Showing 26 - 50 of 647 results
Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing.
From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.
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.
Optogenetic control of integrin-matrix interaction.
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.
Optoregulated Drug Release from an Engineered Living Material: Self-Replenishing Drug Depots for Long-Term, Light-Regulated Delivery.
On-demand and long-term delivery of drugs are common requirements in many therapeutic applications, not easy to be solved with available smart polymers for drug encapsulation. This work presents a fundamentally different concept to address such scenarios using a self-replenishing and optogenetically controlled living material. It consists of a hydrogel containing an active endotoxin-free Escherichia coli strain. The bacteria are metabolically and optogenetically engineered to secrete the antimicrobial and antitumoral drug deoxyviolacein in a light-regulated manner. The permeable hydrogel matrix sustains a viable and functional bacterial population and permits diffusion and delivery of the synthesized drug to the surrounding medium at quantities regulated by light dose. Using a focused light beam, the site for synthesis and delivery of the drug can be freely defined. The living material is shown to maintain considerable levels of drug production and release for at least 42 days. These results prove the potential and flexibility that living materials containing engineered bacteria can offer for advanced therapeutic applications.
Optogenetic Delineation of Receptor Tyrosine Kinase Subcircuits in PC12 Cell Differentiation.
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.
A size-invariant bud-duration timer enables robustness in yeast cell size control.
Cell populations across nearly all forms of life generally maintain a characteristic cell type-dependent size, but how size control is achieved has been a long-standing question. The G1/S boundary of the cell cycle serves as a major point of size control, and mechanisms operating here restrict passage of cells to Start if they are too small. In contrast, it is less clear how size is regulated post-Start, during S/G2/M. To gain further insight into post-Start size control, we prepared budding yeast that can be reversibly blocked from bud initiation. While blocked, cells continue to grow isotropically, increasing their volume by more than an order of magnitude over unperturbed cells. Upon release from their block, giant mothers reenter the cell cycle and their progeny rapidly return to the original unperturbed size. We found this behavior to be consistent with a size-invariant 'timer' specifying the duration of S/G2/M. These results indicate that yeast use at least two distinct mechanisms at different cell cycle phases to ensure size homeostasis.
Luminescence-activated nucleotide cyclase regulates spatial and temporal cAMP synthesis.
cAMP is a ubiquitous second messenger that regulates cellular proliferation, differentiation, attachment, migration, and several other processes. It has become increasingly evident that tight regulation of cAMP accumulation and localization confers divergent yet specific signaling to downstream pathways. Currently, few tools are available that have sufficient spatial and temporal resolution to study location-biased cAMP signaling. Here, we introduce a new fusion protein consisting of a light-activated adenylyl cyclase (bPAC) and luciferase (nLuc). This construct allows dual activation of cAMP production through temporally precise photostimulation or chronic chemical stimulation that can be fined-tuned to mimic physiological levels and duration of cAMP synthesis to trigger downstream events. By targeting this construct to different compartments, we show that cAMP produced in the cytosol and nucleus stimulates proliferation in thyroid cells. The bPAC-nLuc fusion construct adds a new reagent to the available toolkit to study cAMP-regulated processes in living cells.
Using Synthetic Biology to Engineer Spatial Patterns.
Synthetic biology has emerged as a multidisciplinary field that provides new tools and approaches to address longstanding problems in biology. It integrates knowledge from biology, engineering, mathematics, and biophysics to build—rather than to simply observe and perturb—biological systems that emulate natural counterparts or display novel properties. The interface between synthetic and developmental biology has greatly benefitted both fields and allowed to address questions that would remain challenging with classical approaches due to the intrinsic complexity and essentiality of developmental processes. This Progress Report provides an overview of how synthetic biology can help to understand a process that is crucial for the development of multicellular organisms: pattern formation. It reviews the major mechanisms of genetically encoded synthetic systems that have been engineered to establish spatial patterns at the population level. Limitations, challenges, applications, and potential opportunities of synthetic pattern formation are also discussed.
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.
Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.
Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. The c-di-GMP-targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications such as biofilm-based biocatalysis.
Development of a Wireless-Controlled LED Array for the Tunable Optogenetic Control of Cellular Activities.
Abstract not available.
Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.
Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.
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.
An Open-Source Plate Reader.
Microplate readers are foundational instruments in ex-perimental biology and bioengineering that enable mul-tiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construc-tion, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs <$3500, a fraction of the cost of commer-cial plate readers, and can detect the fluorescence of common dyes down to ~10 nanomolar concentration. Functional capabilities were demonstrated in context of synthetic biology, optoge¬netics, and photosensory biol-ogy: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing, and by flavin photocycling kinetic measure-ments of a LOV (light-oxygen-voltage) domain photo-receptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and au-tomating it using the custom Python-based API (Appli-cation Program Interface) are provided. This work con-tributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid proto-typing capabilities and low-cost electronics, optoelec-tronics, and microcomputers.
Mechanobiology of Protein Droplets: Force Arises from Disorder.
The use of optogenetic approaches has revealed new roles for intracellular protein condensates
described in two papers in this issue of Cell (Bracha et. al., 2018; Shin et al., 2018). These results
show that growing condensates are able to exert mechanical forces resulting in chromatin
rearrangement, establishing a new role for liquid-liquid phase separation in the mechanobiology
of the cell.
Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.
Liquid-liquid phase separation plays a key role in the
assembly of diverse intracellular structures. However,
the biophysical principles by which phase separation
can be precisely localized within subregions
of the cell are still largely unclear, particularly for
low-abundance proteins. Here, we introduce an oligomerizing
biomimetic system, ‘‘Corelets,’’ and utilize
its rapid and quantitative light-controlled
tunability to map full intracellular phase diagrams,
which dictate the concentrations at which phase
separation occurs and the transition mechanism, in
a protein sequence dependent manner. Surprisingly,
both experiments and simulations show that while
intracellular concentrations may be insufficient for
global phase separation, sequestering protein ligands
to slowly diffusing nucleation centers can
move the cell into a different region of the phase diagram,
resulting in localized phase separation. This
diffusive capture mechanism liberates the cell from
the constraints of global protein abundance and is
likely exploited to pattern condensates associated
with diverse biological processes.
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 control of morphogenesis goes 3D.
The generation of form in living embryos, a process termed “morphogenesis” from the Greek word lοqφοcέmerg, is one of the most fascinating unsolved problems in biology. In embryonic epithelia, most attention has been paid to events occurring at the apical surface of epithelia, particularly the regulation of actomyosin contractility during morphogenetic change. In a new report, De Renzis and colleagues demonstrate a key role for regulated actomyosin contractility at the basal surface of the epithelium during formation of the first epithelial fold in Drosophila (the “ventral furrow”) (Krueger et al, 2018).
Mitotic Spindle: Illuminating Spindle Positioning with a Biological Lightsaber.
In metazoans, positioning of the mitotic spindle is controlled by the microtubule-dependent motor protein dynein, which associates with the cell cortex. Using optogenetic tools, two new studies examine how the levels and activity of dynein are regulated at the cortex to ensure proper positioning of the mitotic spindle.
Guided by light: optogenetic control of microtubule gliding assays.
Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays, in which surface-immobilized motor proteins drive microtubule propulsion, are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, bio-computation and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ~5-fold enrichment within 6 seconds upon illumination. Subsequently, proteins are released with a half-life of 13 seconds when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of non-uniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.
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.
Downregulation of basal myosin-II is required for cell shape changes and tissue invagination.
Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin-mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. Here we use optogenetics to quantitatively control myosin-II levels at the basal surface of invaginating cells during Drosophila gastrulation. We show that while basal myosin-II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre-invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube-like structure. Collectively, these data demonstrate the requirement of myosin-II polarization and basal relaxation throughout the entire invagination process.
Engineering Improved Photoswitches for the Control of Nucleocytoplasmic Distribution.
Optogenetic techniques use light-responsive proteins to study dynamic processes in living cells and organisms. These techniques typically rely on repurposed naturally occurring light-sensitive proteins to control sub-cellular localization and activity. We previously engineered two optogenetic systems, the Light Activated Nuclear Shuttle (LANS) and the Light-Inducible Nuclear eXporter (LINX), by embedding nuclear import or export sequence motifs into the C-terminal helix of the light-responsive LOV2 domain of Avena sativa phototropin 1, thus enabling light-dependent trafficking of a target protein into and out of the nucleus. While LANS and LINX are effective tools, we posited that mutations within the LOV2 hinge-loop, which connects the core PAS domain and the C-terminal helix, would further improve the functionality of these switches. Here, we identify hinge-loop mutations that favourably shift the dynamic range (the ratio of the on- to off-target subcellular accumulation) of the LANS and LINX photoswitches. We demonstrate the utility of these new optogenetic tools to control gene transcription and epigenetic modifications, thereby expanding the optogenetic 'tool kit' for the research community.
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.
Diverse light responses of cyanobacteria mediated by phytochrome superfamily photoreceptors.
Cyanobacteria are an evolutionarily and ecologically important group of prokaryotes. They exist in diverse habitats, ranging from hot springs and deserts to glaciers and the open ocean. The range of environments that they inhabit can be attributed in part to their ability to sense and respond to changing environmental conditions. As photosynthetic organisms, one of the most crucial parameters for cyanobacteria to monitor is light. Cyanobacteria can sense various wavelengths of light and many possess a range of bilin-binding photoreceptors belonging to the phytochrome superfamily. Vital cellular processes including growth, phototaxis, cell aggregation and photosynthesis are tuned to environmental light conditions by these photoreceptors. In this Review, we examine the physiological responses that are controlled by members of this diverse family of photoreceptors and discuss the signal transduction pathways through which these photoreceptors operate. We highlight specific examples where the activities of multiple photoreceptors function together to fine-tune light responses. We also discuss the potential application of these photosensing systems in optogenetics and synthetic biology.