases, and aging). Therefore, the research for methods to detect their levels via light-sensitive fluorescent probes has been extensively studied over the years. However, this is not the only link between light and ROS. In fact, the modulation of ROS mediated by light has been exploited already for a long time. In this review, we report the state of the art, as well as recent developments, in the field of photostimulation of oxidative stress, from photobiomodulation (PBM) mediated by naturally expressed light-sensitive proteins to the most recent optogenetic approaches, and finally, we describe the main methods of exogenous stimulation, in particular highlighting the new insights based on optically driven ROS modulation mediated by polymeric materials.
Optogenetic manipulation of stomatal kinetics improves carbon assimilation, water use, and growth.
Stomata serve dual and often conflicting roles, facilitating carbon dioxide influx into the plant leaf for photosynthesis and restricting water efflux via transpiration. Strategies for reducing transpiration without incurring a cost for photosynthesis must circumvent this inherent coupling of carbon dioxide and water vapor diffusion. We expressed the synthetic, light-gated K+ channel BLINK1 in guard cells surrounding stomatal pores in Arabidopsis to enhance the solute fluxes that drive stomatal aperture. BLINK1 introduced a K+ conductance and accelerated both stomatal opening under light exposure and closing after irradiation. Integrated over the growth period, BLINK1 drove a 2.2-fold increase in biomass in fluctuating light without cost in water use by the plant. Thus, we demonstrate the potential of enhancing stomatal kinetics to improve water use efficiency without penalty in carbon fixation.
Direct observation and analysis of the dynamics of the photoresponsive transcription factor GAL4.
We report direct visualization of the dynamic behavior of transcription factor GAL4 with photo-switching function (GAL4-VVD) in the DNA origami structure. Using high-speed atomic force microscopy (HS-AFM), we observed photo-induced complex formation of GAL4-VVD and substrate DNAs. Dynamic behaviors of GAL4-VVD such as binding, sliding, stalling, and dissociation with two substrate DNA strands, containing specific GAL4 binding sites, were observed. We also observed inter-strand hopping on two double-stranded (ds) DNAs. On a long substrate DNA strand that contained five binding sites, a series of GAL4-VVD/DNA interactions including binding, sliding, stalling, and dissociation could be identified while interacting with the surface. We also found the clear difference in the movement of GAL4-VVD between sliding and stalling in the AFM images. Detailed analysis revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping for rapidly searching specific binding sites, and then stalled to the specific sites for the stable complex formation. The results suggest the existence of the different conformational mode of the protein for sliding and stalling. This single-molecule imaging system at the nanoscale resolution provides the insight of the searching mechanism of the DNA binding proteins.
Synthetic cell-like membrane interfaces for probing dynamic protein-lipid interactions.
The ability to rapidly screen interactions between proteins and membrane-like interfaces would aid in establishing the structure-function of protein-lipid interactions, provide a platform for engineering lipid-interacting protein tools, and potentially inform the signaling mechanisms and dynamics of membrane-associated proteins. Here, we describe the preparation and application of water-in-oil (w/o) emulsions with lipid-stabilized droplet interfaces that emulate the plasma membrane inner leaflet with tunable composition. Fluorescently labeled proteins are easily visualized in these synthetic cell-like droplets on an automated inverted fluorescence microscope, thus allowing for both rapid screening of relative binding and spatiotemporally resolved analyses of for example, protein-interface association and dissociation dynamics and competitive interactions, using commonplace instrumentation. We provide protocols for droplet formation, automated imaging assays and analysis, and the production of the positive control protein BcLOV4, a natural photoreceptor with a directly light-regulated interaction with anionic membrane phospholipids that is useful for optogenetic membrane recruitment.
A yeast system for discovering optogenetic inhibitors of eukaryotic translation initiation.
The precise spatiotemporal regulation of protein synthesis is essential for many complex biological processes such as memory formation, embryonic development and tumor formation. Current methods used to study protein synthesis offer only a limited degree of spatiotemporal control. Optogenetic methods, in contrast, offer the prospect of controlling protein synthesis non-invasively within minutes and with a spatial scale as small as a single synapse. Here, we present a hybrid yeast system where growth depends on the activity of human eukaryotic initiation factor 4E (eIF4E) that is suitable for screening optogenetic designs for the down-regulation of protein synthesis. We used this system to screen a diverse initial panel of 15 constructs designed to couple a light switchable domain (PYP, RsLOV, LOV, Dronpa) to 4EBP2 (eukaryotic initiation factor 4E binding protein 2), a native inhibitor of translation initiation. We identified cLIPS1 (circularly permuted LOV inhibitor of protein synthesis 1), a fusion of a segment of 4EBP2 and a circularly permuted version of the LOV2 domain from Avena sativa, as a photo-activated inhibitor of translation. Adapting the screen for higher throughput, we tested small libraries of cLIPS1 variants and found cLIPS2, a construct with an improved degree of optical control. We show that these constructs can both inhibit translation in yeast harboring a human eIF4E in vivo, and bind human eIF4E in vitro in a light-dependent manner. This hybrid yeast system thus provides a convenient way for discovering optogenetic constructs that can regulate of human eIF4E-depednednt translation initiation in a mechanistically defined manner.
Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions.
Optogenetics enables the spatio-temporally precise control of cell and animal behaviour. Many optogenetic tools are driven by light-controlled protein-protein-interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPI to new target proteins is challenging because it is difficult to predict whether one the many available LSDs will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Optically inducible membrane recruitment and signaling systems.
Optical induction of intracellular signaling by membrane-associated and integral membrane proteins allows spatiotemporally precise control over second messenger signaling and cytoskeletal rearrangements that are important to cell migration, development, and proliferation. Optogenetic membrane recruitment of a protein-of-interest to control its signaling by altering subcellular localization is a versatile means to these ends. Here, we summarize the signaling characteristics and underlying structure-function of RGS-LOV photoreceptors as single-component membrane recruitment tools that rapidly, reversibly, and efficiently carry protein cargo from the cytoplasm to the plasma membrane by a light-regulated electrostatic interaction with the membrane itself. We place the technology-relevant features of these recently described natural photosensory proteins in context of summarized protein engineering and design strategies for optically controlling membrane protein signaling.
Membrane-Associated, Not Cytoplasmic or Nuclear, FGFR1 Induces Neuronal Differentiation.
The intracellular transport of receptor tyrosine kinases results in the differential activation of various signaling pathways. In this study, optogenetic stimulation of fibroblast growth factor receptor type 1 (FGFR1) was performed to study the effects of subcellular targeting of receptor kinases on signaling and neurite outgrowth. The catalytic domain of FGFR1 fused to the algal light-oxygen-voltage-sensing (LOV) domain was directed to different cellular compartments (plasma membrane, cytoplasm and nucleus) in human embryonic kidney (HEK293) and pheochromocytoma (PC12) cells. Blue light stimulation elevated the pERK and pPLCγ1 levels in membrane-opto-FGFR1-transfected cells similarly to ligand-induced receptor activation; however, no changes in pAKT levels were observed. PC12 cells transfected with membrane-opto-FGFR1 exhibited significantly longer neurites after light stimulation than after growth factor treatment, and significantly more neurites extended from their cell bodies. The activation of cytoplasmic FGFR1 kinase enhanced ERK signaling in HEK293 cells but not in PC12 cells and did not induce neuronal differentiation. The stimulation of FGFR1 kinase in the nucleus also did not result in signaling changes or neurite outgrowth. We conclude that FGFR1 kinase needs to be associated with membranes to induce the differentiation of PC12 cells mainly via ERK activation.
Bacteriophytochromes - from informative model systems of phytochrome function to powerful tools in cell biology.
Bacteriophytochromes are a subfamily of the diverse light responsive phytochrome photoreceptors. Considering their preferential interaction with biliverdin IXα as endogenous cofactor, they have recently been used for creating optogenetic tools and engineering fluorescent probes. Ideal absorption characteristics for the activation of bacteriophytochrome-based systems in the therapeutic near-infrared window as well the availability of biliverdin in mammalian tissues have resulted in tremendous progress in re-engineering bacteriophytochromes for diverse applications. At the same time, both the structural analysis and the functional characterization of diverse naturally occurring bacteriophytochrome systems have unraveled remarkable differences in signaling mechanisms and have so far only touched the surface of the evolutionary diversity within the family of bacteriophytochromes. This review highlights recent findings and future challenges.
Neurotrophin receptor tyrosine kinases regulated with near-infrared light.
Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.
Controlling protein conformation with light.
Optogenetics, genetically encoded engineering of proteins to respond to light, has enabled precise control of the timing and localization of protein activity in live cells and for specific cell types in animals. Light-sensitive ion channels have become well established tools in neurobiology, and a host of new methods have recently enabled the control of other diverse protein structures as well. This review focuses on approaches to switch proteins between physiologically relevant, naturally occurring conformations using light, accomplished by incorporating light-responsive engineered domains that sterically and allosterically control the active site.
Photodimerization systems for regulating protein-protein interactions with light.
Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.
Physical Plasma Membrane Perturbation Using Subcellular Optogenetics Drives Integrin-Activated Cell Migration.
Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.
Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.
The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.