Name: visualizing cellular gibberellin levels using the nlsgps1 förster resonance energy transfer (fret) biosensor
Uploaded: 2019-01-12T14:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Visualizing Cellular Gibberellin Levels Using the nlsGPS1 FÃ¶rster Resonance Energy Transfer FRET Biosensor at JoVE.com

This work has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (grant agreement n&#176; 759282).

<ol>
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C., Li, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GUS+reporter-aided+analysis+of+the+promoter+activities+of+Arabidopsis+ACC+synthase+genes+AtACS4,+AtACS5,+and+AtACS7+induced+by+hormones+and+stresses.">The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses.</a> Journal of Experimental Botany. 56 (413), 909-920 (2005).</li><li>Alabadi, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibberellins+Repress+Photomorphogenesis+in+Darkness.">Gibberellins Repress Photomorphogenesis in Darkness.</a> Plant Physiology. 134 (3), 1050-1057 (2004).</li><li>De Lucas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+framework+for+light+and+gibberellin+control+of+cell+elongation.">A molecular framework for light and gibberellin control of cell elongation.</a> Nature. 451 (7177), 480-484 (2008).</li><li>Strasser, B., Sanchez-Lamas, M., Yanovsky, M. 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J., Gilroy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+Ca2+,+redox+status,+ROS+and+pH+using+GFP+biosensors.">High-resolution imaging of Ca2+, redox status, ROS and pH using GFP biosensors.</a> Plant Journal. 70 (1), 118-128 (2012).</li><li>Lanquar, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+cytosolic+zinc+in+Arabidopsis+roots+combining+FRET+sensors+and+RootChip+technology.">Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology.</a> New Phytologist. 202 (1), 198-208 (2014).</li><li>Krebs, M., Schumacher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+of+cytoplasmic+and+nuclear+Ca2++dynamics+in+Arabidopsis+roots.">Live cell imaging of cytoplasmic and nuclear Ca2+ dynamics in Arabidopsis roots.</a> Cold Spring Harbor. 2013 (8), 776-780 (2013).</li></ol>

The authors have nothing to disclose.

The FRET-based GA biosensor nlsGPS1 provides a quantitative method to report and measure GA hormone gradients in multicellular plants. FRET-based biosensors can quantify dynamics with an improved spatiotemporal resolution over direct detection by mass spectrometry and indirect measurement by transcriptional reporters or signaling-protein-degradation-based methods12,13. High-resolution cellular imaging in diverse tissue-types can yield meaningful insights into GA biology and spark new hypotheses regarding the regulation and function of GA accumulations in a multicellular context. For example, monitoring changes in the nlsGPS1 biosensor in specific GA biosynthetic, catabolic, and transport mutants, as well as during spatiotemporally induced perturbations, could be very informative to test specifically how GA gradients are established in the root and address root cell responses to GA gradients. The sensor could be used in other model and crop species to test the conservation of the mechanisms that control the GA-mediated control of seed germination, cellular elongation, and flowering.
The critical steps in the FRET-based imaging of the nlsGPS1 biosensor are that, 1) the pixels should not be saturated during the quantitative FRET analysis, 2) imaging parameters such as &#34;detector gain&#34; should be kept constant for the donor emission (DxDm) and acceptor emission (DxAm) acquisitions, 3) control nlsGPS1-NR lines should be used to rule out artefacts, and 4) samples should be prepared to minimize drift and focal-change issues. Additionally, the environmental conditions in which samples are grown are important to control since GA levels are sensitive to environmental conditions such as light duration and light intensity14,15,16,17. A key limitation of this type of analysis is that a high signal-to-noise ratio is required for imaging due to the increase in noise inherent in ratiometric imaging. Thus, nlsGPS1 imaging will not be useful for tissues and organs that are not amenable to ratiometric fluorescence microscopy using cyan and yellow fluorescent proteins&#8212;for example, deeper tissues where fluorescent proteins are poorly detected. On the other hand, ratiometric readouts are often preferred over intensiometric readouts, because an internal control is helpful to rule out artefacts stemming from changes in biosensor expression, stability, brightness, or detectability in a given cell, tissue, or condition. For example, FRET biosensor imaging and image analyses have also been used to study a variety of ligands in a variety of tissues5,6,18,19,20,. The imaging experiments and image analyses reported here can be modified to suit new imaging methods, such as light sheet microscopy, that could yield novel insights in, for example, deeper root tissue-types.
The first-generation nlsGPS1 biosensor is a high-affinity sensor that provides a high-resolution map of GA gradients that can also report on intracellular increases in GA following exogenous GA treatments. One of the current limitations of nlsGPS1 is that the sensor is not rapidly reversible and, thus, reports not on steady-state GA levels but, likely, on the maximum recent GA concentration in the solution of interest. The precise turnover rate for the sensor is also not known and this, combined with low reversibility, precludes detection of endogenous GA depletions that might be happening within a minutes to few a hours in some tissue-types. It is also important to note that nlsGPS1 has a high affinity for GA4 (Kd = 24 nM) compared to other GA forms (GA3&#160;Kd= 240 nM, GA1&#160;Kd = 110 nM) when imaging other bioactive GAs7. Future generations of GA biosensors can be engineered to increase reversibility while maintaining high affinity or to exhibit different specificities for the various precursor, bioactive, or catabolite GAs.

Plant hormones play a fundamental role in plant growth and development. These small, mobile signaling molecules are typically regulated at several levels, such as biosynthesis, catabolism, and short- and long-distance transport1,2,3,4. The understanding of hormone signaling pathways and downstream transcriptional responses has sharpened over the years. However, to link the diverse cellular responses of the hormone signaling pathways with the regulatory inputs directing hormone distributions, we require a spatiotemporal quantification of hormone levels at a cellular scale. FRET-based biosensors that can detect phytohormones can advance scientists&#39; ability to quantify hormone levels at a cellular scale. FRET-based biosensors consist of a FRET pair (donor and acceptor fluorescent proteins) linked to a sensory domain that binds a specific ligand or responds to a biological stimulus. For small molecule biosensors, ligand binding triggers a conformational change of the sensory domain that results in a change of distance and/or orientation between the two fluorescent proteins of the FRET pair. A ratiometric analysis of a FRET biosensor is accomplished by exciting the donor and measuring the fluorescence emission ratio of acceptor over donor5,6. Ligand binding is detectable as a change in this emission ratio7.
We recently developed a FRET-based biosensor for the plant hormone GA. GAs are a class of hormones that can promote seed germination, cellular elongation, and the developmental transition from vegetative to flowering phases. The nlsGPS1 biosensor is nuclear localized and provides spatiotemporal insights into GA dynamics in diverse plant tissues. In Arabidopsis cells, GA binds to soluble receptors, gibberellin-insensitive dwarf (GID), and the complex induces the degradation of DELLA proteins that act as negative regulators of GA signalling2. The GA sensory domain of nlsGPS1 consists of the Arabidopsis GA receptor (AtGID1C) linked to a 74-amino acid truncation of a DELLA protein (AtGAI) and a FRET pair consisting of enhanced dimerization variants of Cerulean as the donor fluorescent protein and Aphrodite (a codon-diversified Venus) as the acceptor fluorescent protein8. The nlsGPS1 biosensor is a high-affinity sensor for the bioactive GA4 (Kd = 24 nM for GA4) and it can be utilized in diverse tissue-types to map and quantify GA gradients. To avoid misinterpretation of the Arabidopsis GA levels in vivo, we have also developed a nonresponsive variant of nlsGPS1 (nlsGPS1-NR) to use as a negative control. The nlsGPS1-NR protein carries mutations in the GA-binding pocket that disrupt the binding of GA and mutations in the DELLA protein that disrupt the interaction with GID receptor proteins7,9. Emission ratio patterns or changes observed in both nlsGPS1 and nlsGPS1-NR lines can be considered artefacts not directly related to GA-binding events. It is also important to note that nlsGPS1 binding to GA4 is not rapidly reversible, and therefore, cellular nlsGPS1 emission ratios should be interpreted as representing the highest recent concentration of GA in a given nucleus rather than the real-time steady-state levels. As a consequence, an analysis of falling GA levels is not possible with nlsGPS1.
Here we provide a detailed protocol for utilizing a nlsGPS1 biosensor in cells of the model plant Arabidopsis, using confocal imaging-based approaches at a high-resolution. The protocol provides information on imaging plant roots and hypocotyls both at steady state and over time-courses. The nlsGPS1 sensor could potentially be utilized in diverse tissue-types, as well as across plant species, to map and quantify GA distributions.

<table><tbody><tr><td>nlsGPS1 Col0 Arabidopsis seeds</td><td>NASC</td><td>N2107734</td><td></td></tr><tr><td>nlsGPS-NR Col0 Arabidopsis seeds</td><td>NASC</td><td>N2107735</td><td></td></tr><tr><td>Gibberellin A&lt;sub&gt;4&lt;/sub&gt; (GA&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>G7276</td><td>dissolve in EtH 70% , and keep at -20 &amp;deg;C</td></tr><tr><td>sodium hypochlorite solution (Bleach)</td><td>Fisher S/5040</td><td>HSRA 064</td><td></td></tr><tr><td>Hydrogen cloride HCl</td><td>Sigma</td><td>31434</td><td></td></tr><tr><td>Micropore tape</td><td>3M</td><td>1530-1</td><td></td></tr><tr><td>ibidi sticky-slide</td><td>Ibidi</td><td>81128</td><td>Luer 0.1 for root imaging</td></tr><tr><td>ibidi sticky-slide</td><td>Ibidi</td><td>80168</td><td>Luer 0.2 for hypocotyl imaging</td></tr><tr><td>glass coverslip for sticky slides</td><td>Ibidi</td><td>10812</td><td></td></tr><tr><td>Elbow Luer Connectors</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>silicone tubing</td><td>Ibidi</td><td>108401</td><td></td></tr><tr><td>Luer Lock Connector</td><td>Ibidi</td><td>10826</td><td></td></tr><tr><td>programmable syringe pump</td><td>World Precision Instruments</td><td>AL-1000</td><td></td></tr><tr><td>Vacuum grease</td><td>Sigma</td><td>18405</td><td></td></tr><tr><td>Murashige and Skoog Basal Salts</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Agar plant, 1 kg</td><td>Melford</td><td>P1001</td><td></td></tr><tr><td>Microscope slide ground edges, 76 mm x 26 mm, 1.0 mm to 1.2 mm thick</td><td>Fisher Scientific</td><td>12383118</td><td></td></tr><tr><td>Cover slip No.1 1/2 glass 22 mm x 22 mm</td><td>Fisher Scientific</td><td>12363138</td><td></td></tr><tr><td>Luer-slip Syringe 20 mL</td><td>Fisher Scientific</td><td>10785126</td><td></td></tr><tr><td>3M Micropor Surgical Paper Tape</td><td>Fisher Scientific</td><td>12787597</td><td></td></tr><tr><td>Potassium Hydroxide, 500 g</td><td>Sigma Aldrich</td><td>221473-500G-D</td><td></td></tr><tr><td>Absolute Ethanol</td><td>Fisher Scientific</td><td>10428671</td><td></td></tr><tr><td>Forceps Watchmaker 5 StSteel</td><td>Scientific Laboratory Supplies</td><td>INS4340</td><td></td></tr><tr><td>Scissors, 125 mm, stainless steel</td><td>Fisher Scientific</td><td>12338099</td><td></td></tr><tr><td>Fitting reducer 0.5 to 1.6</td><td>Ibidi</td><td>10829</td><td></td></tr><tr><td>Leica SP8</td><td></td><td></td><td>Confocal laser microscope 1</td></tr><tr><td>Zeiss LSM 780</td><td></td><td></td><td>Confocal laser microscope 2</td></tr><tr><td>Imaris</td><td>Bitplane</td><td></td><td>3D visualization and Analysis software</td></tr><tr><td>Fiji</td><td></td><td></td><td>image analysis software</td></tr><tr><td>OriginPro</td><td>Origin Lab</td><td></td><td>Statistical Analysis Software</td></tr></tbody></table>

visualizing cellular gibberellin levels using the nlsgps1 f&#246;rster resonance energy transfer (fret) biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.

Using nlsGPS1, it is possible to measure cellular GA4 levels in tissues amenable to fluorescence imaging, including root tips and dark-grown hypocotyls (Figure 2). In the Arabidopsis root, the nlsGPS1 emission ratio gradient is indicative of low GA levels in the meristematic and division zones and high GA levels in the late elongation zone (Figure&#160;2A and 2B). In contrast, an emission ratio gradient was not observed in nlsGPS1-NR roots, suggesting that the endogenous GA gradient is not an artefact (Figure&#160;2C and 2D).A nlsGPS1 emission ratio gradient was also formed in dark-grown hypocotyls, with low levels in the cotyledons and the apical hook and high levels in the rapidly elongating basal region of the hypocotyl (Figure&#160;2E and 2F). In contrast, an emission ratio gradient was not observed in the nlsGPS1-NR hypocotyls (Figure&#160;2G and 2H). In both Arabidopsis roots and dark-grown hypocotyl cells, endogenous GA accumulation correlated with cellular elongation rate.
Furthermore, exogenously supplied GA4 accumulates preferentially in the elongation zone compared to the division zone of the Arabidopsis root (Figure 3), indicating that nlsGPS1 can be used to study endogenous and exogenous GA patterning.
During time course experiments, nlsGPS1 seedlings were placed in sticky-slide chambers and perfused with &#188; MS liquid, followed by a treatment with 0.1 &#181;M GA4 for 30 min. The video shows a faster accumulation of exogenous GA4 in the root elongation zone compared to the division zone (Video 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58739/58739fig1.jpg" /> 
Figure 1: Sample preparation for confocal imaging. These panels show a schematic representation of the sample preparation for (A) a steady-state experiment, (B) before-and-after exogenous GA4 treatments, and for (C) a treatment time course experiment using sticky-slides (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/58739fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58739/58739fig2.jpg" /> 
Figure 2: The GA gradient in Arabidopsis roots and dark-grown hypocotyls. Two-dimensional images of (A) nlsGPS1 and (C) nlsGPS1-NR roots were analyzed using ImageJ software, and three-dimensional images of (B) nlsGPS1 and (D) nlsGPS1-NR were analyzed using a commercial three-dimensional image analysis software. Both analyses showed an endogenous GA4 gradient in Arabidopsis roots. Two-dimensional images of (E) nlsGPS1 and (G) nlsGPS1-NR dark-grown hypocotyl were analyzed using ImageJ software, and three-dimensional images of (F) nlsGPS1 and (H) nlsGPS1-NR were analyzed using the commercial three-dimensional image analysis software. Both analyses showed an endogenous GA4 gradient in dark-grown hypocotyls. The LUT bar displays the false coloration of nlsGPS1 emission ratios. YFP images are reported as expression controls. Hypocotyl images were acquired using two stage positions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/58739fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58739/58739fig3.jpg" /> 
Figure 3: The exogenous GA gradient in roots. The first two panels show (A) two-dimensional and (B) three-dimensional images of a nlsGPS1 root before and 20 min after the treatment of exogenous GA4 (1 &#181;M). YFP images are reported as expression controls. The last two panels show (C) the mean and standard deviation and (D) beeswarm and box plot of nlsGPS1 emission ratios for nuclei of the elongation zone (the region which is defined with a white frame). In the elongation zone, the nlsGPS1 emission ratio was significantly higher after GA4 treatment (Mann-Whitney U test, *** P-value &#60; 0.0001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/58739fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/58739/58739movie1.jpg" /> 
Video 1: Perfusion experiment of nlsGPS1 root using sticky-slide. This video shows three-dimensional images of nlsGPS1 perfused with &#188; MS liquid and treated with 0.1 &#181;M GA4 for 30 min. In the time course, imaging was acquired every 10 min for 3 h with the following intervals: 30 min of mock solution (frame t = 1, t = 2, t = 3), 30 min of GA4 treatment (frame t = 4, t = 5, t = 6), 2 h of mock solution (frame t = 7 to t = 18) solution. Prior to the acquisition, the sample was perfused with mock solution for 2 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58739/Movie1.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Visualizing Cellular Gibberellin Levels Using the nlsGPS1 FÃ¶rster Resonance Energy Transfer FRET Biosensor at JoVE.com

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 F&#246;rster Resonance Energy Transfer (FRET) Biosensor

Gibberellin Perception Sensor 1 (GPS1) is the first F&#246;rster resonance energy transfer-based biosensor for measuring the cellular levels of gibberellin phytohormones with a high spatiotemporal resolution. This protocol reports on the method to visualize and quantify cellular gibberellin levels using the genetically encoded nlsGPS1 biosensor in Arabidopsis hypocotyls and root tips.

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental ...

visualizing-cellular-gibberellin-levels-using-nlsgps1-frster

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.6K Views.  University of Cambridge. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van plantenontwikkeling met betrekking tot de verdeling van de groei regulerende fytohormonen. Door gebruik te maken van genetisch gecodeerde FRET biosensoren, bijvoorbeeld nlsGPS1 die gibberellins detecteert, is het mogelijk om de dynamische verdeling van belangrijke verbindingen in Arabidopsis weefsels te meten op de cellulaire oplossing. Over het algemeen zullen individuen die nieuw zijn in deze zaak het moei...

Video: Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Bioengineering

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor

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