Curated Optogenetic Publication Database

Abstract: The type III secretion system (T3SS) enables direct injection of bacterial effector proteins into eukaryotic cells. We found that the dynamic cytosolic interface of the system allows Yersinia enterocolitica to suppress premature secretion at low pH, ensuring rapid activation at the site of action. Exploiting this principle, we developed a light-controlled T3SS based on optogenetic interaction switches, which provides unprecedented spatiotemporal control of protein secretion and translocation.

Abstract: Optogenetic tools have been proven to be useful in regulating cellular processes via an external signal. Light can be applied with high spatial and temporal precision as well as easily modulated in quantity and quality. Natural photoreceptors of the light oxygen voltage (LOV) domain family have been characterized in depth, especially the LOV2 domain of Avena sativa (As) phototropin 1 and its derivatives. Information on the behavior of LOV2 variants with changes in the photocycle or the light response has been recorded. Here, we applied well-described photocycle mutations on the AsLOV2 domain of a photosensitive transcription factor (psTF) as well as its variant that is part of the photosensitive degron (psd) psd3 in Saccharomyces cerevisiae. In vivo and in vitro measurements revealed that each photoreceptor component of the light-sensitive transcription factor and the psd3 module can be modulated in its light sensitivity by mutations that are known to prolong or shorten the dark-reversion time of AsLOV2. Yet, only two of the mutations showed differences in the in vivo behavior in the context of the psd3 module. For the AsLOV2 domain in the context of the psTF, we observed different characteristics for all four variants. Molecular dynamics simulations showed distinct influences of the shortened Jα helix and the V416L mutation in the context of the psd3 photoreceptor. In conclusion, we demonstrated the tunability of two optogenetic tools with a set of mutations that affect the photocycle of the inherent photoreceptors. As these optogenetic tools are concurrent in their action, pleiotropic effects on target protein abundance are achievable with the simultaneous action of the diverse photoreceptor variants.

Abstract: Optogenetics holds the promise of controlling biological processes with superb temporal and spatial resolution at minimal perturbation. Although many of the light-reactive proteins used in optogenetic systems are derived from prokaryotes, applications were largely limited to eukaryotes for a long time. In recent years, however, an increasing number of microbiologists use optogenetics as a powerful new tool to study and control key aspects of bacterial biology in a fast and often reversible manner. After a brief discussion of optogenetic principles, this review provides an overview of the rapidly growing number of optogenetic applications in bacteria, with a particular focus on studies venturing beyond transcriptional control. To guide future experiments, we highlight helpful tools, provide considerations for successful application of optogenetics in bacterial systems, and identify particular opportunities and challenges that arise when applying these approaches in bacteria.

Abstract: Cells employ intracellular signaling pathways to sense and respond to changes in their external environment. In recent years, live-cell biosensors have revealed complex pulsatile dynamics in many pathways, but studies of these signaling dynamics are limited by the necessity of live-cell imaging at high spatiotemporal resolution. Here, we describe an approach to infer pulsatile signaling dynamics from a single measurement in fixed cells using a pulse-detecting gene circuit. We computationally screened for circuits with the capability to selectively detect signaling pulses, revealing an incoherent feedforward topology that robustly performs this computation. We implemented the motif experimentally for the Erk signaling pathway using a single engineered transcription factor and fluorescent protein reporter. Our "recorder of Erk activity dynamics" (READer) responds sensitively to spontaneous and stimulus-driven Erk pulses. READer circuits open the door to permanently labeling transient, dynamic cell populations to elucidate the mechanistic underpinnings and biological consequences of signaling dynamics.

Abstract: Chimeric antigen receptor (CAR) T cell-based immunotherapy, approved by the US Food and Drug Administration, has shown curative potential in patients with haematological malignancies. However, owing to the lack of control over the location and duration of the anti-tumour immune response, CAR T cell therapy still faces safety challenges arising from cytokine release syndrome and on-target, off-tumour toxicity. Herein, we present the design of light-switchable CAR (designated LiCAR) T cells that allow real-time phototunable activation of therapeutic T cells to precisely induce tumour cell killing. When coupled with imaging-guided, surgically removable upconversion nanoplates that have enhanced near-infrared-to-blue upconversion luminescence as miniature deep-tissue photon transducers, LiCAR T cells enable both spatial and temporal control over T cell-mediated anti-tumour therapeutic activity in vivo with greatly mitigated side effects. Our nano-optogenetic immunomodulation platform not only provides a unique approach to interrogate CAR-mediated anti-tumour immunity, but also sets the stage for developing precision medicine to deliver personalized anticancer therapy.

Abstract: How T lymphocytes tune their responses to different strengths of stimulation is a fundamental question in immunology. Recent work using new optogenetic, single-cell genomic, and live-imaging approaches has revealed that stimulation strength controls the rate of individual cell responses within a population. Moreover, these responses have been found to use shared molecular programs, regardless of stimulation strength. However, additional data indicate that stimulation duration or cytokine feedback can impact later gene expression phenotypes of activated cells. In-depth molecular studies have suggested mechanisms by which stimulation strength might modulate the probability of T cell activation. This emerging model allows activating T cells to achieve a wide range of population responses through probabilistic control within individual cells.

Abstract: Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Abstract: Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.

Abstract: Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.

Abstract: Biomolecular condensates are membrane-less cellular compartments that form via phase separation. They serve a multitude of functions in all types of cells. Important insights into the composition, architecture and dynamics of biomolecular condensates have been obtained by harnessing the power of fluorescence-based technologies. In this chapter, methods will be discussed for (1) fluorescent labelling of macromolecules, (2) spatial and temporal mapping and tracking of target molecules in cellular and in vitro settings, (3) controlling formation and dissolution of biomolecular condensates, and (4) fluorescence-based condensate-targeted drug discovery.

Abstract: We re-engineered a commonly-used light-sensing protein, AsLOV2, using a circular permutation strategy to allow photoswitchable control of the C-terminus of a peptide. We demonstrate that the circularly permuted AsLOV2 can be used on its own or together with the original AsLOV2 for enhanced caging. In summary, circularly permuted AsLOV2 could expand the engineering capabilities of optogenetic tools.

Abstract: Lipids are highly dynamic molecules that, due to their hydrophobicity, are spatially confined to membrane environments. From these locations, certain privileged lipids serve as signaling molecules. For understanding the biological functions of subcellular pools of signaling lipids, induced proximity tools have been invaluable. These methods involve controlled heterodimerization, by either small-molecule or light triggers, of functional proteins. In the arena of lipid signaling, induced proximity tools can recruit lipid-metabolizing enzymes to manipulate lipid signaling and create artificial tethers between organelle membranes to control lipid trafficking pathways at membrane contact sites. Here, we review recent advances in methodology development and biological application of chemical-induced and light-induced proximity tools for manipulating lipid metabolism, trafficking, and signaling.

Abstract: Cell–cell adhesions are fundamental in regulating multicellular behavior and lie at the center of many biological processes from embryoid development to cancer development. Therefore, controlling cell–cell adhesions is fundamental to gaining insight into these phenomena and gaining tools that would help in the bioartificial construction of tissues. For addressing biological questions as well as bottom-up tissue engineering the challenge is to have multiple cell types self-assemble in parallel and organize in a desired pattern from a mixture of different cell types. Ideally, different cell types should be triggered to self-assemble with different stimuli without interfering with the other and different types of cells should sort out in a multicellular mixture into separate clusters. In this chapter, we will summarize the developments in photoregulation cell–cell adhesions using non-neuronal optogenetics. Among the concepts, we will cover is the control of homophylic and heterophilic cell–cell adhesions, the independent control of two different types with blue or red light and the self-sorting of cells into distinct structures and the importance of cell–cell adhesion dynamics. These tools will give an overview of how the spatiotemporal regulation of cell–cell adhesion gives insight into their role and how tissues can be assembled from cells as the basic building block.

Abstract: Liquid-liquid phase separation is a fundamental mechanism underlying subcellular organization. Motivated by the striking observation that optogenetically generated droplets in the nucleus display suppressed coarsening dynamics, we study the impact of chromatin mechanics on droplet phase separation. We combine theory and simulation to show that cross-linked chromatin can mechanically suppress droplets' coalescence and ripening, as well as quantitatively control their number, size, and placement. Our results highlight the role of the subcellular mechanical environment on condensate regulation.

Abstract: Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.

Abstract: Cells commonly communicate with each other through diffusible molecules but nonchemical communication remains elusive. While bioluminescent organisms communicate through light to find prey or attract mates, it is still under debate if signaling through light is possible at the cellular level. Here, we demonstrate that cell to cell signaling through light is possible in artificial cell communities derived from biomimetic vesicles. In our design, artificial sender cells produce an intracellular light signal, which triggers the adhesion to receiver cells. Unlike soluble molecules, the light signal propagates fast, independent of diffusion and without the need for a transporter across membranes. To obtain a predator-prey relationship, the luminescence predator cells is loaded with a secondary diffusible poison, which is transferred to the prey cell upon adhesion and leads to its lysis. This design provides a blueprint for light based intercellular communication, which can be used for programing artificial and natural cell communities.

Abstract: Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Abstract: Cell shape regulation is important but the mechanisms that govern shape are not fully understood, in part due to limited experimental models where cell shape changes and underlying molecular processes can be rapidly and non-invasively monitored in real time. Here, we use an optogenetic tool to activate RhoA in the middle of mononucleated macrophages to induce contraction, resulting in a side with the nucleus that retains its shape and a non-nucleated side which was unable to maintain its shape and collapsed. In cells overexpressing focal adhesion kinase (FAK), the non-nucleated side exhibited a wide flat morphology and was similar in adhesion area to the nucleated side. In cells overexpressing fascin, an actin bundling protein, the non-nucleated side assumed a spherical shape and was similar in height to the nucleated side. This effect of fascin was also observed in fibroblasts even without inducing furrow formation. Based on these results, we conclude that FAK and fascin work together to maintain cell shape by regulating adhesion area and height, respectively, in different cell types.

Abstract: Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in Drosophila, a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene huckebein (hkb) as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene snail is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.

Abstract: Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

Abstract: Light-Oxygen-Voltage (LOV) domains are responsible for detecting blue light (BL) and regulating the activities of effector domains in various organisms. Photozipper (PZ), an N-terminally truncated aureochrome-1 protein, contains a LOV domain and a basic leucin zipper (bZIP) domain and plays a role as a light-activatable transcription factor. PZ is monomeric in the dark state and undergoes non-covalent dimerization upon illumination with BL, subsequently increasing its affinity for the target DNA. To clarify the molecular mechanism of aureochromes, we prepared site-directed mutants of PZ and performed quantitative analyses in the dark and light states. Although the amino acid substitutions in the hinge region between the LOV core and A'α helix had minor effects on the dimerization and DNA-binding properties of PZ, the substitutions in the β-sheet region of the LOV core and in the A'α helix significantly affected these properties. We found that light signals are transmitted from the LOV core to the effector bZIP domain via the hydrophobic residues on the β-sheet. The light-induced conformational change possibly deforms the hydrophobic regions of the LOV core and induces the detachment of the A'α helix to expose the dimerization surface, likely activating the bZIP domain in a light-dependent manner.

Abstract: The SpyCatcher/SpyTag protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. The utility of this system could be expanded by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage domain 2 of Avena sativa (AsLOV2) undergoes a dramatic conformational change in its c-terminal Jα-helix in response to blue light. By inserting SpyTag into the different locations of the Jα-helix, we created a blue light inducible SpyTag system (BLISS). In this design, the SpyTag is blocked from reacting with the SpyCatcher in the dark, but upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We tested several insertion sites and characterized the kinetics. We found three variants with dynamic ranges over 15, which were active within different concentration ranges. These could be tuned using SpyCatcher variants with different reaction kinetics. Further, the reaction could be instantaneously quenched by removing light. We demonstrated the spatial aspect of this light control mechanism through photopatterning of two fluorescent proteins. This system offers opportunities for many other biofabrication and optogenetics applications.

Abstract: Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the synthesis and application of blue-light-generating synthetic cells using bioluminescence, dismissing the need for an external light source. First, the lipid membrane and internal composition of light-producing synthetic cells were optimized to enable high-intensity emission. Next, we show these cells’ capacity for triggering bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride in a quorum-sensing like mechanism. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of activating engineered processes inside tissues.

Abstract: Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Abstract: Plant-based photosensors, such as the light-oxygen-voltage sensing domain 2 (LOV2) from oat phototropin 1, can be modularly wired into cell signaling networks to remotely control protein activity and physiological processes. However, the applicability of LOV2 is hampered by the limited choice of available caging surfaces and its preference to accommodate the effector domains downstream of the C-terminal Jα helix. Here, we engineered a set of LOV2 circular permutants (cpLOV2) with additional caging capabilities, thereby expanding the repertoire of genetically encoded photoswitches to accelerate the design of optogenetic devices. We demonstrate the use of cpLOV2-based optogenetic tools to reversibly gate ion channels, antagonize CRISPR-Cas9-mediated genome engineering, control protein subcellular localization, reprogram transcriptional outputs, elicit cell suicide and generate photoactivatable chimeric antigen receptor T cells for inducible tumor cell killing. Our approach is widely applicable for engineering other photoreceptors to meet the growing need of optogenetic tools tailored for biomedical and biotechnological applications.