Genetically-encoded Biosensors Research Articles

Genetically-encoded biosensors to monitor intracellular cysteine levels

Genetically-encoded biosensors to monitor intracellular cysteine levels

Smart genetically-encoded biosensors for the chemical monitoring of living systems.

Genetically-encoded biosensors provide the all-optical and non-invasive visualization of dynamic biochemical events within living systems, which has allowed the discovery of profound new insights. Twenty-five years of biosensor development has steadily improved their performance and has provided us with an ever increasing biosensor repertoire. In this feature article, we present recent advances made in biosensor development and provide a perspective on the future direction of the field.

Resource for FRET-Based Biosensor Optimization.

After the development of Cameleon, the first fluorescence resonance energy transfer (FRET)-based calcium indicator, a variety of FRET-based genetically encoded biosensors (GEBs) have visualized numerous target players to monitor their cell physiological dynamics spatiotemporally. Many attempts have been made to optimize GEBs, which require labor-intensive effort, novel approaches, and precedents to develop more sensitive and versatile biosensors. However, researchers face considerable trial and error in upgrading biosensors because examples and methods of improving FRET-based GEBs are not well documented. In this review, we organize various optimization strategies after assembling the existing cases in which the non-fluorescent components of biosensors are upgraded. In addition, promising areas to which optimized biosensors can be applied are briefly discussed. Therefore, this review could serve as a resource for researchers attempting FRET-based GEB optimization.

Absolute measurement of cellular activities using photochromic single-fluorophore biosensors and intermittent quantification

Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca2+ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca2+ concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.

Transcription Factors as Evolvable Biosensors.

One of the most prominent features of genetically encoded biosensors (GEBs) is their evolvability-the ability to invent new sensory functions using mutations. Among the GEBs, the transcription factor-based biosensors (TF-biosensors) is the focus of this review. We also discuss how this class of sensors can be highly evolvable and how we can exploit it. With an established platform for directed evolution, researchers can create, or evolve, new TF-biosensors. Directed evolution experiments have revealed the TF-biosensors' evolvability, which is based partially on their characteristic physicochemical properties.

Anti-cancer potential of persimmon (Diospyros kaki) leaves via the PDGFR-Rac-JNK pathway

Persimmon leaves are known to have some beneficial effects, including ROS elimination, lipid circulation, and neuronal protection. However, their anti-cancer properties and the underlying mechanisms remain unclear. Herein, we show that treatment with the ethanol extract of persimmon, Diospyros kaki, leaves (EEDK) induces cancer cell death and inhibits cell proliferation. Using fluorescence resonance energy transfer (FRET) technology with genetically-encoded biosensors, we first found that EEDK stimulates a PDGFR-Rac signaling cascade in live cells. Moreover, we found that downstream of the PDGFR-Rac pathway, JNKs are activated by EEDK. In contrast, JNK-downstream inhibitors, such as CoCl2, T-5224, and pepstatin A, attenuated EEDK-induced cell death. Thus, we illustrate that the PDGFR-Rac-JNK signaling axis is triggered by EEDK, leading to cancer cell death, suggesting the extract of persimmon leaves may be a promising anti-cancer agent.

Machine learning linked evolutionary biosensor array for highly sensitive and specific molecular identification

Bacteria initiate complicated signaling cascades from the detection of intracellular metabolites or exogenous substances by hundreds of transcription factors, which have been widely investigated as genetically-encoded biosensors for molecular recognition. However, the limited number of transcription factors and their broad substrate specificity result in ambiguity in small molecule identification. This study presents a new small molecule fingerprinting technique using evolutionary biosensor arrays with a machine learning technique that can capture highly specific substrate signals. Employing multiple mutant transcription factors derived from a single transcription factor has effectively circumvented the limited availability of transcription factors induced by a small molecule of our interest. This method achieved up to 95.3% true positive rate for identifying small molecules, and the high-resolution protein engineering technique improved the limit of detection 75-fold. The signal trade-offs with background noises caused by the complex cellular biochemistry of mutant transcription factors enable the biosensor arrays to be more informative in terms of statistical variance. The machine learning technology, coupled with the single transcription factor-driven evolutionary biosensor array, will open new avenues for molecular fingerprinting technologies.

Expanding the Toolkit of Fluorescent Biosensors for Studying Mitogen Activated Protein Kinases in Plants

Mitogen-activated protein kinases (MAPKs) are key regulators of numerous biological processes in plants. To better understand the mechanisms by which these kinases function, high resolution measurement of MAPK activation kinetics in different biological contexts would be beneficial. One method to measure MAPK activation in plants is via fluorescence-based genetically-encoded biosensors, which can provide real-time readouts of the temporal and spatial dynamics of kinase activation in living tissue. Although fluorescent biosensors have been widely used to study MAPK dynamics in animal cells, there is currently only one MAPK biosensor that has been described for use in plants. To facilitate creation of additional plant-specific MAPK fluorescent biosensors, we report the development of two new tools: an in vitro assay for efficiently characterizing MAPK docking domains and a translocation-based kinase biosensor for use in plants. The implementation of these two methods has allowed us to expand the available pool of plant MAPK biosensors, while also providing a means to generate more specific and selective MAPK biosensors in the future. Biosensors developed using these methods have the potential to enhance our understanding of the roles MAPKs play in diverse plant signaling networks affecting growth, development, and stress response.

Combining genetically-encoded biosensors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica

Erythritol is an important sweetener ingredient and chemical precursor for synthesizing materials with phase transition behavior. Commercial erythritol is primarily produced by industrial fermentation. Further strain engineering necessitates the development of high throughput screening method for rapid detection and screening of mutant strain libraries. In this work, we took advantage of the erythritol-responsive transcription factor EryD, and constructed a sensor-regulator system for rapid screening and characterization of erythritol overproducers. We configured the optimal architecture of the EryD sensor-regulator construct with improved sensitivity, specificity and dynamic response range. Coupled with mutagenesis and strain screening based on biosensors, we rapidly screened and characterized a strain library containing 1152 mutants derived from combined UV and ARTP mutagenesis, in a relatively short period of time (1 week). The optimal strain produced more than 148 g/L erythritol in bench-top reactors. This work provides a reference for other metabolic engineering researchers to develop industrially-relevant strains. The reported framework enables us to rapidly improve strain performance and engineer efficient microbial cell factories for industrial applications.

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion.

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.

Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise

Reactive oxygen species (ROS) act as intracellular compartmentalized second messengers, mediating metabolic stress-adaptation. In skeletal muscle fibers, ROS have been suggested to stimulate glucose transporter 4 (GLUT4)-dependent glucose transport during artificially evoked contraction ex vivo, but whether myocellular ROS production is stimulated by in vivo exercise to control metabolism is unclear. Here, we combined exercise in humans and mice with fluorescent dyes, genetically-encoded biosensors, and NADPH oxidase 2 (NOX2) loss-of-function models to demonstrate that NOX2 is the main source of cytosolic ROS during moderate-intensity exercise in skeletal muscle. Furthermore, two NOX2 loss-of-function mouse models lacking either p47phox or Rac1 presented striking phenotypic similarities, including greatly reduced exercise-stimulated glucose uptake and GLUT4 translocation. These findings indicate that NOX2 is a major myocellular ROS source, regulating glucose transport capacity during moderate-intensity exercise.

Molecular and Cellular Immuno-Engineering

Genetically-encoded biosensors based on fluorescence proteins (FPs) and fluorescence resonance energy transfer (FRET) have enabled the specific targeting and visualization of signaling events in live cells with high spatiotem... | Find, read and cite all the research you need on Tech Science Press

Engineering Zap70 Biosensor Through Directed Evolution for Applications in Single-Cell Imaging and Immunotherapy

Genetically-encoded biosensors based on Fluorescence Resonance Energy Transfer (FRET biosensors) have been widely used to dynamically track the activity of Protein Tyrosine Kinases (PTKs) in living cells because of their sens... | Find, read and cite all the research you need on Tech Science Press

Current Progress in Production of Flavonoids using Systems and Synthetic Biology Platforms

Flavonoid is an industrially-important compound due to its high pharmaceutical and cosmeceutical values. However, conventional methods in extracting and synthesizing flavonoids are costly, laborious and not sustainable due to small amount of natural flavonoids, large amounts of chemicals and space used. Biotechnological production of flavonoids represents a viable and sustainable route especially through the use of metabolic engineering strategies in microbial production hosts. In this review, we will highlight recent strategies for the improving the production of flavonoids using synthetic biology approaches in particular the innovative strategies of genetically-encoded biosensors for in vivo metabolite analysis and high-throughput screening methods using fluorescence-activated cell sorting (FACS). Implementation of transcription factor based-biosensor for microbial flavonoid production and integration of systems and synthetic biology approaches for natural product development will also be discussed.

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT) Activity Selectively Targets Human Acute Myeloid Leukemia Stem Cells

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow associated with poor clinical outcomes. Conventional chemotherapies are effective in debulking the leukemic burden in most AML patients. However, a small population of disease-sustaining leukemic stem cells (LSCs) frequently persists and contributes to relapsed disease. Novel therapies that eradicate LSCs have the potential to improve clinical outcomes in AML.To discover novel anti-LSC agents, we performed a high-throughput flow cytometry-based drug screen of 1,220 compounds against a primary AML sample (8227). This sample harbors distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation is restricted to the CD34+CD38- fraction. Through this screen, we identified compounds that selectively depleted the CD34+CD38- fraction including four structurally-unrelated NAMPT inhibitors (FK866, STF-118804, GMX1778, and KPT-9274). These inhibitors also depleted the LSC-enriched CD34+CD38- fraction in two other primary AML samples, indicating that the effect was not unique to 8227 cells. To further evaluate their impact on LSCs in 8227, we measured the expression of 104 genes that were previously found to be differentially expressed between LSC+ and LSC- cell fractions isolated from patient samples. Treatment with NAMPT inhibitors reduced the correlation between the measured LSC gene signature and the LSC+ reference profile, providing additional evidence for their anti-LSC activity.To determine whether the selective loss of CD34+CD38- cells was due to cell death or differentiation, we sorted subsets of 8227 cells based on CD34 and CD38 expression and treated each fraction with FK866. NAMPT inhibition preferentially triggered apoptosis as measured by Annexin V staining in the CD34+CD38- and CD34+CD38+ fractions over the CD34- fraction. We did not observe significant changes in the expression of CD34, CD38, or other myeloid differentiation markers (CD14 and CD15) in the remaining viable cells.Our subsequent mechanistic studies focused on KPT-9274 because it is the furthest along in clinical development. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), a direct NAD+ precursor. To confirm a decrease in intracellular NAD+ with KPT-9274 treatment, we introduced expression of genetically-encoded biosensors for measuring NAD+ levels in different cellular compartments in an AML cell line. KPT-9274 treatment for 15 hours lowered the free NAD+ pool in the cytosol and mitochondria but not in nucleus. To determine whether the drop in NAD+ was necessary for the effects of KPT-9274 on LSCs, we supplemented the primary AML samples with nicotinamide riboside (NR) which can be directly converted to NMN, thereby bypassing the requirement for NAMPT activity to generate NAD+. The addition of NR completely rescued the effects of KPT-9274 on the CD34+CD38- fraction. Niacin can also generate NAD+ through an alternative pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT). However, niacin supplementation failed to rescue the effects of NAMPT inhibition which correlated with the lack of NAPRT expression in LSC-enriched CD34+CD38- cells.Next, we studied the effects of KPT-9274 on normal CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from human cord blood. Although HSPCs were sensitive to the pro-apoptotic effects of KPT-9274, their survival was fully rescued by both NR and niacin. The rescue by niacin correlated with a higher expression of NAPRT in HSPCs. As the blood concentration of niacin is ~1,000 fold higher than that of NR, KPT-9274 is predicted to have a favorable therapeutic window in vivo. To demonstrate its in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with a luciferase-tagged AML cell line (OCI-AML3) with KPT-9274 at a dose of 150 or 250 mg/kg/day or vehicle control for 50 consecutive days by oral administration. KPT-9274 treatment significantly lowered leukemia burden and prolonged survival in both dosing cohorts.In summary, our results indicate that NAMPT inhibition represents an effective approach to target human LSCs through reduction in intracellular NAD+ levels and induction of apoptosis. Our data provide the preclinical rationale for investigating the use of KPT-9274 in AML clinical trials. DisclosuresChan:Genentech: Research Funding; Celgene: Research Funding; AbbVie: Research Funding.

NOX2 is a major ROS source in exercising muscle regulating glucose uptake

Reactive oxygen species (ROS) are increasingly recognized as key regulators of cellular metabolism. In skeletal muscle, ROS has been linked to glucose transport in isolated electrically stimulated muscles but the exact source of ROS in the context of in vivo exercise and whether it controls in vivo glucose uptake by glucose transporter 4 (GLUT4) is unknown. Here, we utilized a combination of genetically-encoded biosensors, fluorescent dyes and mouse models lacking the NADPH oxidase 2 (NOX2) regulatory subunits p47phox and Rac1 to demonstrate that NOX2 is the predominant source of ROS during in vivo treadmill exercise in skeletal muscle. Furthermore, p47phox-deficient mice phenocopy key aspects of metabolic dysregulation in Rac1 mKO mice, including decreased in vivo glucose uptake and GLUT4 translocation. These findings provide strong evidence that NOX2 is a major ROS source during in vivo exercise and its activity is crucial to the translocation of GLUT4 to increase glucose uptake.

Revealing Subcellular Redox Dynamics with Multiplex Imaging of Compartment‐Specific Redox Probes

Reactive oxygen species (ROS), redox enzymes, and redox buffers are central to the propagation of redox signals and oxidative stress between cells in the tissue environment and between organelles in the cellular environment. For example, hydrogen peroxide generated in mitochondria can alter the thiol oxidation states on proteins to alter their function, and subsequently redox active biomolecules such as glutathione can act as reducing equivalents in the face of oxidative stress. In some cases, it has become apparent that ROS mediate physiologically important signaling across compartments, such as in mitonuclear retrograde signaling. However, it has remained technically challenging to quantitatively correlate compartment‐specific redox dynamics with subcellular resolution within live single cells. In order to address this challenge, we are developing genetically‐encoded biosensors that enable the visualization of redox dynamics simultaneously within multiple compartments within the same cell. Our approach utilizes Förster‐type resonance energy transfer in a spectral relay strategy to extend the fluorescence emission of the redox‐sensitive green fluorescent protein (roGFP) into orange and red emission wavelengths. Furthermore, we are developing multiplexed, multiphoton imaging methods to study redox coupling between organelles and compartment‐specific dynamics in thick neural tissues.Support or Funding InformationWe gratefully acknowledge support from the Ralph W. and Grace M. Showalter Trust, the Purdue Research Foundation, and from National Institutes of Health grants R21 NS092010 and R21 EY026425.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcription Factor-Based Biosensors in High-Throughput Screening: Advances and Applications.

The molecular mechanisms that cells use to sense changes in the intra- and extracellular environment are increasingly utilized in synthetic biology to build genetic reporter constructs for various applications. Although in nature sensing can be RNA-mediated, most existing genetically-encoded biosensors are based on transcription factors (TF) and cognate DNA sequences. Here, the recent advances in the integration of TF-based biosensors in metabolic and protein engineering screens whereas distinction is made between production-driven and competitive screening systems for enzyme evolution under physiological conditions are discussed. Furthermore, the advantages and disadvantages of existing TF-based biosensors are examined with respects to dynamic range, sensitivity, and robustness, and compared to other screening approaches. The application examples discussed in this review demonstrate the promising potential TF-based biosensors hold as screening tools in laboratory evolution of proteins and metabolic pathways, alike.

A FRET Biosensor for ROCK Based on a Consensus Substrate Sequence Identified by KISS Technology.

Genetically-encoded biosensors based on Förster/fluorescence resonance energy transfer (FRET) are versatile tools for studying the spatio-temporal regulation of signaling molecules within not only the cells but also tissues. Perhaps the hardest task in the development of a FRET biosensor for protein kinases is to identify the kinase-specific substrate peptide to be used in the FRET biosensor. To solve this problem, we took advantage of kinase-interacting substrate screening (KISS) technology, which deduces a consensus substrate sequence for the protein kinase of interest. Here, we show that a consensus substrate sequence for ROCK identified by KISS yielded a FRET biosensor for ROCK, named Eevee-ROCK, with high sensitivity and specificity. By treating HeLa cells with inhibitors or siRNAs against ROCK, we show that a substantial part of the basal FRET signal of Eevee-ROCK was derived from the activities of ROCK1 and ROCK2. Eevee-ROCK readily detected ROCK activation by epidermal growth factor, lysophosphatidic acid, and serum. When cells stably-expressing Eevee-ROCK were time-lapse imaged for three days, ROCK activity was found to increase after the completion of cytokinesis, concomitant with the spreading of cells. Eevee-ROCK also revealed a gradual increase in ROCK activity during apoptosis. Thus, Eevee-ROCK, which was developed from a substrate sequence predicted by the KISS technology, will pave the way to a better understanding of the function of ROCK in a physiological context.

Intein-mediated protein engineering for biosensor fabrication

Intein-mediated protein engineering has become a valuable tool for biosensing applications. Expressed protein ligation and protein trans-splicing were used to generate proteins with necessary tags or cyclic peptides that can be used to build various biosensing platforms such as biochips or sensor proteins. Especially the use of split-inteins that carry out conditional protein splicing reaction enabled the fabrication of various genetically-encoded biosensors that can monitor various signaling events in vivo including protein- protein interactions and protein translocalization. Biological targets activated split-inteins to generate functioning reporter molecules, such as luciferase and autofluorescent proteins. We here review various sensing platforms that utilize intein-mediated protein engineering technology mainly focusing on cell-based biosensors.