This work was supported by ERC starting grants (ERC-2011-StG 280255 and ERC-2013-StG 337479) and by the Netherlands Organization for Health Research and Development (Vidi 91713319).

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The authors have nothing to disclose.

כאן אנו מציגים פרוטוקול מפורט לשימוש בחיישן של חומצות מרה רומן מקודדים גנטי מסוגל ניטור דינמיקת spatiotemporal תחבורת חומצות מרה בתאים חיים. biosensor זה מורכב מחלבוני ניאון תכלת וסיטרין שהם התמזגו לFXR-LBD, וכך יוצרים חיישן מבוסס סריג חומצות מרה (BAS). <p class="jove_content" style=";text-align:right;di...

העברת פורסטר תהודת אנרגיה (סריג) היא בשימוש נרחב כדי להשיג הבנה טובה יותר של תפקודים תאיים בתאים חיים עם רזולוציה של זמן ומרחב גבוה 1. בסריג, אנרגיה מfluorophore תורם נרגש מועברת לfluorophore acceptor. סריג יעילות הוא תלוי מאוד על המרחק בין התורם fluorophore acceptor והאוריינטציה שלהם, ולכן קריאת נתונים רגישות של שינויי קונפורמציה המשפיעים על שני fluorophores. תופעה זו מנוצלת ליצירת חיישנים מבוססי סריג להדמיה של מולקולות קטנות. שינויים בריכוזם יכולים להיות במעקב עם עלייה / ירידה בשיעור של עוצמת פליטה של acceptor לעומת fluorophore התורם 2. לדוגמא, חיישני סידן מבוססי סריג מאפשרים זיהוי מהיר ויציב של ריכוזי סידן חופשיים בתאים חיים 3. יתרונות אחרים של חיישנים מבוססי סריג הם הדמיה בתאי חיים יחידים, לאיורש אינה הפולשנות, היכולת שלהם להיות ממוקד לסוגי תאים שונים ותאים סלולריים 4. היבטים של דינמיקה של חומצות מרה תאיים רבים עדיין הם הבינו היטב. לדוגמא, מעט מאוד ידוע על בסיס תקנת המנגנון של תחבורת חומצות מרה מצומדות וunconjugated. קיימים טכניקות כדי לפקח על תחבורה זה בעיקר לעשות שימוש בכתבים מבוסס לוציפראז, חומצות מרה רדיואקטיבי, או אנלוגים של חומצות מרה ניאון. האחרון דורש שינוי של חומצות מרה, אולי משפיע על תכונותיהם. יש עיתונאים המבוסס על לוציפראז רזולוציה זמן עני. חוץ מזה, בטכניקות אלה לגרום לאובדן של המדגם ואינם ישימים להדמיה בתאים בודדים. לכן, זה יהיה מועיל להשתמש בשיטות המאפשרות הדמיה תא בודדת חי של פעילות תחבורה באמצעות חיישני סריג, במיוחד משום שהיא כוללת את היתרון של זיהוי 5 ratiometric, 6. בעוד גרסאות של CFP / טופס YFP הנפוץ ביותר בשימוש זוגות סריג, אסטרטגיות חדשות באמצעות Morange וmCherry נושא מוטציות עמותה עצמית-התרמה הובילה להרחבת ארגז הכלים סריג עם חיישני רומן, כוללים חיישן חומצות מרה אדום העביר 7. יצרנו חיישן גנטי מקודד סריג מרה חומצה (BAS), שמורכב מfluorophore תורם (תכלת) וfluorophore acceptor (סיטרין) שהתמזגו עם תחום קולט X farnesoid (FXR) יגנד מחייב בעבר (FXR-LBD) ופפטיד המכיל מוטיב LXXLL 8. מקורבים זה פפטיד עם FXR-LBD באופן חומצה תלויה מרה. עם הפעלת FXR, המרחק בין סיטרין והתכלת ישנה עקב שינוי קונפורמציה. בשורות תאי יונקים, תוצאות FXR הפעלה בעלייה להבחין בבירור בסיטרין / היחס תכלת, בעוד החיישן המטוהר עובד בכיוון ההפוך וגורם ליחס סריג ירד על הפעלת FXR. חיישן זה (CytoBAS)מאפשר ניטור של דינמיקה של חומצות מרה cytosolic. על ידי תוספת carboxyl הטרמינל של מוטיבים מיקוד subcellular, מבנה BAS יכול להיות ממוקד לגרעין (NucleoBAS) וperoxisomes (PeroxiBAS), המאפשר מדידות של ריכוזי חומצות מרה בתאים סלולריים שונים. למרות התוספת של מוטיב מיקוד peroxisomal אינה פוגעת היענותה לחומצות מרה, FXR-ligands החדיר תאים לא לגרום לכל סריג שינויים של PeroxiBAS בתוך peroxisomes 8. כטבעו של פער זה אינו ידוע, הפרוטוקול להלן מתמקד בCytoBAS וNucleoBAS. השימוש בחיישן סריג מקודדים גנטי זה הודגם לאחרונה בתאים המכילים את מובילי כבד חומצות מרה Na פוליפפטיד שיתוף הובלה / taurocholate + (NTCP) ואלפא טרנספורטר המומס אורגני / בטא (OSTαβ) 8. NTCP היא היבואנית של חומצת מרה בכבד הקרן וOSTαβ הוא מרה מעיים basolateralטרנספורטר חומצה שיכול לתפקד גם כיבואן ויצואן תלוי בשיפוע של החומצות מרה אלקטרוכימיים הריכוז 9, 10. נתונים אחרונים הראו כי על תחבורת חומצות מרה על ידי NTCP ו / או OSTαβ, ניתן לצפות תגובות חזקות ומהירה ביחס סריג כתוצאה מאינטראקצית יגנד-FXR-LBD. כאן אנו מתארים פרוטוקולים מפורטים לשיטות למדידת סריג כגון ניתוח confocal מיקרוסקופי ותא מופעל הקרינה מיון (FACS), לסמן את השלבים קריטיים, לטפל בבעיות פוטנציאליות ולדון שיטות חלופיות. שימוש בחיישן סריג מקודדים גנטי זה, אינטראקציה עם חומצות מרה FXR-LBD ניתן לכמת ופיקוח ישיר בתאים חיים ומספקת שיטה מהירה ופשוטה של ​​הדמיה תחבורת חומצות מרה ודינמיקה בזמן אמת. פלסמידים ביטוי יונקים קידוד CytoBAS וNucleoBAS זמינים מסחרי. לכן, biosensor זה יכול עוד לתרום להבנה של מובילי חומצות מרה או תרכובות המפעילות FXR ולספק הבנה עמוקה יותר של ביולוגיה של חומצות מרה ואיתות.

<table><tbody><tr><td>CytoBAS&amp;nbsp;</td><td>Addgene</td><td>62860</td><td></td></tr><tr><td>NucleoBAS&amp;nbsp;</td><td>Addgene</td><td>62861</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagles media (DMEM)</td><td>Lonza</td><td>BE12-614F</td><td>High glucose without L-glutamine</td></tr><tr><td>Penicillin-Streptomycin (pen/strep)</td><td>Lonza</td><td>17-602E</td><td></td></tr><tr><td>L-glutamine (200mM)</td><td>Lonza</td><td>17-605E</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Invitrogen</td><td>102-70</td><td></td></tr><tr><td>Trypsin-EDTA (10x)</td><td>Lonza</td><td>CC-5012</td><td></td></tr><tr><td>T-25 cell culture flask</td><td>VWR international</td><td>392-0253</td><td>Laminin coated</td></tr><tr><td>T-175 cell culture flask</td><td>VWR international</td><td>392-0238</td><td>Laminin coated</td></tr><tr><td>6-well plate</td><td>VWR international</td><td>734-0229</td><td>Poly-L-lysine and Laminin coated</td></tr><tr><td>10 cm dish</td><td>VWR international</td><td>392-0243</td><td>Laminin coated</td></tr><tr><td>Diethylaminoethyl (DEAE) - Dextran</td><td>Sigma-Aldrich</td><td>D9885</td><td></td></tr><tr><td>Polyethylenimine (PEI)&amp;nbsp;</td><td>Brunschwig</td><td>23966-2</td><td></td></tr><tr><td>G418 (geneticin) 50 mg/ml</td><td>Invitrogen</td><td>10131-027</td><td></td></tr><tr><td>Hygromycin B, 50 mg/ml</td><td>Invitrogen</td><td>10687-010</td><td></td></tr><tr><td>Cloning cylinder (6 x 8 mm)</td><td>Bellco</td><td>2090-00608</td><td></td></tr><tr><td>L-15 Leibovitz culture medium</td><td>Invitrogen</td><td>21083-027</td><td>No phenol red</td></tr><tr><td>Polystyrene round bottom tube (5 ml) Facs tube</td><td>Falcon BD</td><td>352008</td><td>No cap, non-sterile</td></tr><tr><td>Falcon 2,063 tubes (5 ml)</td><td>Falcon BD</td><td>352063</td><td>Snap cap, sterile</td></tr><tr><td>Nunc Lab-Tek 8 well coverglass</td><td>Thermo scientific</td><td>155409</td><td>Sterile</td></tr><tr><td>Charcoal-filtered FBS</td><td>Life technologies</td><td>12676011</td><td></td></tr><tr><td>GW4064</td><td>Sigma-Aldrich</td><td>G5172</td><td></td></tr><tr><td>TCDCA</td><td>Sigma-Aldrich</td><td>T6260</td><td></td></tr><tr><td>CDCA</td><td>Sigma-Aldrich</td><td>C9377</td><td></td></tr><tr><td>Other chemicals</td><td>Sigma-Aldrich</td><td>n.v.t.</td><td></td></tr></tbody></table>

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.

חיישן סריג-BAS מוצג מבוסס על יגנד המחייב תחום של FXR (LBD-FXR) מחובר לשני fluorophores סיטרין ותכלת) ומוטיב LXXLL. חיישן זה מאפשר חקירות תחבורת חומצות מרה בתאים חיים עם רזולוציה מרחב ובזמן גבוהה (איור 1). מוטציות בתכלת וסיטרין יושמו כדי לקדם את הקמת...

Watch this Scientific Journal Video about Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor at JoVE.com

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

ניטור בזמן אמת של תאיים חומצות מרה Dynamics שימוש חומצות מרה חיישן מבוסס סריג גנטי מקודד

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. ...

real-time-monitoring-intracellular-bile-acid-dynamics-using

Research

JoVE Journal

Bioengineering

9.8K Views.  Academic Medical Center. 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. 

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

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a F&ouml;rster resonance energy transfer (FRET) probe in which the &epsilon; subunit of the FoF1-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the &epsilon; subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.

We describe the use of two ratiometric, genetically encoded biosensors, which are based on GFP, to monitor mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells.

Biosensors רציומטרי שלמדוד את מצב חיזור מיטוכונדריאלי וה-ATP בתאי שמרי חיים

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell ...

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.

FIBS מאופשר פולשנית מטבולית פרופיל

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 ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

גלוטמין שטף הדמיה באמצעות חיישני מקודדים גנטיים

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

ניטור שינויים דינמיים רמות הסידן מיטוכונדריאלי במהלך אפופטוזיס באמצעות חיישן סידן מקודד גנטית

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Biology

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

מלח מרה-induced ביופילמים בתוך המעית פתוגנים: טכניקות וכימות

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Immunology and Infection

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

שימוש רב-פלואור מרת חומצות<em&gt; In vivo</em&gt; דימות תהודה מגנטית כדי למדוד חומצות מרה התחבורה

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Medicine

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.

סריג מיקרוסקופי לניטור בזמן אמת של אירועי איתות בתאים חיים באמצעות Biosensors Unimolecular

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

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.

בזמן אמת In Vivo הקלטה של תודרנית סידן אותות שהאכילה חרקים באמצעות ביוסנסור פלורסנט

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

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

ניטור בזמן אמת של אורורה קינאז הפעלה באמצעות Biosensors FRET קונפורמציה בתאים חיים

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

שדה רחב, הדמיה בזמן אמת של אותות פצע מקומיים ומערכתיים ב Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...