Curated Optogenetic Publication Database

Abstract: Optogenetic systems use genetically encoded light-sensitive proteins to control cellular processes. This provides the potential to orthogonally control cells with light; however, these systems require many design-build-test cycles to achieve a functional design and multiple illumination variables need to be laboriously tuned for optimal stimulation. We combine laboratory automation and a modular cloning scheme to enable high-throughput construction and characterization of optogenetic split transcription factors in Saccharomyces cerevisiae. We expand the yeast optogenetic toolkit to include variants of the cryptochromes and enhanced Magnets, incorporate these light-sensitive dimerizers into split transcription factors, and automate illumination and measurement of cultures in a 96-well microplate format for high-throughput characterization. We use this approach to rationally design and test an optimized enhanced Magnet transcription factor with improved light-sensitive gene expression. This approach is generalizable to the high-throughput characterization of optogenetic systems across a range of biological systems and applications.

Abstract: Biomolecular condensates, including membraneless organelles, are ubiquitously observed in subcellular compartments. However, the accumulation and dynamic properties of arbitrarily in-duced condensates remain elusive. Here, we show the size, amount, and dynamic properties of subcellular condensates using various fluorescence spectroscopic imaging analyses. Spatial image correlation spectroscopy showed that the size of blue-light-induced condensates of cryptochrome 2-derived oligomerization tag (CRY2olig) tagged with a red fluorescent protein in the nucleus was not different from that in the cytoplasm. Fluorescence intensity measurements showed that the condensates in the nucleus were more prone to accumulation than those in the cytoplasm. Sin-gle-particle tracking analysis showed that the condensates in the nucleus are predisposed to be stationary dynamics compared to those in the cytoplasm. Therefore, the subcellular compartment may, in part, affect the characteristics of self-recruitment of biomolecules in the condensates and their movement property.

Abstract: The inflammasome is a conserved structure for the intracellular detection of danger or pathogen signals. As a large intracellular multiprotein signaling platform, it activates downstream effectors that initiate a rapid necrotic programmed cell death (PCD) termed pyroptosis and activation and secretion of pro-inflammatory cytokines to warn and activate surrounding cells. However, inflammasome activation is difficult to control experimentally on a single-cell level using canonical triggers. We constructed Opto-ASC, a light-responsive form of the inflammasome adaptor protein ASC (Apoptosis-Associated Speck-Like Protein Containing a CARD) which allows tight control of inflammasome formation in vivo. We introduced a cassette of this construct under the control of a heat shock element into zebrafish in which we can now induce ASC inflammasome (speck) formation in individual cells of the skin. We find that cell death resulting from ASC speck formation is morphologically distinct from apoptosis in periderm cells but not in basal cells. ASC-induced PCD can lead to apical or basal extrusion from the periderm. The apical extrusion in periderm cells depends on Caspb and triggers a strong Ca2+ signaling response in nearby cells.

Abstract: Dynamic metabolic engineering is a strategy to switch key metabolic pathways in microbial cell factories from biomass generation to accumulation of target products. Here, we demonstrate that optogenetic intervention in the cell cycle of budding yeast can be used to increase production of valuable chemicals, such as the terpenoid β-carotene or the nucleoside analog cordycepin. We achieved optogenetic cell-cycle arrest in the G2/M phase by controlling activity of the ubiquitin-proteasome system hub Cdc48. To analyze the metabolic capacities in the cell cycle arrested yeast strain, we studied their proteomes by timsTOF mass spectrometry. This revealed widespread, but highly distinct abundance changes of metabolic key enzymes. Integration of the proteomics data in protein-constrained metabolic models demonstrated modulation of fluxes directly associated with terpenoid production as well as metabolic subsystems involved in protein biosynthesis, cell wall synthesis, and cofactor biosynthesis. These results demonstrate that optogenetically triggered cell cycle intervention is an option to increase the yields of compounds synthesized in a cellular factory by reallocation of metabolic resources.

Abstract: In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging, and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition, and TGF-β signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish an hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

Abstract: RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light-inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

Abstract: Collective cell migration during embryonic development, wound healing, and cancer metastasis entails the emergence of leader cells at the migration front. These cells with conspicuous lamellipodial structures provide directional guidance to the collective. Despite their physiological relevance, the mechanisms underlying the emergence of leader cells remain elusive. Here we report that in diverse model systems for wound healing, including cultured epithelial monolayer, Drosophila embryo, and mouse embryonic skin, leader cells display a peripheral accumulation of lysosomes. This accumulation appears essential for leader cell emergence, involves lysosomal movement along microtubules, and depends on the actomyosin contractility-generated cellular forces. Peripheral lysosomes associate with inactive Rac1 molecules to remove them from the leading periphery, which increases local Rac1-activity, triggering actin polymerization and promoting lamellipodium formation. Taken together, we demonstrate that beyond their catabolic role, lysosomes act as the intracellular platform that links mechanical and biochemical signals to control the emergence of leader cells.

Abstract: An increasingly 24/7 connected and urbanised world has created a silent pandemic of noise-induced hearing loss. Ensuring survival to children born (extremely) preterm is crucial. The incubator is a closed medical device, modifying the internal climate, and thus providing an environment for the child, as safe, warm, and comfortable as possible. While sound outside the incubator is managed and has decreased over the years, managing the noise inside the incubator is still a challenge.

Abstract: The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.

Abstract: The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.

Abstract: In addition to biochemical and electrochemical signaling, cells also rely extensively on mechanical signaling to regulate their behavior. While a number of tools have been adapted from physics and engineering to manipulate cell mechanics, they typically require specialized equipment or lack spatiotemporal precision. Alternatively, a recent, more elegant approach is to use light itself to modulate the mechanical equilibrium inside the cell. This approach leverages the power of optogenetics, which can be controlled in a fully reversible manner in both time and space, to tune RhoA signaling, the master regulator of cellular contractility. We review here the fundamentals of this approach, including illustrating the tunability and flexibility that optogenetics offers, and demonstrate how this tool can be used to modulate both internal cytoskeletal flows and contractile force generation. Together these features highlight the advantages that optogenetics offers for investigating mechanical interactions in cells.

Abstract: Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.

Abstract: Optogenetic techniques offer a high spatiotemporal resolution to manipulate cellular activity. For instance, Channelrhodopsin-2 with global light illumination is the most widely used to control neuronal activity at the cellular level. However, the cellular scale is much larger than the diffraction limit of light (<1 μm) and does not fully exploit the features of the "high spatial resolution" of optogenetics. For instance, until recently, there were no optogenetic methods to induce synaptic plasticity at the level of single synapses. To address this, we developed an optogenetic tool named photoactivatable CaMKII (paCaMKII) by fusing a light-sensitive domain (LOV2) to CaMKIIα, which is a protein abundantly expressed in neurons of the cerebrum and hippocampus and essential for synaptic plasticity. Combining photoactivatable CaMKII with two-photon excitation, we successfully activated it in single spines, inducing synaptic plasticity (long-term potentiation) in hippocampal neurons. We refer to this method as "Local Optogenetics", which involves the local activation of molecules and measurement of cellular responses. In this review, we will discuss the characteristics of LOV2, the recent development of its derivatives, and the development and application of paCaMKII.

Abstract: Angiogenesis and tissue repair in chronic non-healing diabetic wounds remain critical clinical problems. Engineered MSC-derived exosomes have significant potential for the promotion of wound healing. Here, we discuss the effects and mechanisms of eNOS-rich umbilical cord MSC exosomes (UCMSC-exo/eNOS) modified by genetic engineering and optogenetic techniques on diabetic chronic wound repair.

Abstract: The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.

Abstract: Necroptosis is programmed cell death that involves active cytokine production and membrane ruptures. Whereas intracellular necroptosis has been extensively studied, intercellular propagation of necroptosis is much less understood. Pharmacological induction of necroptosis cannot delineate whether a necroptotic cell can propagate the death signal to its neighbor because of the confounding effect from the exogenously administrated death-inducers. To address this challenge, we develop an optogenetic system to enable ligand-free, optical induction of necroptosis at the single-cell level. This system, termed Light-activatable Receptor-Interacting Protein Kinase 3 or La-RIPK3, utilizes CRY2olig, a variant of the photoactivatable protein cryptochrome, to induce oligomerization of RIPK3 under blue light stimulation. Kinetic analysis La-RIPK3-activated cells shows that cytokine production and membrane rupture follows distinct kinetics. Moreover, membrane rupture requires a higher threshold of RIPK3 kinase activity than cytokine production. Intriguingly, intercellular propagation of necroptosis requires at least two proximal necroptotic cells, and a single necroptotic cell rarely induces such propagation. These results imply that RIPK3 acts as a gatekeeper to define the threshold of distinct functional outcomes of intracellular and intercellular necroptosis. Such a thresholding mechanism could allow cells to make informed decisions by evaluating the severity of environmental stress when walking a tightrope between committing an immunogenic suicidal fate and maintaining membrane integrity. This study highlights the role of RIPK3-containing necrosomes in regulating intracellular and intercellular necroptosis and offers an optimized optogenetic tool for investigating RIPK3-dependent necroptotic pathways.

Abstract: During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFβ pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.

Abstract: Living cells utilize signaling pathways to sense, transduce, and process information. As the extracellular stimulation often has rich temporal characteristics which may govern dynamic cellular responses, it is important to quantify the rate of information flow through the signaling pathways. In this study, we used an epithelial cell line expressing a light-activatable FGF receptor and an ERK activity reporter to assess the ability of the MAPK/ERK pathway to transduce signal encoded in a sequence of pulses. By stimulating the cells with random light pulse trains, we demonstrated that the MAPK/ERK channel capacity is at least 6 bits per hour. The input reconstruction algorithm detects the light pulses with 1-min accuracy 5 min after their occurrence. The high information transmission rate may enable the pathway to coordinate multiple processes including cell movement and respond to rapidly varying stimuli such as chemoattracting gradients created by other cells.

Abstract: Cellular membranes contain numerous lipid species, and efforts to understand the biological functions of individual lipids have been stymied by a lack of approaches for controlled modulation of membrane composition in situ. Here we present a strategy for editing phospholipids, the most abundant lipids in biological membranes. Our membrane editor is based on a bacterial phospholipase D (PLD), which exchanges phospholipid head groups through hydrolysis or transphosphatidylation of phosphatidylcholine with water or exogenous alcohols. Exploiting activity-dependent directed enzyme evolution in mammalian cells, we have developed and structurally characterized a family of 'superPLDs' with up to a 100-fold enhancement in intracellular activity. We demonstrate the utility of superPLDs for both optogenetics-enabled editing of phospholipids within specific organelle membranes in live cells and biocatalytic synthesis of natural and unnatural designer phospholipids in vitro. Beyond the superPLDs, activity-based directed enzyme evolution in mammalian cells is a generalizable approach to engineer additional chemoenzymatic biomolecule editors.

Abstract: Ras signaling is typically associated with cell growth, but not direct regulation of motility or polarity. By optogenetically targeting different nodes in the Ras/PI3K/Akt network in differentiated human HL-60 neutrophils, we abruptly altered protrusive activity, bypassing the chemoattractant receptor/G-protein network. First, global recruitment of active KRas4B/HRas isoforms or a RasGEF, RasGRP4, immediately increased spreading and random motility. Second, activating Ras at the cell rear generated new protrusions, reversed pre-existing polarity, and steered sustained migration in neutrophils or murine RAW 264.7 macrophages. Third, recruiting a RasGAP, RASAL3, to cell fronts extinguished protrusions and changed migration direction. Remarkably, persistent RASAL3 recruitment at stable fronts abrogated directed migration in three different chemoattractant gradients. Fourth, local recruitment of the Ras-mTORC2 effector, Akt, in neutrophils or Dictyostelium amoebae generated new protrusions and rearranged pre-existing polarity. Overall, these optogenetic effects were mTORC2-dependent but relatively independent of PI3K. Thus, receptor-independent, local activations of classical growth-control pathways directly control actin assembly, cell shape, and migration modes.

Abstract: Optogenetics is a technique employing natural or genetically engineered photoreceptors in transgene organisms to manipulate biological activities with light. Light can be turned on or off, and adjusting its intensity and duration allows optogenetic fine-tuning of cellular processes in a noninvasive and spatiotemporally resolved manner. Since the introduction of Channelrhodopsin-2 and phytochrome-based switches nearly 20 years ago, optogenetic tools have been applied in a variety of model organisms with enormous success, but rarely in plants. For a long time, the dependence of plant growth on light and the absence of retinal, the rhodopsin chromophore, prevented the establishment of plant optogenetics until recent progress overcame these difficulties. We summarize the recent results of work in the field to control plant growth and cellular motion via green light-gated ion channels and present successful applications to light-control gene expression with single or combined photoswitches in plants. Furthermore, we highlight the technical requirements and options for future plant optogenetic research.

Abstract: Immunotherapy emerged as a paradigm shift in cancer treatments, which can effectively inhibit cancer progression by activating the immune system. Remarkable clinical outcomes have been achieved through recent advances in cancer immunotherapy, including checkpoint blockades, adoptive cellular therapy, cancer vaccine, and tumor microenvironment modulation. However, extending the application of immunotherapy in cancer patients has been limited by the low response rate and side effects such as autoimmune toxicities. With great progress being made in nanotechnology, nanomedicine has been exploited to overcome biological barriers for drug delivery. Given the spatiotemporal control, light-responsive nanomedicine is of great interest in designing precise modality for cancer immunotherapy. Herein, we summarized current research utilizing light-responsive nanoplatforms to enhance checkpoint blockade immunotherapy, facilitate targeted delivery of cancer vaccines, activate immune cell functions, and modulate tumor microenvironment. The clinical translation potential of those designs is highlighted and challenges for the next breakthrough in cancer immunotherapy are discussed.

Abstract: ERK and AKT signaling control pluripotent cell self-renewal versus differentiation. ERK pathway activity over time (i.e., dynamics) is heterogeneous between individual pluripotent cells, even in response to the same stimuli. To analyze potential functions of ERK and AKT dynamics in controlling mouse embryonic stem cell (ESC) fates, we developed ESC lines and experimental pipelines for the simultaneous long-term manipulation and quantification of ERK or AKT dynamics and cell fates. We show that ERK activity duration or amplitude or the type of ERK dynamics (e.g., transient, sustained, or oscillatory) alone does not influence exit from pluripotency, but the sum of activity over time does. Interestingly, cells retain memory of previous ERK pulses, with duration of memory retention dependent on duration of previous pulse length. FGF receptor/AKT dynamics counteract ERK-induced pluripotency exit. These findings improve our understanding of how cells integrate dynamics from multiple signaling pathways and translate them into cell fate cues.

Abstract: Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

Abstract: An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.