Curated Optogenetic Publication Database

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: 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: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.

Abstract: This review adds the bilin-binding phytochromes to the Chemical Reviews thematic issue "Optogenetics and Photopharmacology". The work is structured into two parts. We first outline the photochemistry of the covalently bound tetrapyrrole chromophore and summarize relevant spectroscopic, kinetic, biochemical, and physiological properties of the different families of phytochromes. Based on this knowledge, we then describe the engineering of phytochromes to further improve these chromoproteins as photoswitches and review their employment in an ever-growing number of different optogenetic applications. Most applications rely on the light-controlled complex formation between the plant photoreceptor PhyB and phytochrome-interacting factors (PIFs) or C-terminal light-regulated domains with enzymatic functions present in many bacterial and algal phytochromes. Phytochrome-based optogenetic tools are currently implemented in bacteria, yeast, plants, and animals to achieve light control of a wide range of biological activities. These cover the regulation of gene expression, protein transport into cell organelles, and the recruitment of phytochrome- or PIF-tagged proteins to membranes and other cellular compartments. This compilation illustrates the intrinsic advantages of phytochromes compared to other photoreceptor classes, e.g., their bidirectional dual-wavelength control enabling instant ON and OFF regulation. In particular, the long wavelength range of absorption and fluorescence within the "transparent window" makes phytochromes attractive for complex applications requiring deep tissue penetration or dual-wavelength control in combination with blue and UV light-sensing photoreceptors. In addition to the wide variability of applications employing natural and engineered phytochromes, we also discuss recent progress in the development of bilin-based fluorescent proteins.

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: Cells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modeling, optogenetics, and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane–associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and that coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.

Abstract: One of the major cellular mechanisms to ensure protein homeostasis is the endoplasmic reticulum (ER) stress response. This pathway is typically triggered by accumulation of misfolded proteins in the ER lumen. Here we describe activation of ER stress via protein aggregation in the cell nucleus. We find in the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) activation of ER stress due to the aggregation of the diseases-causing progerin protein at the nuclear envelope. The presence of nucleoplasmic protein aggregates is sensed and signaled to the ER lumen via immobilization and clustering of theinner nuclear membrane protein SUN2, leading to activation of the Unfolded Protein Response (UPR). These results identify a nuclear trigger of ER stress and they provide insight into the molecular disease mechanisms of HGPS.

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: Precise spatial and temporal regulation of dynamic morphogen signals during human development governs the processes of cell proliferation, migration, and differentiation to form organized tissues and organs. Tissue patterns spontaneously emerge in various human pluripotent stem cell (hPSC) models. However, the lack of molecular methods for precise control over signal dynamics limits the reproducible production of tissue patterns and a mechanistic understanding of self-organization. We recently implemented an optogenetic-based OptoWnt platform for light-controllable regulation of Wnt/β-catenin signaling in hPSCs for in vitro studies. Using engineered illumination devices to generate light patterns and thus precise spatiotemporal control over Wnt activation, here we triggered spatially organized transcriptional changes and mesoderm differentiation of hPSCs. In this way, the OptoWnt system enabled robust endothelial cell differentiation and cardiac tissue patterning in vitro. Our results demonstrate that spatiotemporal regulation of signaling pathways via synthetic OptoWnt enables instructive stem cell fate engineering and tissue patterning.

Abstract: Exosomes are emerging as a novel drug delivery system for the treatment of numerous diseases, including acute kidney injury. In this issue of Kidney International, Kim et al. use a novel optogenetically engineered exosome technology, "EXPLOR," to deliver the exosomal repressor of nuclear factor-κB into mice before and after renal ischemia-reperfusion. They report that these exosomes downregulated renal nuclear factor-κB signaling and ameliorated acute kidney injury. This study deserves attention for its significant scientific and potential clinical value in acute kidney injury.

Abstract: Endocytosis is an important process by which many signaling receptors reach their intracellular effectors. Accumulating evidence suggests that internalized receptors play critical roles in triggering cellular signaling, including transforming growth factor β (TGFβ) signaling. Despite intensive studies on the TGFβ pathway over the last decades, the necessity of TGFβ receptor endocytosis for downstream TGFβ signaling responses is a subject of debate. In this study, mathematical modeling and synthetic biology approaches are combined to re-evaluate whether TGFβ receptor internalization is indispensable for inducing Smad signaling. It is found that optogenetic systems with plasma membrane-tethered TGFβ receptors can induce fast and sustained Smad2 activation upon light stimulations. Modeling analysis suggests that endocytosis is precluded for the membrane-anchored optogenetic TGFβ receptors. Therefore, this study provides new evidence to support that TGFβ receptor internalization is not required for Smad2 activation.

Abstract: The intrinsic genetic programme of a cell is not sufficient to explain all of the cell’s activities. External mechanical stimuli are increasingly recognized as determinants of cell behaviour. In the epithelial folding event that constitutes the beginning of gastrulation in Drosophila, the genetic programme of the future mesoderm leads to the establishment of a contractile actomyosin network that triggers apical constriction of cells, and thereby, furrow formation. However, some cells do not constrict but instead stretch, even though they share the same genetic programme as their constricting neighbours. We show here that tissue-wide interactions force these cells to expand even when an otherwise sufficient amount of apical, active actomyosin is present. Models based on contractile forces and linear stress-strain responses are not sufficient to reproduce experimental observations, but simulations in which cells behave as ductile materials with non-linear mechanical properties do. Our models show that this behaviour is a general emergent property of supracellular actomyosin networks, in accordance with our experimental observations of actin reorganisation within stretching cells.

Abstract: Upconversion-mediated optogenetics is an emerging powerful technique to remotely control and manipulate the deep-tissue protein functions and signaling pathway activation. This technique uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and through near-infrared light to indirectly activate the traditional optogenetic proteins. With the merits of high spatiotemporal resolution and minimal invasiveness, this technique enables cell-type specific manipulation of cellular activities in deep tissues as well as in living animals. In this review, we introduce the latest development of optogenetic modules and UCNPs, with emphasis on the integration of UCNPs with cellular optogenetics and their biomedical applications on the control of neural/brain activity, cancer therapy and cardiac optogenetics in vivo. Furthermore, we analyze the current developed strategies to optimize and advance the upconversion-mediated optogenetics and discuss the remaining challenges of its further applications in biomedical study and clinical translational research. STATEMENT OF SIGNIFICANCE: Optogenetics harnesses photoactivatable proteins to optically stimulate and control intracellular activities. UCNPs-mediated NIR-activatable optogenetics uses lanthanide upconversion nanoparticles (UCNPs) as light transducers and utilizes near-infrared (NIR) light to indirectly activate the traditional optogenetic proteins. The integration of UCNPs with cellular optogenetics has showed great promise in biomedical applications in regulating neural/brain activity, cancer therapy and cardiac optogenetics in vivo. The evolution and optimization of functional UCNPs and the discovery and engineering of novel optogenetic modules would both contribute to the advance of such unique hybrid technology, which may lead to discoveries in biomedical research and provide new treatments for human diseases.

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: Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.

Abstract: Biological information can be encoded within the dynamics of signaling components, which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions-modulating the pulse frequency, amplitude, and pulse width of light-regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed tunable gene expression and filtering behaviors and provided a quantification of the limit to the amount of information that can be reliably transferred across a single promoter as ∼1.7 bits. Using a library of over 100 orthogonal chromatin regulators, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic gene regulation.

Abstract: The microtubule-associated protein tau oligomerizes, but the actions of oligomeric tau (oTau) are unknown. We have used Cry2-based optogenetics to induce tau oligomers (oTau-c). Optical induction of oTau-c elicits tau phosphorylation, aggregation, and a translational stress response that includes stress granules and reduced protein synthesis. Proteomic analysis identifies HNRNPA2B1 as a principle target of oTau-c. The association of HNRNPA2B1 with endogenous oTau was verified in neurons, animal models, and human Alzheimer brain tissues. Mechanistic studies demonstrate that HNRNPA2B1 functions as a linker, connecting oTau with N6-methyladenosine (m6A) modified RNA transcripts. Knockdown of HNRNPA2B1 prevents oTau or oTau-c from associating with m6A or from reducing protein synthesis and reduces oTau-induced neurodegeneration. Levels of m6A and the m6A-oTau-HNRNPA2B1 complex are increased up to 5-fold in the brains of Alzheimer subjects and P301S tau mice. These results reveal a complex containing oTau, HNRNPA2B1, and m6A that contributes to the integrated stress response of oTau.

Abstract: Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.

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: Migratory cells navigate through crowded 3D microenvironments in vivo. Amoeboid cells, such as immune cells and some cancer cells, are thought to do so by deforming their bodies to squeeze through tight spaces.1 Yet large populations of nearly spherical amoeboid cells migrate2–4 in microenvironments too dense5,6 to move through without extensive shape deformations. How they do so is unknown. We used high-resolution light-sheet microscopy to visualize metastatic melanoma cells in dense environments, finding that cells maintain a round morphology as they migrate and create a path through which to move via bleb-driven mechanical degradation and subsequent macropinocytosis of extracellular matrix components. Proteolytic degradation of the extracellular matrix via matrix metalloproteinases is not required. Membrane blebs are short-lived relative to the timescale of migration, and thus persistence in their polarization is critical for productive ablation of the extracellular matrix. Interactions between small but long-lived cortical adhesions and collagen at the cell front induce PI-3 Kinase signaling that drive bleb enlargement via branched actin polymerization. Large blebs in turn abrade collagen, creating a feedback between extracellular matrix structure, cell morphology, and cell polarization that results in both path generation and persistent cell movement.

Abstract: The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved in a process called abscission. During pre-abscission, we identified that the centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge in human cells grown in culture and in an in vivo vertebrate model, Danio rerio (zebrafish). Rab11-endosomes are dynamically organized in a Rab11-GTP dependent manner at the centrosome during pre-abscission and this organization is required for the centrosome protein, pericentrin, to be enriched at the centrosome. Using zebrafish embryos, we found that reduction in pericentrin expression or optogenetically disrupting Rab11-endosome function inhibited centrosome movement towards the cytokinetic bridge and abscission resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.

Abstract: Optogenetics has opened new insights into biomedical research with the ability to manipulate and control cellular activity using light in combination with genetically engineered photosensitive proteins. By stimulating with light, this method provides high spatiotemporal and high specificity resolution, which is in contrast to conventional pharmacological or electrical stimulation. Optogenetics was initially introduced to control neural activities but was gradually extended to other biomedical fields.

Abstract: The ability to stimulate mammalian cells with light, brought along by optogenetic control, has significantly broadened our understanding of electrically excitable tissues. Backed by advanced (bio)materials, it has recently paved the way towards novel biosensing concepts supporting bio-analytics applications transversal to the main biomedical stream. The advancements concerning enabling biomaterials and related novel biosensing concepts involving optogenetics are reviewed with particular focus on the use of engineered cells for cell-based sensing platforms and the available toolbox (from mere actuators and reporters to novel multifunctional opto-chemogenetic tools) for optogenetic-enabled real-time cellular diagnostics and biosensor development. The key advantages of these modified cell-based biosensors concern both significantly faster (minutes instead of hours) and higher sensitivity detection of low concentrations of bioactive/toxic analytes (below the threshold concentrations in classical cellular sensors) as well as improved standardization as warranted by unified analytic platforms. These novel multimodal functional electro-optical label-free assays are reviewed among the key elements for optogenetic-based biosensing standardization. This focused review is a potential guide for materials researchers interested in biosensing based on light-responsive biomaterials and related analytic tools.

Abstract: Inducible biomolecular condensates play fundamental roles in cellular responses to intracellular and environmental cues. Knowledge about their composition is crucial to understand the functions that arise specifically from the assembly of condensates. This protocol combines an optogenetic and an efficient proximity labeling approach to analyze protein modifications driven by protein condensation in cultured cells. Low endogenous biotin level ensures sharp signals. For complete details on the use and execution of this protocol, please refer to Frattini et al. (2021).

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.