Curated Optogenetic Publication Database

Abstract: Circumventing the limitations of current bioassays, we introduce a light-controlled assay, OptoAssay, toward wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bidirectional movement of assay components, only by changing the wavelength of light. Demonstrating exceptional versatility, the OptoAssay showcases its efficacy on various substrates, delivering a dynamic bioassay format. The applicability of the OptoAssay is successfully demonstrated by the calibration of a competitive model assay, resulting in a superior limit of detection of 8 pg ml-1, which is beyond those of conventional ELISA tests. In the future, combined with smartphones, OptoAssays could obviate the need for external flow control systems such as pumps or valves and signal readout devices, enabling on-site analysis in resource-limited settings.

Abstract: Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.

Abstract: RNA molecules play a vital role in linking genetic information with various cellular processes. In recent years, a variety of optogenetic tools have been engineered for regulating cellular RNA metabolism and functions. These highly desirable tools can offer non-intrusive control with spatial precision, remote operation, and biocompatibility. Here, we would like to review these currently available approaches that can regulate RNAs with light: from non-genetically encodable chemically modified oligonucleotides to genetically encoded RNA aptamers that recognize photosensitive small-molecule or protein ligands. Some key applications of these optogenetic tools will also be highlighted to illustrate how they have been used for regulating all aspects of the RNA life cycle: from RNA synthesis, maturation, modification, and translation to their degradation, localization, and phase separation control. Some current challenges and potential practical utilizations of these RNA optogenetic tools will also be discussed.

Abstract: The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis of action spectra. Current methods to determine action spectra only allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validated the RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

Abstract: Photon upconversion (UC) from red or near-infrared (NIR) light to blue light is promising for in vivo optogenetics. However, the examples of in vivo optogenetics have been limited to lanthanide inorganic UC nanoparticles, and there have been no examples of optogenetics without using heavy metals. Here the first example of in vivo optogenetics using biocompatible heavy metal-free TTA-UC nanoemulsions is shown. A new organic TADF sensitizer, a boron difluoride curcuminoid derivative modified with a bromo group, can promote intersystem crossing to the excited triplet state, significantly improving TTA-UC efficiency. The TTA-UC nanoparticles formed from biocompatible surfactants and methyl oleate acquire water dispersibility and remarkable oxygen tolerance. By combining with genome engineering technology using the blue light-responding photoactivatable Cre-recombinase (PA-Cre), TTA-UC nanoparticles promote Cre-reporter EGFP expression in neurons in vitro and in vivo. The results open new opportunities toward deep-tissue control of neural activities based on heavy metal-free fully organic UC systems.

Abstract: Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense, and compacted environment of the cytoplasm remains challenging. Here we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow which combines thin cells grown on a mechanically defined substratum to rapidly analyse organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles otherwise positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

Abstract: The regulatory complexity of the eukaryotic cell cycle poses technical challenges in experiment design and data interpretation, leaving gaps in our understanding of how cells coordinate cell cycle-related processes. Traditional methods, such as knockouts and deletions are often ineffective to compensatory interactions in the cell cycle control network, while chemical agents that cause cell cycle arrest can have undesired pleiotropic effects. Synthetic inducible systems targeting specific cell cycle regulators offer potential solutions but are limited by the need for external inducers, which make fast reversibility technically challenging. To address these issues, we developed an optogenetic tool (OPTO-Cln2) that enables light-controlled and reversible regulation of G1 progression in budding yeast. Through extensive validation and benchmarking via time-lapse microscopy, we verify that OPTO-Cln2-carrying strains can rapidly toggle between normal and altered G1 progression. By integrating OPTO-Cln2 with a readout of nutrient-sensing pathways (TORC1 and PKA), we show that the oscillatory activity of these pathways is tightly coordinated with G1 progression. Finally, we demonstrate that the rapid reversibility of OPTO-Cln2 facilitates multiple cycles of synchronous arrest and release of liquid cell cultures. Our work provides a powerful new approach for studying cell cycle dynamics and the coordination of growth- with division-related processes.

Abstract: Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesized that inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generated a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packed mitochondria into a tight ball and efficiently moved the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.

Abstract: How do cellular forces propagate through tissue to allow large-scale morphogenetic events? To investigate this question, we use an in vivo optogenetic approach to reversibly manipulate actomyosin contractility at depth within the developing zebrafish neural rod. Contractility was induced along the lateral cortices of a small patch of developing neural epithelial progenitor cells, resulting in a shortening of these cells along their mediolateral axis. Imaging the immediate response of surrounding tissue uncovered a long-range, tangential, and elastic tissue deformation along the anterior-posterior axis. Unexpectedly, this was highly asymmetric, propagating in either the anterior or the posterior direction in response to local gradients in optogenetic activation. The degree of epithelialisation did not have a significant impact on the extent of force propagation via lateral cortices. We also uncovered a dynamic oscillatory expansion and contraction of the tissue along the anterior-posterior axis, with wavelength matching rhombomere length. Together, this study suggests dynamic and wave-like propagation of force between rhombomeres along the anterior-posterior axis. It also suggests that cell generated forces are actively propagated over long distances within the tissue, and that local anisotropies in tissue organisation and contractility may be sufficient to drive directional force propagation.

Abstract: The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.

Abstract: Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

Abstract: Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin’s involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.

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. In this study, using optogenetics, we achieved chemical-free poly-PR condensation/aggregation in cultured cells including human motor neurons, with spatial and temporal control. Strikingly, nuclear poly-PR condensates had anisotropic, hollow-center appearance, resembling TDP-43 anisosomes, and their growth was limited by RNA. These condensates induced abnormal TDP-43 granulation in the nucleus without stress response activation. Cytoplasmic poly-PR aggregates forming under prolonged opto-stimulation were more persistent than its nuclear condensates, selectively sequestered TDP-43 in a demixed state and surrounded spontaneous stress granules. Thus, poly-PR condensation accompanied by nuclear TDP-43 dysfunction may constitute an early pathological event in C9-ALS/FTD. Anisosome-type condensates of disease-linked proteins may represent a common molecular species in neurodegenerative disease.

Abstract: Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors.

Abstract: Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications.

Abstract: Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

Abstract: Biochemical reaction networks adapt to environmental conditions by sensing chemical or physical stimuli and using tightly controlled mechanisms. While most signals come from molecules, many cells can also sense and respond to light. Among the biomolecular structures that enable light sensing, we selected a light-oxygen-voltage (LOV) domain in a previous study that tested the engineering of novel regulatory mechanisms into a nucleic acid polymerase. In this follow-up study, we studied the activities of previously selected variants in kinetic detail, and we generated additional LOV-polymerase fusion variants based on further insertion criteria. Our results provide mechanistic insights into how LOV domain insertion influences polymerase activity in a light-responsive manner: All active and photoresponsive enzyme variants studied by us to date were partially inhibited (i.e., "turned off") after irradiation with blue light at 470 nm, which can be explained by specific obstructions of the polymerase entry or exit structures (substrate entry channels or product exit channels, or both). Although the effects observed are moderate, we anticipate further engineering strategies that could be used to improve the extent of switchability and possibly to develop a "turn-on mode" insertion.

Abstract: Establishing a highly efficient photoactivatable Cre recombinase PA-Cre3.0 can allow spatiotemporal control of Cre recombinase activity. This technique may help to elucidate cell lineages, as well as facilitate gene and cell function analysis during development. This study examined the blue light-mediated optical regulation of Cre-loxP recombination using PA-Cre3.0 transgenic early mouse pre-implantation embryos. We found that inducing PA-Cre3.0 expression in the heterozygous state did not show detectable recombination activation with blue light. Conversely, in homozygous embryos, DNA recombination by PA-Cre3.0 was successfully induced by blue light and resulted in the activation of the red fluorescent protein reporter gene, while almost no leaks of Cre recombination activity were detected in embryos without light illumination. Thus, we characterize the conditions under which the PA-Cre3.0 system functions efficiently in early mouse embryos. These results are expected to provide a new optogenetic tool for certain biological studies, such as developmental process analysis and lineage tracing in early mouse embryos.

Abstract: During animal development, the spatiotemporal properties of molecular events largely determine the biological outcomes. Conventional gene analysis methods lack the spatiotemporal resolution for precise dissection of developmental mechanisms. Although optogenetic tools exist for manipulating designer proteins in cultured cells, few have been successfully applied to endogenous proteins in live animals. Here, we report OptoTrap, a light-inducible clustering system for manipulating endogenous proteins of diverse sizes, subcellular locations, and functions in Drosophila. This system turns on fast, is reversible in minutes or hours, and contains variants optimized for neurons and epithelial cells. By using OptoTrap to disrupt microtubules and inhibit kinesin-1 in neurons, we show that microtubules support the growth of highly dynamic dendrites and that kinesin-1 is required for patterning of low- and high-order dendritic branches in differential spatiotemporal domains. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds promise for studying developmental mechanisms in a wide range of cell types and developmental stages.

Abstract: Recent advancements in plant bioprinting and optogenetic tools have unlocked new avenues to revolutionize plant tissue engineering. Bioprinting of plant cells has the potential to craft intricate 3D structures incorporating multiple cell types, replicating the complex microenvironments found in plants. Concurrently, optogenetic tools enable the control of biological events with spatial, temporal, and quantitative precision. Originally developed for human and microbial systems, these two cutting-edge methodologies are now being adapted for plant research. Although still in the early stages of development, we here review the latest progress in plant bioprinting and optogenetics and discuss compelling opportunities for plant biotechnology and research arising from the combination of the two technologies.

Abstract: We report that the RhoA-specific guanine nucleotide exchange factor ARHGEF17 localizes at the back of a fibroblast’s contractile lamella and regulates its disassembly. This localization emerges through retrograde ARHGEF17 transport together with actomyosin flow that most likely involves interactions with ATP-actin at F-actin barbed ends. During this process, ARHGEF17 increasingly oligomerizes into clusters that co-localize with myosin filaments, and correlate with their disassembly at lamella’s distal edge. ARHGEF17 loss of function leads to decreased RhoA activity at the lamella back and impairs its disassembly. High RhoA activity is however maintained at the lamella front where phosphorylated myosin light chain is observed. We propose that low levels of actomyosin network fracture at the lamella back generates barbed ends leading to generation of ATP-actin and ARHGEF17 binding, local activation of RhoA-dependent contractility, ensuring robust lamella disassembly. ARHGEF17 exemplifies the spatio-temporal complexity of Rho GTPase signaling and the requirement of feedback mechanism for homeostasis of contractile actomyosin networks.

Abstract: Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.

Abstract: Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.

Abstract: Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein. Here, we present cryoelectron microscopy (cryo-EM) structures of the phytochrome from Pseudomonas aeruginosa (PaBphP) in its resting (Pfr) and photoactivated (Pr) state. The kinase-active Pr state has an asymmetric, dimeric structure, whereas the kinase-inactive Pfr state opens up. This behavior is different from other known phytochromes and we explain it with the unusually short connection between the photosensory and output modules. Multiple sequence alignment of this region suggests evolutionary optimization for different modes of signal transduction in sensor proteins. The results establish a new mechanism for light-sensing by phytochrome histidine kinases and provide input for the design of optogenetic phytochrome variants.

Abstract: Oscillations are a recurrent phenomenon in biological systems across scales, but deciphering their fundamental principles is very challenging. Here, we tackle this challenge by redesigning the wellcharacterised synthetic oscillator known as “repressilator” in Escherichia coli and controlling it using optogenetics, creating the “optoscillator”. Bacterial colonies manifest oscillations as spatial ring patterns. When we apply periodic light pulses, the optoscillator behaves as a forced oscillator and we systematically investigate the properties of the rings under various light conditions. Combining experiments with mathematical modeling, we demonstrate that this simple oscillatory circuit can generate complex dynamics that are transformed into distinct spatial patterns. We report the observation of synchronisation, resonance, subharmonic resonance and period doubling. Furthermore, we present evidence of a chaotic regime. This work highlights the intricate spatiotemporal patterns accessible by synthetic oscillators and underscores the potential of our approach in revealing fundamental principles of biological oscillations.