Curated Optogenetic Publication Database

Abstract: Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.

Abstract: Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

Abstract: Subcellular localization of signal molecules is a hallmark in organizing the signaling network. OpEn-Tag is a modular optogenetic endomembrane targeting toolbox that allows alteration of the localization and therefore the activity of signaling processes with the spatiotemporal resolution of optogenetics. OpEn-Tag is a two-component system employing (1) a variety of targeting peptides fused to and thereby dictating the localization of mCherry-labeled cryptochrome 2 binding protein CIBN toward distinct endomembranes and (2) the cytosolic, fluorescence-labeled blue light photoreceptor cryptochrome 2 as a customizable building block that can be fused to proteins of interest. The combination of OpEn-Tag with growth factor stimulation or the use of two membrane anchor sequences allows investigation of multilayered signal transduction processes as demonstrated here for the protein kinase AKT.

Abstract: Optogenetic probes can be powerful tools for dissecting complexity in cell biology, but there is a lack of instrumentation to exploit their potential for automated, high-information-content experiments. This protocol describes the construction and use of the optoPlate-96, a platform for high-throughput three-color optogenetics experiments that allows simultaneous manipulation of common red- and blue-light-sensitive optogenetic probes. The optoPlate-96 enables illumination of individual wells in 96-well microwell plates or in groups of wells in 384-well plates. Its design ensures that there will be no cross-illumination between microwells in 96-well plates, and an active cooling system minimizes sample heating during light-intensive experiments. This protocol details the steps to assemble, test, and use the optoPlate-96. The device can be fully assembled without specialized equipment beyond a 3D printer and a laser cutter, starting from open-source design files and commercially available components. We then describe how to perform a typical optogenetics experiment using the optoPlate-96 to stimulate adherent mammalian cells. Although optoPlate-96 experiments are compatible with any plate-based readout, we describe analysis using quantitative single-cell immunofluorescence. This workflow thus allows complex optogenetics experiments (independent control of stimulation colors, intensity, dynamics, and time points) with high-dimensional outputs at single-cell resolution. Starting from 3D-printed and laser-cut components, assembly and testing of the optoPlate-96 can be accomplished in 3-4 h, at a cost of ~$600. A full optoPlate-96 experiment with immunofluorescence analysis can be performed within ~24 h, but this estimate is variable depending on the cell type and experimental parameters.

Abstract: Over the last decade, multiple studies have shown that signaling proteins activated in different temporal patterns, such as oscillatory, transient, and sustained, can result in distinct gene expression patterns or cell fates. However, the molecular events that ensure appropriate stimulus- and dose-dependent dynamics are not often understood and are difficult to investigate. Here, we used single-cell analysis to dissect the mechanisms underlying the stimulus- and dose-encoding patterns in the innate immune signaling network. We found that Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling dynamics relied on a dose-dependent, autoinhibitory loop that rendered cells refractory to further stimulation. Using inducible gene expression and optogenetics to perturb the network at different levels, we identified IL-1R-associated kinase 1 (IRAK1) as the dose-sensing node responsible for limiting signal flow during the innate immune response. Although the kinase activity of IRAK1 was not required for signal propagation, it played a critical role in inhibiting the nucleocytoplasmic oscillations of the transcription factor NF-κB. Thus, protein activities that may be "dispensable" from a topological perspective can nevertheless be essential in shaping the dynamic response to the external environment.

Abstract: To decipher the kinetic regulation of growth factor signaling outcomes, I will introduce our recently developed non-neuronal optogenetic strategies that enable reversible control of growth factor signaling during cell differentiation and embryonic development.

Abstract: T cells are thought to discriminate self from foreign peptides by converting small differences in ligand binding half-life into large changes in cell signaling. Such a kinetic proofreading model has been difficult to test directly, as existing methods of altering ligand binding half-life also change other potentially important biophysical parameters, most notably the mechanical stability of the receptor-ligand interaction. Here we develop an optogenetic approach to specifically tune the binding half-life of a chimeric antigen receptor without changing other binding parameters and provide direct evidence of kinetic proofreading in T cell signaling. This half-life discrimination is executed in the proximal signaling pathway, downstream of ZAP70 recruitment and upstream of diacylglycerol accumulation. Our methods represent a general tool for temporal and spatial control of T cell signaling and extend the reach of optogenetics to probe pathways where the individual molecular kinetics, rather than the ensemble average, gates downstream signaling.

Abstract: The immune system distinguishes between self and foreign antigens. The kinetic proofreading (KPR) model proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B (PhyB) as a ligand to selectively control the dynamics of ligand binding to the TCR by light. This opto-ligand-TCR system was combined with the unique property of PhyB to continuously cycle between the binding and non-binding states under red light, with the light intensity determining the cycling rate and thus the binding duration. Mathematical modeling of our experimental datasets showed that indeed the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating KPR.

Abstract: The intracellular transport of receptor tyrosine kinases results in the differential activation of various signaling pathways. In this study, optogenetic stimulation of fibroblast growth factor receptor type 1 (FGFR1) was performed to study the effects of subcellular targeting of receptor kinases on signaling and neurite outgrowth. The catalytic domain of FGFR1 fused to the algal light-oxygen-voltage-sensing (LOV) domain was directed to different cellular compartments (plasma membrane, cytoplasm and nucleus) in human embryonic kidney (HEK293) and pheochromocytoma (PC12) cells. Blue light stimulation elevated the pERK and pPLCγ1 levels in membrane-opto-FGFR1-transfected cells similarly to ligand-induced receptor activation; however, no changes in pAKT levels were observed. PC12 cells transfected with membrane-opto-FGFR1 exhibited significantly longer neurites after light stimulation than after growth factor treatment, and significantly more neurites extended from their cell bodies. The activation of cytoplasmic FGFR1 kinase enhanced ERK signaling in HEK293 cells but not in PC12 cells and did not induce neuronal differentiation. The stimulation of FGFR1 kinase in the nucleus also did not result in signaling changes or neurite outgrowth. We conclude that FGFR1 kinase needs to be associated with membranes to induce the differentiation of PC12 cells mainly via ERK activation.

Abstract: Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.

Abstract: The Erk mitogen-activated protein kinase plays diverse roles in animal development. Its widespread reuse raises a conundrum: when a single kinase like Erk is activated, how does a developing cell know which fate to adopt? We combine optogenetic control with genetic perturbations to dissect Erk-dependent fates in the early Drosophila embryo. We find that Erk activity is sufficient to "posteriorize" 88% of the embryo, inducing gut endoderm-like gene expression and morphogenetic movements in all cells within this region. Gut endoderm fate adoption requires at least 1 h of signaling, whereas a 30-min Erk pulse specifies a distinct ectodermal cell type, intermediate neuroblasts. We find that the endoderm-ectoderm cell fate switch is controlled by the cumulative load of Erk activity, not the duration of a single pulse. The fly embryo thus harbors a classic example of dynamic control, where the temporal profile of Erk signaling selects between distinct physiological outcomes.

Abstract: In vivo noninvasively manipulating biological functions by the mediation of biosafe near infrared (NIR) light is becoming increasingly popular. For these applications, upconversion rare-earth nanomaterial holds great promise as a novel photonic element, and has been widely adopted in optogenetics. In this article, an upconversion optogenetic nanosystem that was promised to achieve autophagy up-regulation with spatiotemporal precision was designed. The implantable, wireless, recyclable, less-invasive and biocompatible system worked via two separated parts: blue light-receptor optogenetics-autophagy upregulation plasmids, for protein import; upconversion rods-encapsulated flexible capsule (UCRs-capsule), for converting tissue-penetrative NIR light into local visible blue light. Results validated that this system could achieve up-regulation of autophagy in vitro (in both HeLa and 293T cell lines) and remotely penetrate tissue (∼3.5 mm) in vivo. Since autophagy serves at a central position in intracellular signalling pathways, which is correlative with diverse pathologies, we expect that this method could establish an upconversion material-based autophagy up-regulation strategy for fundamental and clinical applications.

Abstract: Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots.

Abstract: Ras and Rho small GTPases are critical for numerous cellular processes including cell division, migration, and intercellular communication. Despite extensive efforts to visualize the spatiotemporal activity of these proteins, achieving the sensitivity and dynamic range necessary for in vivo application has been challenging. Here, we present highly sensitive intensiometric small GTPase biosensors visualizing the activity of multiple small GTPases in single cells in vivo. Red-shifted sensors combined with blue light-controllable optogenetic modules achieved simultaneous monitoring and manipulation of protein activities in a highly spatiotemporal manner. Our biosensors revealed spatial dynamics of Cdc42 and Ras activities upon structural plasticity of single dendritic spines, as well as a broad range of subcellular Ras activities in the brains of freely behaving mice. Thus, these intensiometric small GTPase sensors enable the spatiotemporal dissection of complex protein signaling networks in live animals.

Abstract: Optogenetic approaches have gathered momentum in precisely modulating and interrogating cellular signalling and gene expression. The use of optogenetics on the outer cell surface to interrogate how cells receive stimuli from their environment, however, has so far not reached its full potential. Here we demonstrate the development of an optogenetically regulated membrane receptor-ligand pair exemplified by the optically responsive interaction of an integrin receptor with the extracellular matrix. The system is based on an integrin engineered with a phytochrome-interacting factor domain (OptoIntegrin) and a red light-switchable phytochrome B-functionalized matrix (OptoMatrix). This optogenetic receptor-ligand pair enables light-inducible and -reversible cell-matrix interaction, as well as the controlled activation of downstream mechanosensory signalling pathways. Pioneering the application of optogenetic switches in the extracellular environment of cells, this OptoMatrix–OptoIntegrin system may serve as a blueprint for rendering matrix–receptor interactions amendable to precise control with light.

Abstract: Nerve growth factor elicits signaling outcomes by interacting with both its high-affinity receptor, TrkA, and its low-affinity receptor, p75NTR. Although these two receptors can regulate distinct cellular outcomes, they both activate the extracellular-signal-regulated kinase pathway upon nerve growth factor stimulation. To delineate TrkA subcircuits in PC12 cell differentiation, we developed an optogenetic system whereby light was used to specifically activate TrkA signaling in the absence of nerve growth factor. By using tyrosine mutants of the optogenetic TrkA in combination with pathway-specific pharmacological inhibition, we find that Y490 and Y785 each contributes to PC12 cell differentiation through the extracellular-signal-regulated kinase pathway in an additive manner. Optogenetic activation of TrkA eliminates the confounding effect of p75NTR and other potential off-target effects of the ligand. This approach can be generalized for the mechanistic study of other receptor-mediated signaling pathways.

Abstract: Abstract not available.

Abstract: Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) is a crucial lipid that regulates PIP3 dynamics. Investigations of how PIP3 is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information of membrane dynamics upon PIP3 production. In the present study, we applied an optogenetic approach using the CRY2–CIBN system. Using this system, we revealed that local PIP3 generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP3-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

Abstract: Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments can often identify the existence of feedback interactions but are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed-loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors, in real time, the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver-on the fly-an optogenetically enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.

Abstract: The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anti-cancer strategies.

Abstract: Epithelial-mesenchymal transition (EMT) is one of the most important mechanisms in the initiation and promotion of cancer cell metastasis. The phosphoinositide 3-kinase (PI3K) signaling pathway has been demonstrated to be involved in TGF-β induced EMT, but the complicated TGF-β signaling network makes it challenging to dissect the important role of PI3K on regulation of EMT process. Here, we applied optogenetic controlled PI3K module (named 'Opto-PI3K'), which based on CRY2 and the N-terminal of CIB1 (CIBN), to rapidly and reversibly control the endogenous PI3K activity in cancer cells with light. By precisely modulating the kinetics of PI3K activation, we found that E-cadherin is an important downstream target of PI3K signaling. Compared with TGF-β treatment, Opto-PI3K had more potent effect in down-regulation of E-cadherin expression, which was demonstrated to be regulated in a light dose-dependent manner. Surprisingly, sustained PI3K activation induced partial EMT state in A549 cells that is highly reversible. Furthermore, we demonstrated that Opto-PI3K only partially mimicked TGF-β effects on promotion of cell migration in vitro. These results reveal the importance of PI3K signaling in TGF-β induced EMT, suggesting other TGF-β regulated signaling pathways are necessary for the full and irreversible promotion of EMT in cancer cells. In addition, our study implicates the great promise of optogenetics in cancer research for mapping input-output relationships in oncogenic pathways.

Abstract: The Ras-Erk (extracellular signal-regulated kinase) pathway encodes information in its dynamics; the duration and frequency of Erk activity can specify distinct cell fates. To enable dynamic encoding, temporal information must be accurately transmitted from the plasma membrane to the nucleus. We used optogenetic profiling to show that both oncogenic B-Raf mutations and B-Raf inhibitors can cause corruption of this transmission, so that short pulses of input Ras activity are distorted into abnormally long Erk outputs. These changes can reshape downstream transcription and cell fates, resulting in improper decisions to proliferate. These findings illustrate how altered dynamic signal transmission properties, and not just constitutively increased signaling, can contribute to cell proliferation and perhaps cancer, and how optogenetic profiling can dissect mechanisms of signaling dysfunction in disease.

Abstract: Nerve growth factor/tropomyosin receptor kinase A (NGF/TrkA) signaling plays a key role in neuronal development, function, survival, and growth. The pathway is implicated in neurodegenerative disorders including Alzheimer's disease, chronic pain, inflammation, and cancer. NGF binds the extracellular domain of TrkA, leading to the activation of the receptor's intracellular kinase domain. TrkA signaling is highly dynamic, thus mechanistic studies would benefit from a tool with high spatial and temporal resolution. Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 (CRY2) and its binding partner CIB1. We demonstrate successful recapitulation of native NGF/TrkA functions by optical induction of plasma membrane recruitment and homo-interaction of the intracellular domain of TrkA. This approach activates PI3K/AKT and Raf/ERK signaling pathways, promotes neurite growth in PC12 cells, and supports the survival of dorsal root ganglion neurons in the absence of NGF. This ability to activate TrkA using light bestows high spatial and temporal resolution for investigating NGF/TrkA signaling.

Abstract: Altered glycolysis is a hallmark of diseases including diabetes and cancer. Despite intensive study of the contributions of individual glycolytic enzymes, systems-level analyses of flux control through glycolysis remain limited. Here, we overexpress in two mammalian cell lines the individual enzymes catalyzing each of the 12 steps linking extracellular glucose to excreted lactate, and find substantial flux control at four steps: glucose import, hexokinase, phosphofructokinase, and lactate export (and not at any steps of lower glycolysis). The four flux-controlling steps are specifically upregulated by the Ras oncogene: optogenetic Ras activation rapidly induces the transcription of isozymes catalyzing these four steps and enhances glycolysis. At least one isozyme catalyzing each of these four steps is consistently elevated in human tumors. Thus, in the studied contexts, flux control in glycolysis is concentrated in four key enzymatic steps. Upregulation of these steps in tumors likely underlies the Warburg effect.

Abstract: Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.