Showing 1 - 25 of 82 results
High-throughput multicolor optogenetics in microwell plates.
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.
Optogenomic Interfaces: Bridging Biological Networks With the Electronic Digital World.
The development of optical nano-bio interfaces is a fundamental step toward connecting biological networks and traditional electronic computing systems. Compared to conventional chemical and electrical nano-bio interfaces, the use of light as a mediator enables new type of interfaces with unprecedented spatial and temporal resolutions. In this paper, the state of the art and future research directions in optogenomic interfaces are discussed. Optogenomic interfaces are light-mediated nano-bio interfaces that allow the control of the genome, i.e., the genes and their interactions in the cell nucleus (and, thus, of all the cell functionalities) with (sub) cellular resolution and high temporal accuracy. Given its fundamental role in the process of cell development, the study is focused on the interactions with the fibroblast growth factor receptor 1 (FGFR1) gene and the integrative nuclear FGFR1 signaling (INFS) module in stem cells and in neuronal cells, whose control opens the door to transformative applications, including reconstructive medicine and cancer therapy. Three stages of optogenomic interfaces are described, ranging from already experimentally validated interfaces activating broad cellular responses and expressing individual genes to more advanced interfaces able to regulate and correct DNA topology, chromatin structure, and cellular development.
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 shows that kinetic proofreading regulates the activity of the T cell receptor.
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.
Light-Controlled Afﬁnity Puriﬁcation of Protein Complexes Exempliﬁed by the Resting ZAP70 Interactome.
Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the know interaction partners, and could filter out all other proteins.
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.
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.
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.
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.
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.
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.
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.
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.
Light-Induced Printing of Protein Structures on Membranes in Vitro.
Reconstituting functional modules of biological systems in vitro is an important yet challenging goal of bottom-up synthetic biology, in particular with respect to their precise spatiotemporal regulation. One of the most desirable external control parameters for the engineering of biological systems is visible light, owing to its specificity and ease of defined application in space and time. Here we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on time scales of seconds, and the pattern density can be precisely controlled by protein concentration, laser power, and activation time. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. Thus, light mediated sequential protein assembly in cell-free systems represents a promising approach to hierarchically building up the next level of complexity toward a minimal cell.
Optogenetic Medicine: Synthetic Therapeutic Solutions Precision-Guided by Light.
Gene- and cell-based therapies are well recognized as central pillars of next-generation medicine, but controllability remains a critical issue for clinical applications. In this context, optogenetics is opening up exciting new opportunities for precision-guided medicine by using illumination with light of appropriate intensity and wavelength as a trigger signal to achieve pinpoint spatiotemporal control of cellular activities, such as transgene expression. In this review, we highlight recent advances in optogenetics, focusing on devices for biomedical applications. We introduce the construction and applications of optogenetic-based biomedical tools to treat neurological diseases, diabetes, heart diseases, and cancer, as well as bioelectronic implants that combine light-interfaced electronic devices and optogenetic systems into portable personalized precision bioelectronic medical tools. Optogenetics-based technology promises the capability to achieve traceless, remotely controlled precision dosing of an enormous range of therapeutic outputs. Finally, we discuss the prospects for optogenetic medicine, as well as some emerging challenges.
Light‐Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.
The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology‐inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light‐controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non‐neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light‐sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light‐controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.
Lighting Up Cancer Dynamics.
Live-cell microscopy has revealed that signaling pathways carry elaborate time-varying activities. Yet, the connection between these dynamics and cellular disease has remained elusive. Recent work leverages cellular optogenetics to analyze the Ras-to-Erk transfer function in cancer cells. These analyses reveal how changes to the filtering properties of a pathway lead to the misperception of extracellular events. Overall, these studies suggest that mutations do not simply hyperactivate pathways but rather can also change their transmission properties in more subtle ways.
Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway.
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.
Generic and reversible opto-trapping of biomolecules.
Molecular traps can control activity and abundance of many biological factors. Here, we report the development of a generic opto-trap to reversibly bind and release biomolecules with high spatiotemporal control by illumination with noninvasive and cell-compatible red and far-red light. We use the Arapidopsis thaliana photoreceptor phytochrome B to regulate the release of diverse proteins from a variety of material scaffolds. Fusion of a short 100 amino acids "PIF-tag", derived from the phytochrome interacting factor 6, renders arbitrary molecules opto-trap-compatible. Reversible opto-trapping of target molecules enables novel possibilities for future developments in diagnostics, therapeutics and basic research.
A compendium of chemical and genetic approaches to light-regulated gene transcription.
On-cue regulation of gene transcription is an invaluable tool for the study of biological processes and the development and integration of next-generation therapeutics. Ideal reagents for the precise regulation of gene transcription should be nontoxic to the host system, highly tunable, and provide a high level of spatial and temporal control. Light, when coupled with protein or small molecule-linked photoresponsive elements, presents an attractive means of meeting the demands of an ideal system for regulating gene transcription. In this review, we cover recent developments in the burgeoning field of light-regulated gene transcription, covering both genetically encoded and small-molecule based strategies for optical regulation of transcription during the period 2012 till present.
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.
Four Key Steps Control Glycolytic Flux in Mammalian Cells.
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.
Independent Control over Multiple Cell Types in Space and Time Using Orthogonal Blue and Red Light Switchable Cell Interactions.
Independent control over multiple cell–material interactions with high spatiotemporal resolution is a key for many biomedical applications and understanding cell biology, as different cell types can perform different tasks in a multicellular context. In this study, the binding of two different cell types to materials is orthogonally controlled with blue and red light providing independent regulation in space and time. Cells expressing the photoswitchable protein cryptochrome 2 (CRY2) on cell surface bind to N‐truncated CRY‐interacting basic helix–loop–helix protein 1 (CIBN)‐immobilized substrates under blue light and cells expressing the photoswitchable protein phytochrome B (PhyB ) on cell surface bind to phytochrome interaction factor 6 (PIF6)‐immobilized substrates under red light, respectively. These light‐switchable cell interactions provide orthogonal and noninvasive control using two wavelengths of visible light. Moreover, both cell–material interactions are dynamically switched on under light and reversible in the dark. The specificity of the CRY2/CIBN and PhyB/PIF6 interactions and their response to different wavelengths of light allow selectively activating the binding of one cell type with blue and the other cell type with red light in the presence of the other cell type.