Curated Optogenetic Publication Database

Abstract: Pulsed actomyosin contractions drive morphogenetic processes, but how cyclic frequencies and amplitudes of contractions are tuned to achieve processive shrinking of cell surfaces remains unclear. In this issue of Developmental Cell, Cavanaugh et al. (2020) use optogenetics and biophysical modeling to demonstrate how cells respond to different oscillatory force profiles.

Abstract: Optogenetics have recently increased in popularity as tools to study behavior in response to the brain and how these trends relate back to a neuronal circuit. Additionally, the high demand for human cerebral tissue in research has led to the generation of a new model to investigate human brain development and disease. Human Pluripotent Stem Cells (hPSCs) have been previously used to recapitulate the development of several tissues such as intestine, stomach and liver and to model disease in a human context, recently new improvements have been made in the field of hPSC-derived brain organoids to better understand overall brain development but more specifically, to mimic inter-neuronal communication. This review aims to highlight the recent advances in these two separate approaches of brain research and to emphasize the need for overlap. These two novel approaches would combine the study of behavior along with the specific circuits required to produce the signals causing such behavior. This review is focused on the current state of the field, as well as the development of novel optogenetic technologies and their potential for current scientific study and potential therapeutic use.

Abstract: Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.

Abstract: Optogenetic tools can provide direct and programmable control of gene expression. Light-inducible recombinases, in particular, offer a powerful method for achieving precise spatiotemporal control of DNA modification. However, to-date this technology has been largely limited to eukaryotic systems. Here, we develop optogenetic recombinases for Escherichia coli that activate in response to blue light. Our approach uses a split recombinase coupled with photodimers, where blue light brings the split protein together to form a functional recombinase. We tested both Cre and Flp recombinases, Vivid and Magnet photodimers, and alternative protein split sites in our analysis. The optimal configuration, Opto-Cre-Vvd, exhibits strong blue light-responsive excision and low ambient light sensitivity. For this system we characterize the effect of light intensity and the temporal dynamics of light-induced recombination. These tools expand the microbial optogenetic toolbox, offering the potential for precise control of DNA excision with light-inducible recombinases in bacteria.

Abstract: Optogenetic activation of receptors has advantages compared with chemical or ligand treatment because of its high spatial and temporal precision. Especially in the brain, the use of a genetically encoded light-tunable receptor is superior to direct infusion or systemic drug treatment. We applied light activatable TrkB receptor in mouse brain with reduced basal activity by incorporating Cry2PHR mutant, Opto-cytTrkB(E281A). Upon AAV mediated gene delivery, this form was expressed at sufficient levels in the mouse hippocampus (HPC) and medial entorhinal cortex (MEC) retaining normal canonical signal transduction by blue light stimulus, even by delivery of non-invasive LED light on the mouse head. Within target cells, where its expression was driven by a cell type-specific promoter, Opto-cytTrkB(E281A)-mediated TrkB signaling could be controlled by adjusting light-stimulation conditions. We further demonstrated that Opto-cytTrkB(E281A) could locally induce TrkB signaling in axon terminals in the MEC-HPC. In summary, Opto-cytTrkB(E281A) will be useful for elucidating time- and region-specific roles of TrkB signaling ranging from cellular function to neural circuit mechanisms.

Abstract: This work establishes a means to exploit genetic networks to create living synthetic composites that change shape in response to specific biochemical or physical stimuli. Baker's yeast embedded in a hydrogel forms a responsive material where cellular proliferation leads to a controllable increase in the composite volume of up to 400%. Genetic manipulation of the yeast enables composites where volume change on exposure to l-histidine is 14× higher than volume change when exposed to d-histidine or other amino acids. By encoding an optogenetic switch into the yeast, spatiotemporally controlled shape change is induced with pulses of dim blue light (2.7 mW/cm2). These living, shape-changing materials may enable sensors or medical devices that respond to highly specific cues found within a biological milieu.

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: 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 this study, we use an optogenetic inhibitor of JNK in dendritic spine sub-compartments of rat hippocampal neurons. JNK inhibition exerts rapid (within seconds) reorganisation of actin in the spine-head. Using real-time FRET to measure JNK activity, we find that either excitotoxic insult (NMDA) or endocrine stress (corticosterone), activate spine-head JNK causing internalization of AMPARs and spine retraction. Both events are prevented upon optogenetic inhibition of JNK, and rescued by JNK inhibition even 2 h after insult. Moreover, we identify that the fast-acting anti-depressant ketamine reduces JNK activity in hippocampal neurons suggesting that JNK inhibition may be a downstream mediator of its anti-depressant effect. In conclusion, we show that JNK activation plays a role in triggering spine elimination by NMDA or corticosterone stress, whereas inhibition of JNK facilitates regrowth of spines even in the continued presence of glucocorticoid. This identifies that JNK acts locally in the spine-head to promote AMPAR internalization and spine shrinkage following stress, and reveals a protective function for JNK inhibition in preventing spine regression.SIGNIFICANCE STATEMENT Identifying mechanisms that underlie dendritic spine elimination is important if we are to understand maladaptive changes that contribute to psychiatric disease. Compartment-specific, fast-acting tools can expedite this endeavor. Here we use a light-activated inhibitor of JNK to control kinase activity specifically in dendritic spines. Light-activation of the JNK inhibitor reduces AMPA receptor removal and spine regression in response to corticosterone and NMDA stress. Furthermore, we find that the anti-depressant drug ketamine lowers JNK activity in hippocampal neurons and prevents spine regression, though direct JNK inhibition is more effective. This study identifies a role for JNK in spine regression and may be relevant for endocrine control of synaptic strength and for conditions where chronic glucocorticoid stress leads to spine elimination.

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: Intracellular signaling forms complicated networks that involve dynamic alterations of the protein-protein interactions occurring inside a cell. To dissect these complex networks, light-inducible optogenetic technologies have offered a novel approach for modulating the function of intracellular machineries in space and time. Optogenetic approaches combine genetic and optical methods to initiate and control protein functions within live cells. In this review, we provide an overview of the optical strategies that can be used to manipulate intracellular signaling proteins and secondary messengers at the molecular level. We briefly address how an optogenetic actuator can be engineered to enhance homo- or hetero-interactions, survey various optical tools and targeting strategies for controlling cell-signaling pathways, examine their extension to in vivo systems and discuss the future prospects for the field.

Abstract: Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.

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: Individual cellular activities fluctuate but are constantly coordinated at the population level via cell-cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM)1,2. This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion3-7. However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe (Lfng) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng-null PSM. However, when Lfng-null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell-cell coupling. Mixed cultures of control and Lfng-null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results-together with mathematical modelling-raised the possibility that Lfng-null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators8. Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng-null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation.

Abstract: Taking advantage of the recent development of genetically-defined photo-activatable actuator molecules, cellular functions, including gene expression, can be controlled by exposure to light. Such optogenetic strategies enable precise temporal and spatial manipulation of targeted single cells or groups of cells at a level hitherto impossible. In this review, we introduce light-controllable gene expression systems exploiting blue or red/far-red wavelengths and discuss their inherent properties potentially affecting induced downstream gene expression patterns. We also discuss recent advances in optical devices that will extend the application of optical gene expression control technologies into many different areas of biology and medicine.

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: The progress in live-cell imaging technologies has revealed diverse dynamic patterns of transcriptional activity in various contexts. The discovery raised a next question of whether the gene expression patterns play causative roles in triggering specific biological events or not. Here, we introduce optogenetic methods that realize optical control of gene expression dynamics in mammalian cells and would be utilized for answering the question, by referring the past, the present, and the future.

Abstract: Cells respond to a wide range of extracellular stimuli, and process the input information through an intracellular signaling system comprised of biochemical and biophysical reactions, including enzymatic and protein-protein interactions. It is essential to understand the molecular mechanisms underlying intracellular signal transduction in order to clarify not only physiological cellular functions but also pathological processes such as tumorigenesis. Fluorescent proteins have revolutionized the field of life science, and brought the study of intracellular signaling to the single-cell and subcellular levels. Much effort has been devoted to developing genetically encoded fluorescent biosensors based on fluorescent proteins, which enable us to visualize the spatiotemporal dynamics of cell signaling. In addition, optogenetic techniques for controlling intracellular signal transduction systems have been developed and applied in recent years by regulating intracellular signaling in a light-dependent manner. Here, we outline the principles of biosensors for probing intracellular signaling and the optogenetic tools for manipulating them.

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: In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.

Abstract: Directional cell motility relies on the ability of single cells to establish a front-rear polarity and can occur in the absence of external cues. The initiation of migration has often been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cell-substrate interaction forces has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics the complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters of NIH-3T3 fibroblasts and RPE1 epithelial cells reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to trigger cell motility in 1D in the absence of pre-established polarity. A theoretical model validates the crucial role of adhesion dynamics, proposing that front-rear polarity can emerge independently of a complex self-polarizing system.

Abstract: Photoactivated adenylyl cyclase (PAC) is a unique protein that, upon blue light exposure, catalyzes cAMP production. The crystal structures of two PACs, from Oscillatoria acuminata (OaPAC) and Beggiatoa sp. (bPAC), have been solved, and they show a high degree of similarity. However, the photoactivity of OaPAC is much lower than that of bPAC, and the regulatory mechanism of PAC photoactivity, which induces the difference in activity between OaPAC and bPAC, has not yet been clarified. Here, we investigated the role of the C-terminal region in OaPAC, the length of which is the only notable difference from bPAC. We found that the photoactivity of OaPAC was inversely proportional to the C-terminal length. However, the deletion of more than nine amino acids did not further increase the activity, indicating that the nine amino acids at the C-terminal critically affect the photoactivity. Besides, absorption spectral features of light-sensing domains (BLUF domains) of the C-terminal deletion mutants showed similar light-dependent spectral shifts as in WT, indicating that the C-terminal region influences the activity without interacting with the BLUF domain. The study characterizes new PAC mutants with modified photoactivities, which could be useful as optogenetics tools.

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: Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.