Curated Optogenetic Publication Database

Abstract: Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.

Abstract: Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)-an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer-and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

Abstract: Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.

Abstract: Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.

Abstract: Migration of meiosis-I (MI) spindle from the cell center to a sub-cortical location is a critical step for mouse oocytes to undergo asymmetric meiotic cell division. In this study, we investigate the mechanism by which formin-2 (FMN2) orchestrates the initial movement of MI spindle. By defining protein domains responsible for targeting FMN2, we show that spindle-periphery localized FMN2 is required for spindle migration. The spindle-peripheral FMN2 nucleates short actin bundles from vesicles derived likely from the endoplasmic reticulum (ER) and concentrated in a layer outside the spindle. This layer is in turn surrounded by mitochondria. A model based on polymerizing actin filaments pushing against mitochondria, thus generating a counter force on the spindle, demonstrated an inherent ability of this system to break symmetry and evolve directional spindle motion. The model is further supported through experiments involving spatially biasing actin nucleation via optogenetics and disruption of mitochondrial distribution and dynamics.

Abstract: Optogenetic approaches for controlling Ca2+ channels provide powerful means for modulating diverse Ca2+-specific biological events in space and time. However, blue light-responsive photoreceptors are, in principle, considered inadequate for deep tissue stimulation unless accompanied by optic fiber insertion. Here, we present an ultra-light-sensitive optogenetic Ca2+ modulator, named monSTIM1 encompassing engineered cryptochrome2 for manipulating Ca2+ signaling in the brain of awake mice through non-invasive light delivery. Activation of monSTIM1 in either excitatory neurons or astrocytes of mice brain is able to induce Ca2+-dependent gene expression without any mechanical damage in the brain. Furthermore, we demonstrate that non-invasive Ca2+ modulation in neurons can be sufficiently and effectively translated into changes in behavioral phenotypes of awake mice.

Abstract: Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

Abstract: The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.

Abstract: The Gram-positive bacterium Bacillus subtilis exhibits complex spatial and temporal gene expression signals. Although optogenetic tools are ideal for studying such processes, none has been engineered for this organism. Here, we port a cyanobacterial light sensor pathway comprising the green/red photoreversible two-component system CcaSR, two metabolic enzymes for production of the chromophore phycocyanobilin (PCB), and an output promoter to control transcription of a gene of interest into B. subtilis. Following an initial non-functional design, we optimize expression of pathway genes, enhance PCB production via a translational fusion of the biosynthetic enzymes, engineer a strong chimeric output promoter, and increase dynamic range with a miniaturized photosensor kinase. Our final design exhibits over 70-fold activation and rapid response dynamics, making it well-suited to studying a wide range of gene regulatory processes. In addition, the synthetic biology methods we develop to port this pathway should make B. subtilis easier to engineer in the future.

Abstract: 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.

Abstract: 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.

Abstract: Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.

Abstract: From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.

Abstract: Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.

Abstract: During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, β2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.

Abstract: The Ras-ERK signaling pathway regulates diverse cellular processes in response to environmental stimuli and contains important therapeutic targets for cancer. Recent single cell studies revealed stochastic pulses of ERK activation, the frequency of which determines functional outcomes such as cell proliferation. Here we show that ERK pulses are initiated by localized protrusive activities. Chemically and optogenetically induced protrusions trigger ERK activation through various entry points into the feedback loop involving Ras, PI3K, the cytoskeleton, and cellular adhesion. The excitability of the protrusive signaling network drives stochastic ERK activation in unstimulated cells and oscillations upon growth factor stimulation. Importantly, protrusions allow cells to sense combined signals from substrate stiffness and the growth factor. Thus, by uncovering the basis of ERK pulse generation we demonstrate how signals involved in cell growth and differentiation are regulated by dynamic protrusions that integrate chemical and mechanical inputs from the environment.

Abstract: Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.

Abstract: Many natural transcription factors are regulated in a pulsatile fashion, but it remains unknown whether synthetic gene expression systems can benefit from such dynamic regulation. Here we find, using a fast-acting, optogenetic transcription factor in Saccharomyces cerevisiae, that dynamic pulsatile signals reduce cell-to-cell variability in gene expression. We then show that by encoding such signals into a single input, expression mean and variability can be independently tuned. Further, we construct a light-responsive promoter library and demonstrate how pulsatile signaling also enables graded multi-gene regulation at fixed expression ratios, despite differences in promoter dose-response characteristics. Pulsatile regulation can thus lead to beneficial functional behaviors in synthetic biological systems, which previously required laborious optimization of genetic parts or the construction of synthetic gene networks.

Abstract: During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. Here we use optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. We show that precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal-ventral or anterior-posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes.

Abstract: The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.

Abstract: Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.

Abstract: The actomyosin cytoskeleton, a key stress-producing unit in epithelial cells, oscillates spontaneously in a wide variety of systems. Although much of the signal cascade regulating myosin activity has been characterized, the origin of such oscillatory behavior is still unclear. Here, we show that basal myosin II oscillation in Drosophila ovarian epithelium is not controlled by actomyosin cortical tension, but instead relies on a biochemical oscillator involving ROCK and myosin phosphatase. Key to this oscillation is a diffusive ROCK flow, linking junctional Rho1 to medial actomyosin cortex, and dynamically maintained by a self-activation loop reliant on ROCK kinase activity. In response to the resulting myosin II recruitment, myosin phosphatase is locally enriched and shuts off ROCK and myosin II signals. Coupling Drosophila genetics, live imaging, modeling, and optogenetics, we uncover an intrinsic biochemical oscillator at the core of myosin II regulatory network, shedding light on the spatio-temporal dynamics of force generation.

Abstract: Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior.

Abstract: Arabidopsis cryptochrome 2 (CRY2) can simultaneously undergo light-dependent CRY2-CRY2 homo-oligomerization and CRY2-CIB1 hetero-dimerization, both of which have been widely used to optically control intracellular processes. Applications using CRY2-CIB1 interaction desire minimal CRY2 homo-oligomerization to avoid unintended complications, while those utilizing CRY2-CRY2 interaction prefer robust homo-oligomerization. However, selecting the type of CRY2 interaction has not been possible as the molecular mechanisms underlying CRY2 interactions are unknown. Here we report CRY2-CIB1 and CRY2-CRY2 interactions are governed by well-separated protein interfaces at the two termini of CRY2. N-terminal charges are critical for CRY2-CIB1 interaction. Moreover, two C-terminal charges impact CRY2 homo-oligomerization, with positive charges facilitating oligomerization and negative charges inhibiting it. By engineering C-terminal charges, we develop CRY2high and CRY2low with elevated or suppressed oligomerization respectively, which we use to tune the levels of Raf/MEK/ERK signaling. These results contribute to our understanding of the mechanisms underlying light-induced CRY2 interactions and enhance the controllability of CRY2-based optogenetic systems.Cryptochrome 2 (CRY2) can form light-regulated CRY2-CRY2 homo-oligomers or CRY2-CIB1 hetero-dimers, but modulating these interactions is difficult owing to the lack of interaction mechanism. Here the authors identify the interactions facilitating homo-oligomers and introduce mutations to create low and high oligomerization versions.

Abstract: A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease.