Curated Optogenetic Publication Database

Abstract: The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.

Abstract: Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

Abstract: We describe a modular computational framework for analyzing cell-wide spatiotemporal signaling dynamics in single-cell microscopy experiments that accounts for the experiment-specific geometric and diffractive complexities that arise from heterogeneous cell morphologies and optical instrumentation. Inputs are unique cell geometries and protein concentrations derived from confocal stacks and spatiotemporally varying environmental stimuli. After simulating the system with a model of choice, the output is convolved with the microscope point-spread function for direct comparison with the observable image. We experimentally validate this approach in single cells with BcLOV4, an optogenetic membrane recruitment system for versatile control over cell signaling, using a three-dimensional non-linear finite element model with all parameters experimentally derived. The simulations recapitulate observed subcellular and cell-to-cell variability in BcLOV4 signaling, allowing for inter-experimental differences of cellular and instrumentation origins to be elucidated and resolved for improved interpretive robustness. This single-cell approach will enhance optogenetics and spatiotemporally resolved signaling studies.

Abstract: We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.

Abstract: We describe single-component optogenetic probes whose activation dynamics depend on both light and temperature. We used the BcLOV4 photoreceptor to stimulate Ras and phosphatidyl inositol-3-kinase signaling in mammalian cells, allowing activation over a large dynamic range with low basal levels. Surprisingly, we found that BcLOV4 membrane translocation dynamics could be tuned by both light and temperature such that membrane localization spontaneously decayed at elevated temperatures despite constant illumination. Quantitative modeling predicted BcLOV4 activation dynamics across a range of light and temperature inputs and thus provides an experimental roadmap for BcLOV4-based probes. BcLOV4 drove strong and stable signal activation in both zebrafish and fly cells, and thermal inactivation provided a means to multiplex distinct blue-light sensitive tools in individual mammalian cells. BcLOV4 is thus a versatile photosensor with unique light and temperature sensitivity that enables straightforward generation of broadly applicable optogenetic tools.

Abstract: Optogenetic tools are created to control RhoA GTPase, a central regulator of actin organization and actomyosin contractility. RhoA GTPase, or its upstream activator ARHGEF11, is fused to BcLOV4, a photoreceptor that can be dynamically recruited to the plasma membrane by a light-regulated protein-lipid electrostatic interaction with the inner leaflet. Direct membrane recruitment of these proteins induces potent contractile signaling sufficient to separate adherens junctions with as little as one pulse of blue light. Induced cytoskeletal morphology changes are dependent on the alignment of the spatially patterned stimulation with the underlying cell polarization. RhoA-mediated cytoskeletal activation drives yes-associated protein (YAP) nuclear localization within minutes and consequent mechanotransduction verified by YAP-transcriptional enhanced associate domain transcriptional activity. These single-transgene tools do not require protein binding partners for dynamic membrane localization and permit spatiotemporally precise control over RhoA signaling to advance the study of its diverse regulatory roles in cell migration, morphogenesis, and cell cycle maintenance.

Abstract: We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment. Opto-Rac1 is a fusion of wildtype human Rac1 small GTPase to the C-terminal region of BcLOV4, a LOV (light-oxygen-voltage) photoreceptor that rapidly binds the plasma membrane upon blue-light activation via a direct electrostatic interaction with anionic membrane phospholipids. Translocation of the fused wildtype Rac1 effector permits its activation by GEFs (guanine nucleotide exchange factors) and consequent actin polymerization and lamellipodia formation, unlike in existing single-chain systems that operate by allosteric photo-switching of constitutively active Rac1 or the heterodimerization-based (i.e. two-component) membrane recruitment of a Rac1-activating GEF. Opto-Rac1 induction of lamellipodia formation was spatially restricted to the patterned illumination field and was efficient, requiring sparse stimulation duty ratios of ∼1-2% (at the sensitivity threshold for flavin photocycling) to cause significant changes in cell morphology. This work exemplifies how the discovery of LOV proteins of distinct signal transmission modes can beget new classes of optogenetic tools for controlling cellular function.

Abstract: 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.

Abstract: 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.

Abstract: Microplate readers are foundational instruments in ex-perimental biology and bioengineering that enable mul-tiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construc-tion, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs <$3500, a fraction of the cost of commer-cial plate readers, and can detect the fluorescence of common dyes down to ~10 nanomolar concentration. Functional capabilities were demonstrated in context of synthetic biology, optoge¬netics, and photosensory biol-ogy: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing, and by flavin photocycling kinetic measure-ments of a LOV (light-oxygen-voltage) domain photo-receptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and au-tomating it using the custom Python-based API (Appli-cation Program Interface) are provided. This work con-tributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid proto-typing capabilities and low-cost electronics, optoelec-tronics, and microcomputers.

Abstract: We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Abstract: As fast terminators of G-protein coupled receptor (GPCR) signaling, regulators of G-protein signaling (RGS) serve critical roles in fine-tuning second messenger levels and, consequently, cellular responses to external stimuli. Here, we report the creation of an optogenetic RGS2 (opto-RGS2) that suppresses agonist-evoked calcium oscillations by the inactivation of Gαq protein. In this system, cryptochrome-mediated hetero-dimerization of the catalytic RGS2-box with its N-terminal amphipathic helix reconstitutes a functional membrane-localized complex that can dynamically suppress store-operated release of calcium. Engineered opto-RGS2 cell lines were used to establish the role of RGS2 as a key inhibitory feedback regulator of the stochasticity of the Gαq-mediated calcium spike timing. RGS2 reduced the stochasticity of carbachol-stimulated calcium oscillations, and the feedback inhibition was coupled to the global calcium elevation by calmodulin/RGS2 interactions. The identification of a critical negative feedback circuit exemplifies the utility of optogenetic approaches for interrogating RGS/GPCR biology and calcium encoding principles through temporally precise molecular gain-of-function.

Abstract: Light-oxygen-voltage sensitive (LOV) flavoproteins are ubiquitous photoreceptors that mediate responses to environmental cues. Photosensory inputs are transduced into signaling outputs via structural rearrangements in sensor domains that consequently modulate the activity of an effector domain or multidomain clusters. Establishing the diversity in effector function and sensor-effector topology will inform what signaling mechanisms govern light-responsive behaviors across multiple kingdoms of life and how these signals are transduced. Here, we report the bioinformatics identification of over 6,700 candidate LOV domains (including over 4,000 previously unidentified sequences from plants and protists), and insights from their annotations for ontological function and structural arrangements. Motif analysis identified the sensors from ∼42 million ORFs, with strong statistical separation from other flavoproteins and non-LOV members of the structurally related Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family. Conserved-domain analysis determined putative light-regulated function and multidomain topologies. We found that for certain effectors, sensor-effector linker length is discretized based on both phylogeny and the preservation of α-helical heptad repeats within an extended coiled-coil linker structure. This finding suggests that preserving sensor-effector orientation is a key determinant of linker length, in addition to ancestry, in LOV signaling structure-function. We found a surprisingly high prevalence of effectors with functions previously thought to be rare among LOV proteins, such as regulators of G protein signaling, and discovered several previously unidentified effectors, such as lipases. This work highlights the value of applying genomic and transcriptomic technologies to diverse organisms to capture the structural and functional variation in photosensory proteins that are vastly important in adaptation, photobiology, and optogenetics.