Showing 26 - 50 of 116 results
Light-induced dimerization approaches to control cellular processes.
Light-inducible approaches provide means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. The new expansions of the toolbox facilitate control of signal transduction, genome editing, 'painting' patterns of active molecules onto cellular membranes and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches has also seen interesting progress. Here we provide an overview of the optogenetic systems and the emerging chemo-optogenetic systems, and discuss recent applications in tackling complex biological problems.
Engineered illumination devices for optogenetic control of cellular signaling dynamics.
Spatially and temporally varying patterns of morphogen signals during development drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. Here we show that optogenetic Wnt/β-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). The optical design of LAVA devices was optimized for uniform illumination of multi-well cell culture plates to enable high-throughput, spatiotemporal optogenetic activation of signaling pathways and protein-protein interactions. Using the LAVA devices, variation in light intensity induced a dose-dependent response in optoWnt activation and downstream Brachyury expression in human embryonic stem cells (hESCs). Furthermore, time-varying and spatially localized patterns of light revealed tissue patterning that models embryonic presentation of Wnt signals in vitro. The engineered LAVA devices thus provide a low-cost, user-friendly method for high-throughput and spatiotemporal optogenetic control of cell signaling for applications in developmental and cell biology.
Regulation of signaling proteins in the brain by light.
In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.
Optogenetic control of Wnt signaling for modeling early embryogenic patterning with human pluripotent stem cells.
The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by dynamic, spatially and temporally varying morphogen signals. Analogous tissue patterns emerge spontaneously in embryonic stem cell (ESC) models for gastrulation, but mechanistic insight into this self-organization is limited by a lack of molecular methods to precisely control morphogen signal dynamics. Here we combine optogenetic stimulation and single-cell imaging approaches to study self-organization of human pluripotent stem cells. Precise control of morphogen signal dynamics, achieved through activation of canonical Wnt/β-catenin signaling over a broad high dynamic range (>500-fold) using an optoWnt optogenetic system, drove broad transcriptional changes and mesendoderm differentiation of human ESCs at high efficiency (>95% cells). Furthermore, activating Wnt signaling in subpopulations of ESCs in 2D and 3D cultures induced cell self-organization and morphogenesis reminiscent of human gastrulation, including changes in cell migration and epithelial to mesenchymal transition. Our findings thus reveal an instructive role for Wnt in directing cell patterning in this ESC model for gastrulation.
Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein-Protein Interactions.
Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein-protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Light-based control of metabolic flux through assembly of synthetic organelles.
To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology.
Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.
Optically inducible membrane recruitment and signaling systems.
Optical induction of intracellular signaling by membrane-associated and integral membrane proteins allows spatiotemporally precise control over second messenger signaling and cytoskeletal rearrangements that are important to cell migration, development, and proliferation. Optogenetic membrane recruitment of a protein-of-interest to control its signaling by altering subcellular localization is a versatile means to these ends. Here, we summarize the signaling characteristics and underlying structure-function of RGS-LOV photoreceptors as single-component membrane recruitment tools that rapidly, reversibly, and efficiently carry protein cargo from the cytoplasm to the plasma membrane by a light-regulated electrostatic interaction with the membrane itself. We place the technology-relevant features of these recently described natural photosensory proteins in context of summarized protein engineering and design strategies for optically controlling membrane protein signaling.
RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43.
TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.
Photodimerization systems for regulating protein-protein interactions with light.
Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.
Continued Activity of the Pioneer Factor Zelda Is Required to Drive Zygotic Genome Activation.
Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.
Synthetic switches and regulatory circuits in plants.
Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical- and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi- and fully synthetic open- and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.
Intensiometric biosensors visualize the activity of multiple small GTPases in vivo.
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.
Perspectives of RAS and RHEB GTPase Signaling Pathways in Regenerating Brain Neurons.
Cellular activation of RAS GTPases into the GTP-binding "ON" state is a key switch for regulating brain functions. Molecular protein structural elements of rat sarcoma (RAS) and RAS homolog protein enriched in brain (RHEB) GTPases involved in this switch are discussed including their subcellular membrane localization for triggering specific signaling pathways resulting in regulation of synaptic connectivity, axonal growth, differentiation, migration, cytoskeletal dynamics, neural protection, and apoptosis. A beneficial role of neuronal H-RAS activity is suggested from cellular and animal models of neurodegenerative diseases. Recent experiments on optogenetic regulation offer insights into the spatiotemporal aspects controlling RAS/mitogen activated protein kinase (MAPK) or phosphoinositide-3 kinase (PI3K) pathways. As optogenetic manipulation of cellular signaling in deep brain regions critically requires penetration of light through large distances of absorbing tissue, we discuss magnetic guidance of re-growing axons as a complementary approach. In Parkinson's disease, dopaminergic neuronal cell bodies degenerate in the substantia nigra. Current human trials of stem cell-derived dopaminergic neurons must take into account the inability of neuronal axons navigating over a large distance from the grafted site into striatal target regions. Grafting dopaminergic precursor neurons directly into the degenerating substantia nigra is discussed as a novel concept aiming to guide axonal growth by activating GTPase signaling through protein-functionalized intracellular magnetic nanoparticles responding to external magnets.
A bright future: optogenetics to dissect the spatiotemporal control of cell behavior.
Cells sense, process, and respond to extracellular information using signaling networks: collections of proteins that act as precise biochemical sensors. These protein networks are characterized by both complex temporal organization, such as pulses of signaling activity, and by complex spatial organization, where proteins assemble structures at particular locations and times within the cell. Yet despite their ubiquity, studying these spatial and temporal properties has remained challenging because they emerge from the entire protein network rather than a single node, and cannot be easily tuned by drugs or mutations. These challenges are being met by a new generation of optogenetic tools capable of directly controlling the activity of individual signaling nodes over time and the assembly of protein complexes in space. Here, we outline how these recent innovations are being used in conjunction with engineering-influenced experimental design to address longstanding questions in signaling biology.
Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome.
Phase transitions involving biomolecular liquids are a
fundamental mechanism underlying intracellular organization.
In the cell nucleus, liquid-liquid phase
separation of intrinsically disordered proteins (IDPs)
is implicated in assembly of the nucleolus, as well
as transcriptional clusters, and other nuclear bodies.
However, it remains unclear whether and how physical
forces associated with nucleation, growth, and
wetting of liquid condensates can directly restructure
chromatin. Here, we use CasDrop, a novel
CRISPR-Cas9-based optogenetic technology, to
show that various IDPs phase separate into liquid
condensates that mechanically exclude chromatin
as they grow and preferentially form in low-density,
largely euchromatic regions. A minimal physical
model explains how this stiffness sensitivity arises
from lower mechanical energy associated with deforming
softer genomic regions. Targeted genomic
loci can nonetheless be mechanically pulled together
through surface tension-driven coalescence. Nuclear
condensates may thus function as mechanoactive
chromatin filters, physically pulling in targeted
genomic loci while pushing out non-targeted regions
of the neighboring genome.
Optogenetic dissection of Rac1 and Cdc42 gradient shaping.
During cell migration, Rho GTPases spontaneously form spatial gradients that define the front and back of cells. At the front, active Cdc42 forms a steep gradient whereas active Rac1 forms a more extended pattern peaking a few microns away. What are the mechanisms shaping these gradients, and what is the functional role of the shape of these gradients? Here we report, using a combination of optogenetics and micropatterning, that Cdc42 and Rac1 gradients are set by spatial patterns of activators and deactivators and not directly by transport mechanisms. Cdc42 simply follows the distribution of Guanine nucleotide Exchange Factors, whereas Rac1 shaping requires the activity of a GTPase-Activating Protein, β2-chimaerin, which is sharply localized at the tip of the cell through feedbacks from Cdc42 and Rac1. Functionally, the spatial extent of Rho GTPases gradients governs cell migration, a sharp Cdc42 gradient maximizes directionality while an extended Rac1 gradient controls the speed.
Programming Bacteria With Light—Sensors and Applications in Synthetic Biology
Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture.
Bringing Light to Transcription: The Optogenetics Repertoire.
The ability to manipulate expression of exogenous genes in particular regions of living organisms has profoundly transformed the way we study biomolecular processes involved in both normal development and disease. Unfortunately, most of the classical inducible systems lack fine spatial and temporal accuracy, thereby limiting the study of molecular events that strongly depend on time, duration of activation, or cellular localization. By exploiting genetically engineered photo sensing proteins that respond to specific wavelengths, we can now provide acute control of numerous molecular activities with unprecedented precision. In this review, we present a comprehensive breakdown of all of the current optogenetic systems adapted to regulate gene expression in both unicellular and multicellular organisms. We focus on the advantages and disadvantages of these different tools and discuss current and future challenges in the successful translation to more complex organisms.
CRAC channel-based optogenetics.
Store-operated Ca²+ entry (SOCE) constitutes a major Ca2+ influx pathway in mammals to regulate a myriad of physiological processes, including muscle contraction, synaptic transmission, gene expression, and metabolism. In non-excitable cells, the Ca²+ release-activated Ca²+ (CRAC) channel, composed of ORAI and stromal interaction molecules (STIM), constitutes a prototypical example of SOCE to mediate Ca2+ entry at specialized membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and the plasma membrane (PM). The key steps of SOCE activation include the oligomerization of the luminal domain of the ER-resident Ca2+ sensor STIM1 upon Ca²+ store depletion, subsequent signal propagation toward the cytoplasmic domain to trigger a conformational switch and overcome the intramolecular autoinhibition, and ultimate exposure of the minimal ORAI-activating domain to directly engage and gate ORAI channels in the plasma membrane. This exquisitely coordinated cellular event is also facilitated by the C-terminal polybasic domain of STIM1, which physically associates with negatively charged phosphoinositides embedded in the inner leaflet of the PM to enable efficient translocation of STIM1 into ER-PM MCSs. Here, we present recent progress in recapitulating STIM1-mediated SOCE activation by engineering CRAC channels with optogenetic approaches. These STIM1-based optogenetic tools make it possible to not only mechanistically recapture the key molecular steps of SOCE activation, but also remotely and reversibly control Ca²+-dependent cellular processes, inter-organellar tethering at MCSs, and transcriptional reprogramming when combined with CRISPR/Cas9-based genome-editing tools.
Synergistic Ensemble of Optogenetic Actuators and Dynamic Indicators in Cell Biology.
Discovery of the naturally evolved fluorescent proteins and their genetically engineered biosensors have enormously contributed to current bio-imaging techniques. These reporters to trace dynamic changes of intracellular protein activities have continuously transformed according to the various demands in biological studies. Along with that, light-inducible optogenetic technologies have offered scientists to perturb, control and analyze the function of intracellular machineries in spatiotemporal manner. In this review, we present an overview of the molecular strategies that have been exploited for producing genetically encoded protein reporters and various optogenetic modules. Finally, in particular, we discuss the current efforts for combined use of these reporters and optogenetic modules as a powerful tactic for the control and imaging of signaling events in cells and tissues.
Illuminating pathogen-host intimacy through optogenetics.
The birth and subsequent evolution of optogenetics has resulted in an unprecedented advancement in our understanding of the brain. Its outstanding success does usher wider applications; however, the tool remains still largely relegated to neuroscience. Here, we introduce selected aspects of optogenetics with potential applications in infection biology that will not only answer long-standing questions about intracellular pathogens (parasites, bacteria, viruses) but also broaden the dimension of current research in entwined models. In this essay, we illustrate how a judicious integration of optogenetics with routine methods can illuminate the host-pathogen interactions in a way that has not been feasible otherwise.
Blue-Light Receptors for Optogenetics.
Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.
Optical activation of TrkA signaling.
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.
Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli.
Protein/RNA clusters arise frequently in spatially regulated biological processes, from the asymmetric distribution of P granules and PAR proteins in developing embryos to localized receptor oligomers in migratory cells. This co-occurrence suggests that protein clusters might possess intrinsic properties that make them a useful substrate for spatial regulation. Here, we demonstrate that protein droplets show a robust form of spatial memory, maintaining the spatial pattern of an inhibitor of droplet formation long after it has been removed. Despite this persistence, droplets can be highly dynamic, continuously exchanging monomers with the diffuse phase. We investigate the principles of biophysical spatial memory in three contexts: a computational model of phase separation; a novel optogenetic system where light can drive rapid, localized dissociation of liquid-like protein droplets; and membrane-localized signal transduction from clusters of receptor tyrosine kinases. Our results suggest that the persistent polarization underlying many cellular and developmental processes could arise through a simple biophysical process, without any additional biochemical feedback loops.