Curated Optogenetic Publication Database

Abstract: Microtubules derived from the Golgi (Golgi MTs) have been implicated to play critical roles in persistent cell migration, but the underlying mechanisms remain elusive, partially due to the lack of direct observation of Golgi MT-dependent vesicular trafficking. Here, using super-resolution stochastic optical reconstruction microscopy (STORM), we discovered that post-Golgi cargos are more enriched on Golgi MTs and also surprisingly move much faster than on non-Golgi MTs. We found that, compared to non-Golgi MTs, Golgi MTs are morphologically more polarized toward the cell leading edge with significantly fewer inter-MT intersections. In addition, Golgi MTs are more stable and contain fewer lattice repair sites than non-Golgi MTs. Our STORM/live-cell imaging demonstrates that cargos frequently pause at the sites of both MT intersections and MT defects. Furthermore, by optogenetic maneuvering of cell direction, we demonstrate that Golgi MTs are essential for persistent cell migration but not for cells to change direction. Together, our study unveils the role of Golgi MTs in serving as a group of "fast tracks" for anterograde trafficking of post-Golgi cargos.

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: 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: 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: 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: 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: 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: 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: 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: 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: 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.

Abstract: Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin-Radixin-Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.

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: The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3'UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3'UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

Abstract: Light-inducible optogenetic systems offer precise spatiotemporal control over a myriad of biologic processes. Unfortunately, current systems are inherently limited by their dependence on external light sources for their activation. Further, the utility of laser/LED-based illumination strategies are often constrained by the need for invasive surgical procedures to deliver such devices and local heat production, photobleaching and phototoxicity that compromises cell and tissue viability. To overcome these limitations, we developed a novel BRET-activated optogenetics (BEACON) system that employs biologic light to control optogenetic tools. BEACON is driven by self-illuminating bioluminescent-fluorescent proteins that generate "spectrally tuned" biologic light via bioluminescence resonance energy transfer (BRET). Notably, BEACON robustly activates a variety of commonly used optogenetic systems in a spatially restricted fashion, and at physiologically relevant time scales, to levels that are achieved by conventional laser/LED light sources.

Abstract: Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.

Abstract: Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

Abstract: Building tissue from cells as the basic building block based on principles of self-assembly is a challenging and promising approach. Understanding how far principles of self-assembly and self-sorting known for colloidal particles apply to cells remains unanswered. In this study, we demonstrate that not just controlling the cell-cell interactions but also their dynamics is a crucial factor that determines the formed multicellular structure, using photoswitchable interactions between cells that are activated with blue light and reverse in the dark. Tuning dynamics of the cell-cell interactions by pulsed light activation, results in multicellular architectures with different sizes and shapes. When the interactions between cells are dynamic compact and round multicellular clusters under thermodynamic control form, while otherwise branched and lose aggregates under kinetic control assemble. These structures parallel what is known for colloidal assemblies under reaction and diffusion limited cluster aggregation, respectively. Similarly, dynamic interactions between cells are essential for cells to self-sort into distinct groups. Using four different cell types, which expressed two orthogonal cell-cell interaction pairs, the cells sorted into two separate assemblies. Bringing concepts of colloidal self-assembly to bottom-up tissue engineering provides a new theoretical framework and will help in the design of more predictable tissue-like structures.