Curated Optogenetic Publication Database

Abstract: Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.

Abstract: In multicellular organisms, living cells cooperate with each other to exert coordinated complex functions by responding to extracellular chemical or physical stimuli via proteins on the plasma membrane. Conventionally, chemical signal transduction or mechano-transduction has been investigated by chemical, genetic, or physical perturbation; however, these methods cannot manipulate biomolecular reactions at high spatiotemporal resolution. In contrast, recent advances in optogenetic perturbation approaches have succeeded in controlling signal transduction with external light. The methods have enabled spatiotemporal perturbation of the signaling, providing functional roles of the specific proteins. In this chapter, we summarize recent advances in the optogenetic tools that modulate the function of a receptor protein. While most optogenetic systems have been devised for controlling ion channel conductivities, the present review focuses on the other membrane proteins involved in chemical transduction or mechano-transduction. We describe the properties of natural or artificial photoreceptor proteins used in optogenetic systems. Then, we discuss the strategies for controlling the receptor protein functions by external light. Future prospects of optogenetic tool development are discussed.

Abstract: Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.

Abstract: Light-sensitive proteins can be used to perturb signaling networks in living cells and animals with high spatiotemporal resolution. We recently engineered a protein heterodimer that dissociates when irradiated with blue light and demonstrated that by fusing each half of the dimer to termini of a protein that it is possible to selectively block binding surfaces on the protein when in the dark. On activation with light, the dimer dissociates and exposes the binding surface, allowing the protein to bind its partner. Critical to the success of this system, called Z-lock, is that the linkers connecting the dimer components to the termini are engineered so that the dimer forms over the appropriate binding surface. Here, we develop and test a protocol in the Rosetta molecular modeling program for designing linkers for Z-lock. We show that the protocol can predict the most effective linker sets for three different light-sensitive switches, including a newly designed switch that binds the Rho-family GTPase Cdc42 on stimulation with blue light. This protocol represents a generalized computational approach to placing a wide variety of proteins under optogenetic control with Z-lock.

Abstract: Light can be controlled with high spatial and temporal accuracy. Therefore, optogenetics is an attractive experimental approach to modulate intracellular cytoskeleton dynamics at much faster timescales than by genetic modification. For example, in mammalian cells, microtubules (MTs) grow tens of micrometers per minute and many intracellular MT functions are mediated by a complex of +TIP proteins that dynamically associate with growing MT plus ends. EB1 is a central component of this +TIP protein network, and we recently developed a photo-inactivated π-EB1 by inserting a blue light-sensitive LOV2/Zdk1 module between the EB1 MT-binding domain and the +TIP adaptor domain. Blue light-induced π-EB1 photodissociation results in disassembly of the +TIP complex and strongly attenuates MT growth in mammalian cells.In this chapter, we discuss theoretical and practical aspects of how to perform high-resolution live-cell microscopy in combination with π-EB1 photodissociation. However, these techniques are broadly applicable to other LOV2-based and likely other blue light-sensitive optogenetics. In addition to being a tool to investigate +TIP functions acutely and with subcellular resolution, because of its dramatic and rapid change in intracellular localization, π-EB1 can serve as a powerful tool to test and characterize optogenetic illumination setups. We describe protocols on how to achieve micrometer-scale intracellular control of π-EB1 activity using patterned illumination, and we introduce a do-it-yourself LED cube design compatible with transmitted light microscopy in multiwell plates.

Abstract: Eukaryotic protein kinases (EPKs) catalyze the transfer of a phosphate group onto another protein in response to appropriate regulatory cues. In doing so, they provide a primary means for cellular information transfer. Consequently, EPKs play crucial roles in cell differentiation and cell-cycle progression, and kinase dysregulation is associated with numerous disease phenotypes including cancer. Nonnative cues for synthetically regulating kinases are thus much sought after, both for dissecting cell signaling pathways and for pharmaceutical development. In recent years advances in protein engineering and sequence analysis have led to new approaches for manipulating kinase activity, localization, and in some instances specificity. These tools have revealed fundamental principles of intracellular signaling and suggest paths forward for the design of therapeutic allosteric kinase regulators.

Abstract: Embryogenesis is coordinated by signaling pathways that pattern the developing organism. Many aspects of this process are not fully understood, including how signaling molecules spread through embryonic tissues, how signaling amplitude and dynamics are decoded, and how multiple signaling pathways cooperate to pattern the body plan. Optogenetic approaches can be used to address these questions by providing precise experimental control over a variety of biological processes. Here, we review how these strategies have provided new insights into developmental signaling and discuss how they could contribute to future investigations.

Abstract: Here we introduce Z-lock, an optogenetic approach for reversible, light-controlled steric inhibition of protein active sites. The light oxygen voltage (LOV) domain and Zdk, a small protein that binds LOV selectively in the dark, are appended to the protein of interest where they sterically block the active site. Irradiation causes LOV to change conformation and release Zdk, exposing the active site. Computer-assisted protein design was used to optimize linkers and Zdk-LOV affinity, for both effective binding in the dark, and effective light-induced release of the intramolecular interaction. Z-lock cofilin was shown to have actin severing ability in vitro, and in living cancer cells it produced protrusions and invadopodia. An active fragment of the tubulin acetylase αTAT was similarly modified and shown to acetylate tubulin on irradiation.

Abstract: Hydrogel materials have become a versatile platform for in vitro cell culture due to their ability to simulate many aspects of native tissues. However, precise spatiotemporal presentation of peptides and other biomolecules has remained challenging. Here we report the use of light-sensing proteins (LSPs), more commonly used in optogenetics research, as light-activated reversible binding sites within synthetic poly(ethylene glycol) (PEG) hydrogels. We used LOVTRAP, a two component LSP system consisting of LOV2, a protein domain that can cycle reversibly between "light" and "dark" conformations in response to blue light, and a z-affibody, Zdark (Zdk), that binds the dark state of LOV2, to spatiotemporally control the presentation of a recombinant protein within PEG hydrogels. By immobilizing LOV2 within PEG gels, we were able to capture a recombinant fluorescent protein (used as a model biomolecule) containing a Zdk domain, and then release the Zdk fusion protein using blue light. Zdk was removed from LOV2-containing PEG gels using focused blue light, resulting in a 30% reduction of fluorescence compared to unexposed regions of the gel. Additionally, the reversible binding capability of LOVTRAP was observed in our system, enabling our LOV2 gels to capture and release Zdk at least three times. By adding a Zdk domain to a recombinant peptide or protein, dynamic, spatially constrained displays of non-diffusing ligands within a PEG gel could feasibly be achieved using LOV2.

Abstract: Natural photoreceptors from plants and microorganisms are used for synthetic approaches to control cell behaviour. Light perception by the photoreceptor, often by a cofactor, induces a conformational change, which is transduced to the effector and regulates its activity. Synthetic combinations of photoreceptors and effectors resulted in a wealth of cellular events that are controlled by optogenetic tools. A general approach is to regulate protein abundance controlling either protein stability, protein biosynthesis or both with optogenetic tools.

Abstract: Optogenetics has demonstrated great potential in the fields of tissue engineering and regenerative medicine, from basic research to clinical applications. Spatiotemporal encoding during individual development has been widely identified and is considered a novel strategy for regeneration. A as a noninvasive method with high spatiotemporal resolution, optogenetics are suitable for this strategy. In this review, we discuss roles of dynamic signal coding in cell physiology and embryonic development. Several optogenetic systems are introduced as ideal optogenetic tools, and their features are compared. In addition, potential applications of optogenetics for tissue engineering are discussed, including light-controlled genetic engineering and regulation of signaling pathways. Furthermore, we present how emerging biomaterials and photoelectric technologies have greatly promoted the clinical application of optogenetics and inspired new concepts for optically controlled therapies. Our summation of currently available data conclusively demonstrates that optogenetic tools are a promising method for elucidating and simulating developmental processes, thus providing vast prospects for tissue engineering and regenerative medicine applications.

Abstract: Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKIN-dependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux. Abbreviations: BSA: bovine serum albumin; CHMP: chromatin-modifying protein; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; ESCRT: endosomal sorting complex required for transport; HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HRP: horseradish peroxidase; ILV: intralumenal vesicle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LOV2: light oxygen voltage 2; MLS: mitochondrial localization sequence; MT-CO2: mitochondrially encoded cytochrome c oxidase II; O+A: oligomycin and antimycin A; PBS: phosphate-buffered saline; PIPES: piperazine-N,N-bis(2-ethanesulfonic acid); PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB: RAS-related in brain; SD: standard deviation; SEM: standard error of the mean; TOMM20: TOMM20: translocase of outer mitochondrial membrane 20; VCL: vinculin; VPS4: vacuolar protein sorting protein 4; Zdk1: Zdark 1; TUBG: Tubulin gamma chain.

Abstract: Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.

Abstract: 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.

Abstract: 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.

Abstract: 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.

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: 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.

Abstract: The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.

Abstract: Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.

Abstract: Optogenetics is a powerful method for studying dynamic processes in living cells and has advanced cell biology research over the recent past. Key to the successful application of optogenetics is the careful design of the light‐sensing module, typically employing a natural or engineered photoreceptor that links the exogenous light input to the cellular process under investigation. Light–oxygen–voltage (LOV) domains, a highly diverse class of small blue light sensors, have proven to be particularly versatile for engineering optogenetic input modules. These can function via diverse modalities, including inducible allostery, protein recruitment, dimerization, or dissociation. This study reviews recent advances in the development of LOV domain‐based optogenetic tools and their application for studying and controlling selected cellular functions. Focusing on the widely employed LOV2 domain from Avena sativa phototropin‐1, this review highlights the broad spectrum of engineering opportunities that can be explored to achieve customized optogenetic regulation. Finally, major bottlenecks in the development of optogenetic methods are discussed and strategies to overcome these with recent synthetic biology approaches are pointed out.

Abstract: In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occuring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.

Abstract: Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

Abstract: The field of optogenetics uses genetically encoded, light-responsive proteins to control physiological processes. This technology has been hailed as the one of the ten big ideas in brain science in the past decade,[1] the breakthrough of the decade,[2] and the method of the year in 2010[3] and again in 2014[4]. The excitement evidenced by these proclamations is confirmed by a couple of impressive numbers. The term "optogenetics" was coined in 2006.[5] As of December 2017, "optogenetics" is found in the title or abstract of almost 1600 currently funded National Institutes of Health grants. In addition, nearly 600 reviews on optogenetics have appeared since 2006, which averages out to approximately one review per week! However, in spite of these impressive numbers, the potential applications and implications of optogenetics are not even close to being fully realized. This is due, in large part, to the challenges associated with the design of optogenetic analogs of endogenous proteins. This review is written from a chemist's perspective, with a focus on the molecular strategies that have been developed for the construction of optogenetic proteins.

Abstract: Signal transductions are the basis for all cellular functions. Previous studies investigating signal transductions mainly relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies do not allow the modulation of protein activity in cells, tissues, and organs in animals with high spatial and temporal precision. Recently, non-channelrhodopsin-type optogenetic tools for regulating signal transduction have emerged. These photoswitches address several disadvantages of previous techniques, and allow us to control a variety of signal transductions such as cell membrane dynamics, calcium signaling, lipid signaling, and apoptosis. In this review, we summarize recent advances in the development of such photoswitches and how these optotools are applied to signaling processes.