Showing 426 - 450 of 1087 results
Physical Plasma Membrane Perturbation Using Subcellular Optogenetics Drives Integrin-Activated Cell Migration.
Cells experience physical deformations to the plasma membrane that can modulate cell behaviors like migration. Understanding the molecular basis for how physical cues affect dynamic cellular responses requires new approaches that can physically perturb the plasma membrane with rapid, reversible, subcellular control. Here we present an optogenetic approach based on light-inducible dimerization that alters plasma membrane properties by recruiting cytosolic proteins at high concentrations to a target site. Surprisingly, this polarized accumulation of proteins in a cell induces directional amoeboid migration in the opposite direction. Consistent with known effects of constraining high concentrations of proteins to a membrane in vitro, there is localized curvature and tension decrease in the plasma membrane. Integrin activity, sensitive to mechanical forces, is activated in this region. Localized mechanical activation of integrin with optogenetics allowed simultaneous imaging of the molecular and cellular response, helping uncover a positive feedback loop comprising SFK- and ERK-dependent RhoA activation, actomyosin contractility, rearward membrane flow, and membrane tension decrease underlying this mode of cell migration.
Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells.
Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells.
Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo.
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.
Developmental Erk Signaling Illuminated.
How a small number of signaling pathways can be re-used in distinct embryonic contexts to control different fates remains unclear. In this issue of Developmental Cell, Johnson and Toettcher (2019) use optogenetic approaches to explore how different dynamic ERK signaling states control specific developmental fates in the Drosophila embryo.
Optogenetic Control of Subcellular Protein Location and Signaling in Vertebrate Embryos.
This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an "anchor" protein and a "bait" protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117-126, 2016) and describe the imaging parameters used to achieve subcellular light patterning.
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 Control of Protein Degradation during Cell Proliferation and Developmental Processes.
Synthetic tools for the control of protein function are valuable for biomedical research to characterize cellular functions of essential proteins or if a rapid switch in protein activity is necessary. The ability to tune protein activity precisely opens another level of investigations that is not available with gene deletion mutants. Control of protein stability is a versatile approach to influence the activity of a target protein by its cellular abundance. Diverse strategies have been developed to achieve efficient proteolysis using external inducers or differentiation-coupled signals. The latter is especially important for the inactivation of a protein during a developmental process. Recently, several approaches to achieve this have been engineered. In this article, we present current synthetic tools for regulation of protein stability that allow fine-tuning of protein abundance, their advantages and disadvantages with an emphasis on methods applicable in the context of cell differentiation and development. We give an outlook toward future developments and discuss main applications of these tools.
Optogenetic Navigation of Routes Leading to Protein Amyloidogenesis in Bacteria.
Modulation of liquid-liquid and liquid-hydrogel phase transitions is central to avoid the cytotoxic aggregation of proteins in eukaryotic cells, but knowledge on its relevance in bacteria is limited. Here the power of optogenetics to engineer proteins as light-responsive switches has been used to control the balance between solubility and aggregation for LOV2-WH1, a chimera between the plant blue light-responsive domain LOV2 and the bacterial prion-like protein RepA-WH1. These proteins were first linked by fusing, as a continuous α-helix, the C-terminal photo-transducer Jα helix in LOV2 with the N-terminal domain-closure α1 helix in RepA-WH1, and then improved for light-responsiveness by including mutations in the Jα moiety. In the darkness and in a crowded solution in vitro, LOV2-WH1 nucleates the irreversible assembly of amyloid fibers into a hydrogel. However, under blue light illumination LOV2-WH1 assembles as soluble oligomers. When expressed in Escherichia coli, LOV2-WH1 forms in the darkness large intracellular amyloid inclusions compatible with bacterial proliferation. Strikingly, under blue light LOV2-WH1 aggregates decrease in size while they become detrimental for bacterial growth. LOV2-WH1 optogenetics governs the assembly of mutually exclusive inert amyloid fibers or cytotoxic oligomers, thus enabling the navigation of the conformational landscape of protein amyloidogenesis to generate potential photo-activated anti-bacterial devices (optobiotics).
Near-infrared light remotely up-regulate autophagy with spatiotemporal precision via upconversion optogenetic nanosystem.
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.
Cell-machine interfaces for characterizing gene regulatory network dynamics.
Gene regulatory networks and the dynamic responses they produce offer a wealth of information about how biological systems process information about their environment. Recently, researchers interested in dissecting these networks have been outsourcing various parts of their experimental workflow to computers. Here we review how, using microfluidic or optogenetic tools coupled with fluorescence imaging, it is now possible to interface cells and computers. These platforms enable scientists to perform informative dynamic stimulations of genetic pathways and monitor their reaction. It is also possible to close the loop and regulate genes in real time, providing an unprecedented view of how signals propagate through the network. Finally, we outline new tools that can be used within the framework of cell-machine interfaces.
Optogenetic tools light up phase separation.
Abstract not available.
Photo‐ECM: A Blue Light Photoswitchable Synthetic Extracellular Matrix Protein for Reversible Control over Cell–Matrix Adhesion.
The dynamic and spatiotemporal control of integrin‐mediated cell adhesion to RGD motifs in its extracellular matrix (ECM) is important for understating cell biology and biomedical applications because cell adhesion fundamentally regulates cellular behavior. Herein, the first photoswitchable synthetic ECM protein, Photo‐ECM, based on the blue light switchable protein LOV2 is engineered. The Photo‐ECM protein includes a RGD sequence, which is hidden in the folded LOV2 protein structure in the dark and is exposed under blue light so that integrins can bind and cells can adhere. The switchable presentation of the RGD motif allows to reversibly mediate and modulate integrin‐based cell adhesions using noninvasive blue light. With this protein cell adhesions in live cells could be reversed and the dynamics at the cellular level is observed. Hence, the Photo‐ECM opens a new possibility to investigate the spatiotemporal regulation of cell adhesions in cell biology and is the first step toward a genetically encoded and light‐responsive ECM.
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.
Phytochrome-Based Extracellular Matrix with Reversibly Tunable Mechanical Properties.
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.
Perspective Tools for Optogenetics and Photopharmacology: From Design to Implementation.
Optogenetics and photopharmacology are two perspective modern
methodologies for control and monitoring of biological processes from an isolated
cell to complex cell assemblies and organisms. Both methodologies use optically
active components that being introduced into the cells of interest allow for optical
control or monitoring of different cellular processes. In optogenetics, genetic
materials are introduced into the cells to express light-sensitive proteins or protein
constructs. In photopharmacology, photochromic compounds are delivered into a
cell directly but not produced inside the cell from a genetic material. The development
of both optogenetics and photopharmacology is inseparable from the design
of improved tools (protein constructs or organic molecules) optimized for specific
applications. Herein, we review the main tools that are used in modern optogenetics
and photopharmaclogy and describe the types of cellular processes that can be
controlled by these tools. Although a large number of different kinds of optogenetic
tools exist, their performance can be evaluated with a limited number of metrics that
have to be optimized for specific applications.We classify thesemetrics and describe
the ways of their improvement.
Blue Light Switchable Cell–Cell Interactions Provide
Reversible and Spatiotemporal Control Towards
Bottom-Up Tissue Engineering.
Controlling cell–cell interactions is central for understanding key cellular
processes and bottom-up tissue assembly from single cells. The challenge is
to control cell–cell interactions dynamically and reversibly with high spati-
otemporal precision noninvasively and sustainably. In this study, cell–cell
interactions are controlled with visible light using an optogenetic approach by
expressing the blue light switchable proteins CRY2 or CIBN on the surfaces of
cells. CRY2 and CIBN expressing cells form specific heterophilic interactions
under blue light providing precise control in space and time. Further, these
interactions are reversible in the dark and can be repeatedly and dynamically
switched on and off. Unlike previous approaches, these genetically encoded
proteins allow for long-term expression of the interaction domains and
respond to nontoxic low intensity blue light. In addition, these interactions
are suitable to assemble cells into 3D multicellular architectures. Overall, this
approach captures the dynamic and reversible nature of cell–cell interactions
and controls them noninvasively and sustainably both in space and time. This
provides a new way of studying cell–cell interactions and assembling cellular
building blocks into tissues with unmatched flexibility.
Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions.
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp-dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.
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.
Characterization and engineering of photoactivated adenylyl cyclases.
Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3', 5'-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.
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.