Curated Optogenetic Publication Database

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: The transition of bacteria from an individualistic to a biofilm lifestyle profoundly alters their biology. During biofilm development, the bacterial cell-cell adhesions are a major determinant of initial microcolonies, which serve as kernels for the subsequent microscopic and mesoscopic structure of the biofilm, and determine the resulting functionality. In this study, the significance of bacterial cell-cell adhesion dynamics on bacterial aggregation and biofilm maturation is elucidated. Using photoswitchable adhesins between bacteria, modifying the dynamics of bacterial cell-cell adhesions with periodic dark-light cycles is systematic. Dynamic cell-cell adhesions with liquid-like behavior improve bacterial aggregation and produce more compact microcolonies than static adhesions with solid-like behavior in both experiments and individual-based simulations. Consequently, dynamic cell-cell adhesions give rise to earlier quorum sensing activation, better intermixing of different bacterial populations, improved biofilm maturation, changes in the growth of cocultures, and higher yields in fermentation. The here presented approach of tuning bacterial cell-cell adhesion dynamics opens the door for regulating the structure and function of biofilms and cocultures with potential biotechnological applications.

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: Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Abstract: Light-dependent adaptations of organismal physiology, development, and behavior abound in nature and depend on sensory photoreceptors. As one class, light–oxygen–voltage (LOV) photoreceptors harness flavin-nucleotide chromophores to sense blue light. Photon absorption drives the LOV receptor to its signaling state, characterized by a metastable thioadduct between the flavin and a conserved cysteine residue. With this cysteine absent, LOV receptors instead undergo photoreduction to the flavin semiquinone which however can still elicit downstream physiological responses. Irrespective of the cysteine presence, the LOV photochemical response thus entails a formal reduction of the flavin. Against this backdrop, we here investigate the reduction midpoint potential E0 in the paradigmatic LOV2 domain from Avena sativa phototropin 1 (AsLOV2), and how it can be deliberately varied. Replacements of residues at different sites near the flavin by methionine consistently increase E0 from its value of around −280 mV by up to 40 mV. Moreover, methionine introduction invariably impairs photoactivation efficiency and thus renders the resultant AsLOV2 variants less light-sensitive. Although individual methionine substitutions also affect the stability of the signaling state and downstream allosteric responses, no clear-cut correlation with the redox properties emerges. With a reduction midpoint potential near −280 mV, AsLOV2 and, by inference, other LOV receptors may be partially reduced inside cells which directly affects their light responsiveness. The targeted modification of the chromophore environment, as presently demonstrated, may mitigate this effect and enables the design of LOV receptors with stratified redox sensitivities.

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: Phospholipase C (PLC) is a key enzyme that regulates physiological processes via lipid and calcium signaling. Despite advances in protein engineering, no tools are available for direct PLC control. Here, we developed a novel optogenetic tool, light-controlled PLCβ (opto-PLCβ). Opto-PLCβ uses a light-induced dimer module, which directs an engineered PLC to the plasma membrane in a light-dependent manner. Our design includes an autoinhibitory capacity, ensuring stringent control over PLC activity. Opto-PLCβ triggers reversible calcium responses and lipid dynamics in a restricted region, allowing precise spatiotemporal control of PLC signaling. Using our system, we discovered that phospholipase D-mediated phosphatidic acid contributes to diacylglycerol clearance on the plasma membrane. Moreover, we extended its applicability in vivo, demonstrating that opto-PLCβ can enhance amygdala synaptic plasticity and associative fear learning in mice. Thus, opto-PLCβ offers precise spatiotemporal control, enabling comprehensive investigation of PLC-mediated signaling pathways, lipid dynamics, and their physiological consequences in vivo.

Abstract: Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. However, the impact of the material properties of biomolecular condensates on important processes, such as the control of gene expression, remains largely elusive. Here, the material properties of optogenetically induced transcription factor condensates are systematically tuned, and probed for their impact on the activation of target promoters. It is demonstrated that transcription factors in rather liquid condensates correlate with increased gene expression levels, whereas stiffer transcription factor condensates correlate with the opposite effect, reduced activation of gene expression. The broad nature of these findings is demonstrated in mammalian cells and mice, as well as by using different synthetic and natural transcription factors. These effects are observed for both transgenic and cell-endogenous promoters. The findings provide a novel materials-based layer in the control of gene expression, which opens novel opportunities in optogenetic engineering and synthetic biology.

Abstract: Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.

Abstract: The ability of cells to perceive and respond to mechanical cues is essential for numerous biological activities. Emerging evidence indicates important contributions of organelles to cellular mechanosensitivity and mechanotransduction. However, whether and how the endoplasmic reticulum (ER) senses and reacts to mechanical forces remains elusive. To fill the knowledge gap, after developing a light-inducible ER-specific mechanostimulator (LIMER), we identify that mechanostimulation of ER elicits a transient, rapid efflux of Ca2+ from ER in monkey kidney COS-7 cells, which is dependent on the cation channels transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and polycystin-2 (PKD2) in an additive manner. This ER Ca2+ release can be repeatedly stimulated and tuned by varying the intensity and duration of force application. Moreover, ER-specific mechanostimulation inhibits ER-to-Golgi trafficking. Sustained mechanostimuli increase the levels of binding-immunoglobulin protein (BiP) expression and phosphorylated eIF2α, two markers for ER stress. Our results provide direct evidence for ER mechanosensitivity and tight mechanoregulation of ER functions, placing ER as an important player on the intricate map of cellular mechanotransduction.

Abstract: Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell-cell communication precisely during necroptosis. Receptor-interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non-canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3-mediated necroptosis. Opsin-free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light-sensitive protein-protein interaction to modulate cell signaling. Compared to chemical-based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand-free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3-mediated necroptosis in colorectal HT-29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system Support Protocol: Quantification of the titer of lentivirus Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT-29 cells Basic Protocol 3: Optimization of optogenetic stimulation conditions Basic Protocol 4: Time-stamped live-cell imaging of HT-29 lytic cell death Basic Protocol 5: Quantification of HT-29 lytic cell death.

Abstract: Biomolecular condensates are dynamic subcellular compartments that lack surrounding membranes and can spatiotemporally organize the cellular biochemistry of eukaryotic cells. However, such dynamic organization has not been realized in prokaryotes that naturally lack organelles, and strategies are urgently needed for dynamic biomolecular compartmentalization. Here we develop a light-switchable condensate system for on-demand dynamic organization of functional cargoes in the model prokaryotic Escherichia coli cells. The condensate system consists of two modularly designed and genetically encoded fusions that contain a condensation-enabling scaffold and a functional cargo fused to the blue light-responsive heterodimerization pair, iLID and SspB, respectively. By appropriately controlling the biogenesis of the protein fusions, the condensate system allows rapid recruitment and release of cargo proteins within seconds in response to light, and this process is also reversible and repeatable. Finally, the system is demonstrated to dynamically control the subcellular localization of a cell division inhibitor, SulA, which enables the reversible regulation of cell morphologies. Therefore, this study provides a new strategy to dynamically control cellular processes by harnessing light-controlled condensates in prokaryotic cells.

Abstract: The ORAI proteins serve as crucial pore-forming subunits of calcium-release-activated calcium (CRAC) channels, pivotal in regulating downstream calcium-related signaling pathways. Dysregulated calcium homeostasis arising from mutations and post-translational modifications in ORAI can lead to immune disorders, myopathy, cardiovascular diseases, and even cancers. Small molecules targeting ORAI present an approach for calcium signaling modulation. Moreover, emerging techniques like optogenetics and optochemistry aim to offer more precise regulation of ORAI. This review focuses on the role of ORAI in cancers, providing a concise overview of their significance in the initiation and progression of cancers. Additionally, it highlights state-of-the-art techniques for ORAI channel modulation, including advanced optical tools, potent pharmacological inhibitors, and antibodies. These novel strategies offer promising avenues for the functional regulation of ORAI in research and may inspire innovative approaches to cancer therapy targeting ORAI.

Abstract: Molecular optogenetics utilizes genetically encoded, light-responsive protein switches to control the function of molecular processes. Over the last two years, there have been notable advances in the development of novel optogenetic switches, their utilization in elucidating intricate signaling pathways, and their progress toward practical applications in biotechnological processes, material sciences, and therapeutic applications. In this review, we discuss these areas, offer insights into recent developments, and contemplate future directions.

Abstract: Inteins are proteins found in nature that execute protein splicing. Among them, split inteins stand out for their versatility and adaptability, presenting creative solutions for addressing intricate challenges in various biological applications. Their exquisite attributes, including compactness, reliability, orthogonality, low toxicity, and irreversibility, make them of interest to various fields including synthetic biology, biotechnology and biomedicine. In this review, we delve into the inherent challenges of using inteins, present approaches for overcoming these challenges, and detail their reliable use for specific cellular tasks. We will discuss the use of conditional inteins in areas like cancer therapy, drug screening, patterning, infection treatment, diagnostics and biocontainment. Additionally, we will underscore the potential of inteins in executing basic logical operations with practical implications. We conclude by showcasing their potential in crafting complex genetic circuits for performing computations and feedback control that achieves robust perfect adaptation.

Abstract: Liquid-liquid phase separation (LLPS) is responsible for the emergence of intracellular membrane-less organelles and the development of coacervate protocells. Benefitting from the advantages of simplicity, precision, programmability, and noninvasiveness, light has become an effective tool to regulate the assembly dynamics of LLPS, and mediate various biochemical processes associated with LLPS. In this review, recent advances in optically controlling membrane-less organelles within living organisms are summarized, thereby modulating a series of biological processes including irreversible protein aggregation pathologies, transcription activation, metabolic flux, genomic rearrangements, and enzymatic reactions. Among these, the intracellular systems (i.e., optoDroplet, Corelet, PixELL, CasDrop, and other optogenetic systems) that enable the photo-mediated control over biomolecular condensation are highlighted. The design of photoactive complex coacervate protocells in laboratory settings by utilizing photochromic molecules such as azobenzene and diarylethene is further discussed. This review is expected to provide in-depth insights into phase separation-associated biochemical processes, bio-metabolism, and diseases.

Abstract: The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment. Specifically, we temporally controlled the activity of Ras homolog family member A (RhoA), which is known to have a central role in mechanotransduction, using our previously developed Arabidopsis thaliana cryptochrome-2-based optoactivation system. Interestingly, pulsed RhoA activation induced Egr1 upregulation in stiff 3D gels only, whereas static light stimulation induced an increase in Egr1 expression across a wide range of 3D gel stiffnesses. Actin assembly inhibition limited Egr1 upregulation upon RhoA activation, implying that RhoA signaling requires an actin-involved process to upregulate Egr1. Consistently, static-light RhoA activation rather than pulsed-light activation restricted neurogenesis in soft gels. Our findings indicate that the dynamics of RhoA activation influence Egr1-mediated stem cell fate within 3D matrices in a matrix stiffness-dependent manner.

Abstract: Only a few short decades have passed since the sequencing of GFP, yet the modern repertoire of transgenically encoded optical tools implies an exponential proliferation of ever improving constructions to interrogate the subcellular environment. A myriad of tags for labeling proteins, RNA, or DNA have arisen in the last few decades, facilitating unprecedented visualization of subcellular components and processes. Development of a broad array of modern genetically encoded sensors allows real-time, in vivo detection of molecule levels, pH, forces, enzyme activity, and other subcellular and extracellular phenomena in ever expanding contexts. Optogenetic, genetically encoded optically controlled manipulation systems have gained traction in the biological research community and facilitate single-cell, real-time modulation of protein function in vivo in ever broadening, novel applications. While this field continues to explosively expand, references are needed to assist scientists seeking to use and improve these transgenic devices in new and exciting ways to interrogate development and disease. In this review, we endeavor to highlight the state and trajectory of the field of in vivo transgenic optical tools.

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: The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as stress granules formed in response to energetic and oxidative stress are closely linked to ageing, cancer, cardiovascular disease, and viral infection. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.

Abstract: Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca2+ entry pathway into the cell, the so-called Ca2+ release-activated Ca2+ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor-ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca2+ ion channel, Orai. Ca2+ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response.

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: Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Abstract: The precise localization and activation of proteins at the cell membrane at a certain time gives rise to many cellular processes, including cell polarization, migration, and division. Thus, methods to recruit proteins to model membranes with subcellular resolution and high temporal control are essential when reproducing and controlling such processes in synthetic cells. Here, a method is described for fabricating light-regulated reversible protein patterns at lipid membranes with high spatiotemporal precision. For this purpose, we immobilize the photoswitchable protein iLID (improved light-inducible dimer) on supported lipid bilayers (SLBs) and on the outer membrane of giant unilamellar vesicles (GUVs). Upon local blue light illumination, iLID binds to its partner Nano (wild-type SspB) and allows the recruitment of any protein of interest (POI) fused to Nano from the solution to the illuminated area on the membrane. This binding is reversible in the dark, which provides dynamic binding and release of the POI. Overall, this is a flexible and versatile method for regulating the localization of proteins with high precision in space and time using blue light.