Curated Optogenetic Publication Database

Abstract: Auditory receptors can be motile to actively amplify their mechanical input. Here we describe a novel and different type of motility that, residing in supporting cells, shapes physiological responses of mechanoreceptor cells. In Drosophila larvae, supporting cap cells transmit mechanical stimuli to proprioceptive chordotonal neurons. We found that the cap cells are strongly pre-stretched at rest to twice their relaxed length. The tension in these cells is modulated by non-muscle myosin-II motors. Activating the motors optogenetically causes contractions of the cap cells. Cap-cell-specific knockdown of the regulatory light chain of myosin-II alters mechanically evoked receptor neuron responses, converting them from phasic to more tonic, impairing sensory adaptation. Hence, two motile mechanisms seem to operate in concert in insect chordotonal organs, one in the sensory receptor neurons, based on dynein, and the other in supporting cells, based on myosin.

Abstract: Liquid-liquid phase separation (LLPS) has emerged as a major organizing principle in cells. Recent work showed that multiple components of integrin-mediated focal adhesions including p130Cas can form LLPS, which govern adhesion dynamics and related cell behaviors. In this study, we found that the focal adhesion protein p130Cas drives formation of structures with the characteristics of LLPS that bud from focal adhesions into the cytoplasm. Condensing concentrated cytoplasm around p130Cas-coated beads allowed their isolation, which were enriched in a subset of focal adhesion proteins, mRNAs and RNA binding proteins, including those implicated in inhibiting mRNA translation. Plating cells on very high concentrations of fibronectin to induce large focal adhesions inhibited message translation which required p130Cas and correlated with droplet formation. Photo-induction of p130Cas condensates using the Cry2 system also reduced translation. These results identify a novel regulatory mechanism in which high adhesion limits message translation via induction of p130Cas-dependent cytoplasmic LLPS. This mechanism may contribute to the quiescent state of very strongly adhesive myofibroblasts and senescent cells.

Abstract: For cells to thrive, they must make appropriate fate decisions based on a myriad of internal and external stimuli. But how do they integrate these different forms of information to contextualise their decisions? Old yeast cells showed an ability to dampen their proliferation as they entered senescence. Conversely, they had an enhanced ability to promote proliferation during escape from pheromone stimulation. A network of nucleoprotein condensation states involving processing bodies (P-bodies) and the prion-like RNA-binding protein, Whi3, controlled these opposing fate decisions. In old but not in young cells, condensation of Whi3 was both necessary and sufficient for senescence entry. In old cells, Whi3 localised to age-dependent P-bodies. Preventing their formation stopped Whi3 condensation from driving senescence entry. Challenging old cells with an external stimulus, pheromone, revealed that the condensates had a second function: potentiating the cell's ability to trigger escape from the mating pheromone response. These findings identify biomolecular condensation as an integrator of contextual information as cells make decisions, enabling them to navigate overlapping life events.

Abstract: During mitosis, the human microtubule depolymerase KIF2C increases the turnover of kinetochore-microtubule attachments. This facilitates the correction of attachment errors. Moreover, BRCA2 phosphorylated at Thr207 by PLK1 (BRCA2-pT207) assembles a complex including PLK1, PP2A and BUBR1 that contributes to the stability of the kinetochore-microtubule attachments. PLK1, together with Aurora B, critically regulate the accurate segregation of chromosomes. Here we demonstrate that KIF2C contains an N-terminal domain that binds directly to several phosphorylated peptides, including BRCA2-pT207. Using an optogenetic platform, we reveal that KIF2C assembles into membrane-less compartments or biomolecular condensates that are located next to microtubules. We provide evidence that condensate assembly depends on the presence of the newly defined N-terminal phospho-binding domain of KIF2C and on the kinase activities of Aurora B and PLK1. Moreover, KIF2C condensates concentrate active PLK1 and colocalize with BRCA2-pT207. We propose that, because of its phospho-dependent binding and oligomerization capacities, KIF2C forms biomolecular condensates that partition PLK1 and locally amplify its kinase activity during mitosis.

Abstract: A crucial step in early embryogenesis is the establishment of spatial patterns of signaling activity. Tools to perturb morphogen signals with high resolution in space and time can help reveal how embryonic cells decode these signals to make appropriate fate decisions. Here, we present new optogenetic reagents and an experimental pipeline for creating designer Nodal signaling patterns in live zebrafish embryos. Nodal receptors were fused to the light sensitive heterodimerizing pair Cry2/CIB1N, and the Type II receptor was sequestered to the cytosol. The improved optoNodal2 reagents eliminate dark activity and improve response kinetics, without sacrificing dynamic range. We adapted an ultra-widefield microscopy platform for parallel light patterning in up to 36 embryos and demonstrated precise spatial control over Nodal signaling activity and downstream gene expression. Patterned Nodal activation drove precisely controlled internalization of endodermal precursors. Further, we used patterned illumination to generate synthetic signaling patterns in Nodal signaling mutants, rescuing several characteristic developmental defects. This study establishes an experimental toolkit for systematic exploration of Nodal signaling patterns in live embryos.

Abstract: Cytokinesis physically separates daughter cells at the end of cell division. This step is particularly challenging for epithelial cells, which are connected to their neighbors and to the extracellular matrix by transmembrane protein complexes. To systematically evaluate the impact of the cell adhesion machinery on epithelial cytokinesis efficiency, we performed an RNAi-based modifier screen in the Drosophila follicular epithelium. Strikingly, this unveiled adhesion molecules and transmembrane receptors that facilitate cytokinesis completion. Among these is Dystroglycan, which connects the extracellular matrix to the cytoskeleton via Dystrophin. Live imaging revealed that Dystrophin and Dystroglycan become enriched in the ingressing membrane, below the cytokinetic ring, during and after ring constriction. Using multiple alleles, including Dystrophin isoform-specific mutants, we show that Dystrophin/Dystroglycan localization is linked with unanticipated roles in regulating cytokinetic ring contraction and in preventing membrane regression during the abscission period. Altogether, we provide evidence that, rather than opposing cytokinesis completion, the machinery involved in cell-cell and cell-matrix interactions has also evolved functions to ensure cytokinesis efficiency in epithelial tissues.

Abstract: Proteinaceous inclusions formed by C9orf72 derived dipeptide-repeat (DPR) proteins are a histopathological hallmark in ∼50% of familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) cases. However DPR aggregation/inclusion formation could not be efficiently recapitulated in cell models for four out of five DPRs. We utilised the Cry2olig optogenetic module to achieve chemical-free and controllable poly-PR condensation/aggregation in cultured cells. This approach revealed that poly-PR forms anisotropic condensates in the nucleus and that RNA limits their fusion-dependent growth. Poly-PR self-assembly induced nuclear TDP-43 condensation without activation of cellular stress response. Poly-PR cytoplasmic redistribution and aggregation could be also achieved with prolonged light stimulation. Cytoplasmic poly-PR assemblies were more persistent than its nuclear condensates, selectively sequestered TDP-43 and surrounded spontaneous stress granules. Our data suggest that poly-PR condensation in the nucleus and cytoplasm, causative of TDP-43 dysfunction, may constitute an early pathological event in C9-ALS/FTD. The opto-DPR platform described here is a useful tool for modelling cytopathologies elicited by DPR aggregation for mechanistic research and drug discovery.

Abstract: Innate immunity protects us in youth but turns against us as we age. The reason for this tradeoff is unclear. Seeking a thermodynamic basis, we focused on death fold domains (DFDs), whose ordered polymerization has been stoichiometrically linked to innate immune signal amplification. We hypothesized that soluble ensembles of DFDs function as phase change batteries that store energy via supersaturation and subsequently release it through nucleated polymerization. Using imaging and FRET-based cytometry to characterize the phase behaviors of all 109 human DFDs, we found that the hubs of innate immune signaling networks encode large nucleation barriers that are intrinsically insulated from cross-pathway activation. We showed via optogenetics that supersaturation drives signal amplification and that the inflammasome is constitutively supersaturated in vivo. Our findings reveal that the soluble “inactive” states of adaptor DFDs function as essential, yet impermanent, kinetic barriers to inflammatory cell death, suggesting a thermodynamic driving force for aging.

Abstract: Directed evolution is a powerful method in biological engineering. Current approaches were devised for evolving steady-state properties such as enzymatic activity or fluorescence intensity. A fundamental problem remains how to evolve dynamic, multi-state, or computational functionalities, e.g., folding times, on-off kinetics, state-specific activity, stimulus-responsiveness, or switching and logic capabilities. These require applying selection pressure on all of the states of a protein of interest (POI) and the transitions between them. We realized that optogenetics and cell cycle oscillations could be leveraged for a novel directed evolution paradigm (‘optovolution’) that is germane for this need: We designed a signaling cascade in budding yeast where optogenetic input switches the POI between off (0) and on (1) states. In turn, the POI controls a Cdk1 cyclin, which in the re-engineered cell cycle system is essential for one cell cycle stage but poisonous for another. Thus, the cyclin must oscillate (1-0-1-0…) for cell proliferation. In this system, evolution can act efficiently on the dynamics, transient states, and input-output relations of the POI in every cell cycle. Further, controlling the pacemaker, light, directs and tunes selection pressures. Optovolution is in vivo, continuous, self-selecting, and genetically robust. We first evolved two optogenetic systems, which relay 0/1 input to 0/1 output: We obtained 25 new variants of the widely used LOV transcription factor El222. These mutants were stronger, less leaky, or green- and red-responsive. The latter was conjectured to be impossible for LOV domains but is needed for multiplexing and lowering phototoxicity. Evolving the PhyB-Pif3 optogenetic system, we discovered that loss of YOR1 makes supplementing the expensive and unstable chromophore phycocyanobilin (PCB) unnecessary. Finally, we demonstrate the generality of the method by creating and evolving a destabilized rtTA transcription factor, which performs an AND operation between transcriptional and doxycycline input. Optovolution makes coveted, difficult-to-change protein functionalities evolvable.

Abstract: Mechanosensitive PIEZO2 ion channels play roles in touch, proprioception, and inflammatory pain. Currently, there are no small molecule inhibitors that selectively inhibit PIEZO2 over PIEZO1. The TMEM120A protein was shown to inhibit PIEZO2 while leaving PIEZO1 unaffected. Here we find that TMEM120A expression elevates cellular levels of phosphatidic acid and lysophosphatidic acid (LPA), aligning with its structural resemblance to lipid-modifying enzymes. Intracellular application of phosphatidic acid or LPA inhibited PIEZO2, but not PIEZO1 activity. Extended extracellular exposure to the non-hydrolyzable phosphatidic acid and LPA analogue carbocyclic phosphatidic acid (ccPA) also inhibited PIEZO2. Optogenetic activation of phospholipase D (PLD), a signaling enzyme that generates phosphatidic acid, inhibited PIEZO2, but not PIEZO1. Conversely, inhibiting PLD led to increased PIEZO2 activity and increased mechanical sensitivity in mice in behavioral experiments. These findings unveil lipid regulators that selectively target PIEZO2 over PIEZO1, and identify the PLD pathway as a regulator of PIEZO2 activity.

Abstract: Signal processing by intracellular kinases control near all biological processes but how precise functions of signal pathways evolve with changed cellular contexts is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) activated in response to a broad range of pathological and physiological stimuli are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK regulatory network and defines JNK signal response to precise stimuli. We showed that nuanced fluctuations in physiological pHi regulates JNK activity in response to cell stress. Interestingly, the relationship between pHi and JNK activity was dependent on specific stimuli and upstream kinases involved in pathway activation. Cytosolic alkalinisation promoted phase transition of upstream ASK1 to augment JNK activation. While increased pHi similarly induced JNK2 to form condensates, this led to attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contribution by JNK2 and ASK1 condensates was sufficient to delineate the strength of JNK signal response to specific stimuli. This new knowledge of pHi regulation with consideration of JNK2 and ASK1 contribution to signal transduction may delineate oncogenic versus tumour suppressive functions of the JNK pathway and cancer cell drug responses.

Abstract: Neuronal tracing methods are essential tools to understand the fundamental architecture of neural circuits and their connection to the overall functional behavior of the brain. Viral vectors used to map these transsynaptic connections are capable of cell-type-specific and directional-specific labeling of the neuronal connections. Herein, we describe a novel approach to guide the transsynaptic spreading of the Rabies Virus (RV) retrograde tracer using light. We built a Baculovirus (BV) as a helper virus to deliver all the functional components necessary and sufficient for a nontoxic RV to spread from neuron to neuron, with a light-actuated gene switch to control the RV polymerase, the L gene. This design should allow for precisely controlled polysynaptic viral tracing with minimal viral toxicity. To use this system in a highly scalable and automated manner, we built optoelectronics for controlling this system in vitro with a large field of view using an off-the-shelf CMOS sensor, OLED display panel, and microcontrollers. We describe the assembly of these genetic circuits using the uLoop DNA assembly method and a library of genetic parts designed for the uLoop system. Combining these tools provides a framework for increasing the capabilities of nontoxic tracing through multiple synapses and increasing the throughput of neural tracing using viruses.

Abstract: Eph receptors are ubiquitous class of transmembrane receptors that mediate cell-cell communication, proliferation, differentiation, and migration. EphA1 receptors specifically play an important role in angiogenesis, fetal development, and cancer progression; however, studies of this receptor can be challenging as its ligand, ephrinA1, binds and activates several EphA receptors simultaneously. Optogenetic strategies could be applied to circumvent this requirement for ligand activation and enable selective activation of the EphA1 subtype. In this work, we designed and tested several iterations of an optogenetic EphA1 - Cryptochrome 2 (Cry2) fusion, investigating their capacity to mimic EphA1-dependent signaling in response to light activation. We then characterized the key cell signaling target of MAPK phosphorylation activated in response to light stimulation. The optogenetic regulation of Eph receptor RTK signaling without the need for external stimulus promises to be an effective means of controlling individual Eph receptor-mediated activities and creates a path forward for the identification of new Eph-dependent functions.

Abstract: Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. Using custom devices for rapid and high-throughput temperature control during live-cell microscopy, we find that the original Melt variant fully switches states between 28-32°C, and state changes can be observed within minutes of temperature changes. Melt was highly modular, permitting thermal control over diverse intracellular processes including signaling, proteolysis, and nuclear shuttling through straightforward end-to-end fusions with no further engineering. Melt was also highly tunable, giving rise to a library of Melt variants with switch point temperatures ranging from 30-40°C. The variants with higher switch points allowed control of molecular circuits between 37°C-41°C, a well-tolerated range for mammalian cells. Finally, Melt could thermally regulate important cell decisions over this range, including cytoskeletal rearrangement and apoptosis. Thus Melt represents a versatile thermogenetic module that provides straightforward, temperature-based, real-time control of mammalian cells with broad potential for biotechnology and biomedicine.

Abstract: Epithelia are polarised layers of cells that line the outer and inner surfaces of organs. At the basal side, the epithelial cell layer is supported by a basement membrane, which is a thin polymeric layer of self-assembled extracellular matrix (ECM) that tightly adheres to the basal cell surface. Proper shaping of epithelial layers is an important prerequisite for the development of healthy organs during the morphogenesis of an organism. Experimental evidence indicates that local degradation of the basement membrane drives epithelial folding. Here, we present a coarse-grained plate theory model of the basement membrane that assumes force balance between i) cell-transduced active forces and ii) deformation-induced elastic forces. We verify key assumptions of this model through experiments in the Drosophila wing disc epithelium and demonstrate that the model can explain the emergence of outward epithelial folds upon local plate degradation. Our model accounts for local degradation of the basement membrane as a mechanism for the generation of epithelial folds in the absence of epithelial growth.

Abstract: Cell-cell communication through diffusible signals allows distant cells to coordinate biological functions. Such coordination depends on the signal landscapes generated by emitter cells and the sensory capacities of receiver cells. In contrast to morphogen gradients in embryonic development, microbial signal landscapes occur in open space with variable cell densities, spatial distributions, and physical environments. How do microbes shape signal landscapes to communicate robustly under such circumstances remains an unanswered question. Here we combined quantitative spatial optogenetics with biophysical theory to show that in the mating system of budding yeast— where two mates communicate to fuse—signal landscapes convey demographic or positional information depending on the spatial organization of mating populations. This happens because α-factor pheromone and its mate-produced protease Bar1 have characteristic wide and narrow diffusion profiles, respectively. Functionally, MATα populations signal their presence as collectives, but not their position as individuals, and Bar1 is a sink of alpha-factor, capable of both density-dependent global attenuation and local gradient amplification. We anticipate that optogenetic control of signal landscapes will be instrumental to quantitatively understand the spatial behavior of natural and engineered cell-cell communication systems.

Abstract: Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is under-explored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcription factor BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci.

Abstract: Anteroposterior (AP) elongation of the vertebrate body plan is driven by convergence and extension (C&E) gastrulation movements in both the mesoderm and neuroectoderm, but how or whether molecular regulation of C&E differs between tissues remains an open question. Using a zebrafish explant model of AP axis extension, we show that C&E of the neuroectoderm and mesoderm can be uncoupled ex vivo, and that morphogenesis of individual tissues results from distinct morphogen signaling dynamics. Using precise temporal manipulation of BMP and Nodal signaling, we identify a critical developmental window during which high or low BMP/Nodal ratios induce neuroectoderm- or mesoderm-driven C&E, respectively. Increased BMP activity similarly enhances C&E specifically in the ectoderm of intact zebrafish gastrulae, highlighting the in vivo relevance of our findings. Together, these results demonstrate that temporal dynamics of BMP and Nodal morphogen signaling activate distinct morphogenetic programs governing C&E gastrulation movements within individual tissues.

Abstract: Phospholipase D (PLD) and phosphatidic acid (PA) play a spatio-temporal role in regulating diverse cellular activities. Although current methodologies enable optical control of the subcellular localization of PLD and by which influence local PLD enzyme activity, the overexpression of PLD elevates the basal PLD enzyme activity and further leads to increased PA levels in cells. In this study, we employed a split protein reassembly strategy and optogenetic techniques to modify superPLD (a PLDPMF variant with a high basal activity). We splited this variants into two HKD domains and fused these domains with optogenetic elements and by which we achieved light-mediated dimerization of the two HKD proteins and then restored the PLD enzymatic activity.

Abstract: The front of migratory cellular clusters during development, wound healing and cancer invasion is typically populated with highly protrusive cells that are called leader cells. Leader cells are thought to physically pull and direct their cohort of followers, but how leaders and followers are mechanically organized to migrate collectively remains controversial. One possibility is that the autonomous local action of a leader cell is sufficient to drive migration of the group. Yet another possibility is that a global mechanical organization is required for the group to move cohesively. Here we show that the effectiveness of leader-follower organization is proportional to the asymmetry of traction and tension within the cellular cluster. By combining hydrogel micropatterning and optogenetic activation of Rac1, we locally generate highly protrusive leaders at the edge of minimal cell groups. We find that the induced leader can robustly drag one follower but is generally unable to direct larger groups. By measuring traction forces and tension propagation in groups of increasing size, we establish a quantitative relationship between group velocity and the asymmetry of the traction and tension profiles. We propose a model of the motile cluster as an active polar fluid that explains this force-velocity relationship in terms of asymmetries in the distribution of active tractions. Our results challenge the notion of autonomous leader cells by showing that collective cell migration requires a global mechanical organization within the cluster.

Abstract: Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function—dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles, a major question in cell biology and drug development. Here we report an optogenetic approach to selectively dissolve a condensate of interest in a reversible and spatially controlled manner. We show that light-gated recruitment of maltose-binding protein (MBP), a commonly used solubilizing domain in protein purification, results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP, showing that disrupting condensation of the oncogenic fusion protein FUS-CHOP results in reversion of FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

Abstract: The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

Abstract: The regulation of synaptic transmission is crucial for plasticity, homeostasis and learning. Chemical synaptic transmission is thus modulated to accommodate different activity levels, which also enables homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cAMP signaling enhances cholinergic neuron output, and these neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish. Optogenetic stimulation by photoactivated adenylyl cyclase (bPAC) resulted in elevated locomotion as measured in behavioural assays. Furthermore, post-synaptic patch-clamp recordings revealed that in bPAC transgenics, the frequency of miniature excitatory postsynaptic currents (mEPSCs) was increased after photostimulation. These results suggested that cAMP-mediated activation of ZF motor neurons leads to increased fusion of SVs, consequently resulting in enhanced neuromuscular activity. We generated mutants lacking the neuropeptide processing enzyme carboxypeptidase E (cpe), and the most abundant neuropeptide precursor in motor neurons, tachykinin (tac1). Both mutants showed exaggerated locomotion after photostimulation. cpe mutants exhibit lower mEPSC frequency during photostimulation and less large-amplitude mEPSCs. In tac1 mutants mEPSC frequency was not affected but amplitudes were significantly smaller. Exaggerated locomotion in the mutants thus reflected upscaling of postsynaptic excitability. cpe and tac1 mutant muscles expressed more nicotinic acetylcholine receptors (nAChR) on their surface. Thus, neuropeptide signaling regulates synaptic transmitter output in zebrafish motor neurons, and muscle cells homeostatically regulate nAChR surface expression, compensating reduced presynaptic input. This mechanism may be widely conserved in the animal kingdom.

Abstract: Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses.

Abstract: Cellular processes can be modulated by physical means, such as light, which offers advantages over chemically inducible systems with respect to spatiotemporal control. Here we introduce an optogenetic gene expression system for Lactococcus lactis that utilizes a split T7 RNA polymerase linked to two variants of the Vivid regulators. Depending on the chosen photoreceptor variant, either ‘Magnets’ or ‘enhanced Magnets’, this system can achieve either high protein expression levels or low basal activity in the absence of light, exhibiting a fold induction close to 30, rapid expression kinetics, and heightened light sensitivity. This system functions effectively in liquid cultures and within cells embedded in hydrogel matrices, highlighting its potential in the development of novel engineered living materials capable of responding to physical stimuli such as light. The optogenetic component of this system is highly customizable, allowing for the adjustment of expression patterns through modifications to the promoters and/or engineered T7 RNA polymerase variants. We anticipate that this system can be broadly adapted to other Gram-positive hosts with minimal modifications required.