Wir sind sehr dankbar, dass Victor GJ Rodgers für wertvolle Ratschläge. Wir danken allen Mitgliedern der Liao-Gruppe für sehr enge gemeinsame Arbeit und für die Hilfe bei dieser Studie. Diese Studie wurde von den National Institutes of Health (Grant AI076504 JL) unterstützt.

Ein. Plasmidkonstrukte <ol><li> Amplifizieren die offenen Leseraster der Gene mittels PCR und Klonierung der PCR-Produkte in PCRII-TOPO-Vektor. </li><li> Bestätigen der Produkte durch Sequenzierung und Klonen der cDNA CyPet-(pre-SUMO1)-YPet, CyPet-SUMO1, YPet und katalytische Domäne in das SENP1 pET28 (b) Vektor mit einer N-terminalen Hexahistidin-Tag. </li></ol> 2. Protein Expression and Purification <ol><li> Transformieren Escherichia coli-Zellen des Stammes BL21 (DE3) mit pET28 (b) Vektoren, codierend CyPet-(pre-SUMO1)-YPet, CyPet-SUMO1, YPet und die katalytischen Domänen von SENP1. </li><li> Angewachsen die transformierten Bakterien in 2xYT Medium für 3 Stunden bei 37 ° C (unter Schütteln bei 250 rpm), um eine optische Dichte von 0,5 bei 600 nm erreicht. </li><li> Fügen Sie 100 uM Isopropyl-β-D-(IPTG) (Endkonzentration), um die Proteinexpression zu induzieren und schütteln für 16 Stunden bei 25 ° C bei 200 Umdrehungen pro Minute. </li><li> Ernten die Bakterien durch centrifugation bei 10.000 rpm bei 4 ° C für 5 min und resuspendiert ihnen in einem Puffer aus 20 mM Tris-HCl (pH 7,4), 50 mM NaCl und 5 mM Imidazol. </li><li> Beschallen die Zellen für 10 Minuten in 5-sec Intervallen bei einer Leistungseinstellung von 25 W mit MiSonics 4000 Sonicator und sammeln sie durch Zentrifugation bei 35.000 × g bei 4 ° C für 30 min. </li><li> Einrichten der Säule mit 500 ml Ni-NTA-Kügelchen für 1 l-Kultur und Transfer des Überstandes in die Säule. </li><li> Waschen des Harzes mit einem Puffer von 20 mM Tris-HCl (pH 7,4), 500 mM NaCl und 10 mM Imidazol zweimal. </li><li> Eluieren Protein mit Puffer von 20 mM Tris-HCl (pH 7,4), 500 mM NaCl und 500 mM Imidazol und Dialyse in einen Puffer von 20 mM Tris-HCl (pH 7,4), 50 mM NaCl und 1 mM Dithiothreitol (DTT) bei 4 ° C über Nacht. </li><li> Die Konzentrationen der gereinigten Proteine ​​durch das Bradford-Assay. Alternatives Verfahren zur Protein-Konzentration Messungen SDS-PAGE-Gelelektrophorese werden und dann mit Coomassie-Blau gefärbt, gefolgt mit bildgebendenquantitativ. </li></ol> 3. Quantitative FRET Spectrum Analysis <ol><li> Die allgemeine Strategie für den Test wurde am FRET Signalisierung (Abbildung 1). Die FRET-Paar wurde CyPet und YPet, an den N-und C-Termini tagged jeweils von Pre-SUMO1. SENP1 spaltet das Fusionsprotein CyPet-(pre-SUMO1)-YPet am Gly-Gly Seite auf SUMO1 der C-Terminus und somit löst die SUMO Schwanz mit YPet. Die FRET-Signal gestört wird, was zu einem Anstieg der Emission aus CyPet und eine dramatische Abnahme der YPet die Emission bei der CyPet Anregungswellenlänge. </li><li> Der absolute FRET-induzierte YPet die Emission CyPet direkte Emission und YPet direkte Emission (Abbildung: wenn es durch Licht mit einer Wellenlänge von 414 nm, die gesamte Fluoreszenzemission CyPet-(pre-SUMO1)-YPet bei 530 nm angeregt werden aus drei Quellen abgeleitet werden 2). </li></ol><img alt="Gleichung 1" src="/files/ftp_upload/4430/4430eq1.jpg" /> wo FL 530/414 &lt;/ Sub&gt; ist die gesamte Fluoreszenz-Emission bei 530 nm, wenn sie bei 414 nm angeregt, FRET FL ist die absolute FRET-Signals, FL ist CyPet (cont) die direkte Emission CyPet wenn sie bei 414 nm angeregt, und FL YPet (cont) ist die YPet direkte Emission, wenn bei 414 nm angeregt. Der Index der (cont) steht für den Beitrag. <ol start="3"><li> Die direkte Emission von CyPet bei 530 nm war proportional zu seiner Emission bei 475 nm, wenn sie bei 414 nm mit einem konstanten Verhältnis von α angeregt. CyPet-SUMO1 wurde bei Konzentrationen von 50, 100, 200, 500 und 750 nM hergestellt und 1 mM und die Emissionen bei 475 und 530 nm wurden nach Anregung bei 414 nm zur α (Abbildung 3-1) zu bestimmen. </li><li> Die direkte Emission von YPet bei 530 nm bei Anregung bei 414 nm war proportional zu seiner Emission bei 530 nm, wenn bei 475 nm mit einem konstanten Verhältnis von β angeregt. YPet bei Konzentrationen von 50, 100, 200, 500, 750 A hergestelltnd 1000 nm, und die Emission bei 530 nm wurde gemessen, wenn die Proben durch Wellenlängen von 414 und 475 nm angeregt wurden β (Abb. 3-2) zu bestimmen. </li><li> Wenn CyPet-(pre-SUMO1)-YPet durch SENP1 verdaut wurde, die Spaltung CyPet-SUMO1 und die SUMO1 Schwanz mit YPet veröffentlicht. Wenn die Verbindung bei 414 nm angeregt wurde, wurde die Fluoreszenz-Emission bei 530 nm (FL &#39;530/414) verringert, aber trotzdem in drei Teile unterteilt werden: </li></ol><img alt="Gleichung 2" src="/files/ftp_upload/4430/4430eq2.jpg" /> wo FL &#39;530/414 die gesamte Fluoreszenzemission bei 530 nm ist nach der Verdauung, wenn bei 414 nm angeregt, FL&#39; FRET ist die verbleibende absolute FRET-Signals ist FL &#39;CyPet (475/414) die CyPet Emission bei 475 nm nach der Verdauung, wenn Anregung bei 414 nm (hier die CyPet Emission ist aus zwei Teilen: unverdaute CyPet-(pre-SUMO1)-YPet und verdauten CyPet-SUMO1) und FL YPet(530/475) ist die Emission YPet wenn bei 475 nm angeregt, die konstant ob CyPet-(pre-SUMO1)-YPet verdaut wird oder nicht. <ol start="6"><li> Nach Verdauung durch SENP1, FRET die verbleibende Emission (FL &#39;FRET) ist: </li></ol><img alt="Gleichung 3" src="/files/ftp_upload/4430/4430eq3.jpg" /> wobei C die Gesamtkonzentration der CyPet-(pre-SUMO1)-YPet und x die Konzentration der verdauten CyPet-(pre-SUMO1)-YPet. <ol start="7"><li> Durch die Kombination aller Elemente ist die nachgewiesene Fluoreszenz-Emission bei 530 nm unter Anregung von 414 nm (FL &#39;530/414): </li></ol><img alt="Gleichung 4" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4. FRET-basierten Protease-Assay für Enzyme Kinetische Studie <ol><li> CyPet-(pre-SUMO1)-YPet wurde mit der katalytischen Domäne von SENP1 bei 37 ° C in einem Puffer von 20 mM Tris-HCl (pH 7,4), 50 mM NaCl, 0,1% inkubiert (v /v) Tween-20 und 1 mM DTT bis zu einem Gesamtvolumen von 80 ml und übertragen in 384-Well-Platte. </li><li> Durchläufe wurden durch Messung der Fluoreszenz-Emission bei 475 und 530 nm nach einer Anregung bei 414 nm in einem Multiwell-Plattenleser Fluoreszenz für 5 min mit 15-sec Intervallen durchgeführt. </li><li> Die Reaktionsgeschwindigkeit (v) wurde mit der Änderung in der Menge an Substrat (S) als korreliert: </li></ol><img alt="Gleichung 5" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li> Die Konzentration des Produkts exponentiell von 0 als [S] 0 (original Substratkonzentration) bei t = 0: </li></ol><img alt="Gleichung 6" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> Wenn t = 0, ist die ursprüngliche Geschwindigkeit (V 0): </li></ol><img alt="Gleichung 7" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li> Die concentration des SENP1 wurde fixiert, und die Konzentration des CyPet-(pre-SUMO1)-YPet wurde aus 100-mal mehr als die Konzentration des Enzyms, das durch Michaelis-Menten-Gleichung erforderlich ist erhöht. </li><li> All der fluoreszierenden Lesungen wurden von der quantitative FRET-Analyse-Methode analysiert und dargestellt in GraphPad Prism V Software, um die Michaelis-Menten-Gleichung passen. Die nichtlineare Regression kann auch mit Hilfe von mehr gemeinsamen Software-Pakete wie Tabellenkalkulations-Programmen (z. B. Microsoft Excel) durchgeführt werden. </li></ol> 5. Repräsentative Ergebnisse Reifung der Pre-SUMO1 durch SENP1 kann durch Überwachen der Änderungen des Fluoreszenzsignals bei 475 und 530 nm während des Verfahrens bestimmt werden. Das Ergebnis zeigte, dass die Geschwindigkeit des Vor-SUMO1 Verdauung durch SENP1 in einem Substrat-dosisabhängigen Weise (Abbildung 4). Dies legt nahe, dass die katalytische Domäne von SENP1 zeigt ausgezeichnete Aktivität zur pre-SUMO1 der Reifung. Die anfängliche Reaktiontion Geschwindigkeiten wurden durch die obige Analyse mit verschiedenen Substratkonzentrationen (Tabelle 1) berechnet. Der k cat / K M-Verhältnis wird im Allgemeinen verwendet, um die Effizienz verschiedener Enzyme mit einem Substrat oder einem bestimmten Enzyms mit verschiedenen Substraten zu vergleichen. K M und V max aus der Michaelis-Menten-Gleichung durch Auftragen der verschiedenen Anfangsgeschwindigkeiten, entsprechend erhalten werden zu den verschiedenen Konzentrationen von CyPet-(pre-SUMO1)-YPet (Abbildung 5) k cat wurde als erhalten.: <img alt="Gleichung 8" src="/files/ftp_upload/4430/4430eq8.jpg" /> Gemäß der obigen Analyse betrug die berechnete K M 0,21 ± 0,04 pM, war der k cat 6,90 ± 0,28 s -1, und die k cat / K M betrug (3,2 ± 0,55) x 10 7 M -1 </sup<html> &gt; S -1. <img alt="Abbildung 1" src="/files/ftp_upload/4430/4430fig1.jpg" /> Abbildung 1. Graph der FRET-basierten Protease Assay für SENP die pre-sumos Reifung. <img alt="Abbildung 2" src="/files/ftp_upload/4430/4430fig2.jpg" /> Abbildung 2. Quantitative Analyse von Fluoreszenz-Signal in Form von Beiträgen durch den Donor, Akzeptor und FRET. Dissektion der Emissionsspektren CyPet-(pre-SUMO1)-YPet unter Anregung bei 414 nm. FL CyPet (530/414) wird CyPet die Emission bei 530 nm unter Anregung von 414 nm, FRET FL ist die FRET-induzierte YPet Emission bei 530 nm unter Anregung von 414 nm und FL YPet (530/414) YPet die Emission bei 530 nm unter Anregung von 414 nm. load/4430/4430fig3.jpg &quot;/&gt; Abbildung 3. Berechnung der Richtung Emissionsfaktor α und β. <img alt="Abbildung 4" src="/files/ftp_upload/4430/4430fig4.jpg" /> Abbildung 4. Quantitative Analyse von CyPet-(pre-SUMO1)-YPet durch unterschiedliche Übersetzungsverhältnisse der katalytischen Domäne von SENP1 verdaut. Reaktionen wurden innerhalb der ersten 5 min verfolgt. <img alt="Abbildung 5" src="/files/ftp_upload/4430/4430fig5.jpg" /> Abbildung 5. Michaelis-Menten grafische Analyse der CyPet-(pre-SUMO1)-YPet die Verdauung durch SENP1. Daten wurden aufgetragen und mittels GraphPad Prism V und nichtlineare Regression. <table border="1"><tbody><tr><td> [S] (uM) </td><td> V 0 (uM / s) </td></tr><tr><td> 0,115 </td><td> 0,0023 ± 0,00005 </td></tr&gt; <tr><td> 0,214 </td><td> 0,0028 ± 0,00004 </td></tr><tr><td> 0,407 </td><td> 0,0033 ± 0,00007 </td></tr><tr><td> 0,594 </td><td> 0,0037 ± 0,00013 </td></tr><tr><td> 0,725 </td><td> 0,0043 ± 0,00012 </td></tr><tr><td> 1,471 </td><td> 0,0051 ± 0,00036 </td></tr><tr><td> 1,899 </td><td> 0,0050 ± 0,00031 </td></tr><tr><td> 2,300 </td><td> 0,0050 ± 0,00062 </td></tr></tbody></table> Tabelle 1. Anfangsgeschwindigkeiten Bestimmung der pre-SUMO1 die Reifung durch SENP1. In jeder Substratkonzentration, wurden vier Proben verwendet werden, um die Verdauung zu messen. Die Standardabweichung kam aus den Variationen dieser vier Proben. <table border="1"><tbody><tr><td> K M (uM) </td><td> k cat (s-1) </td><td> k cat / k M (M -1 • s-1) </td></tr><tr><td> 0,21 ± 0,04 </td><td> 6,90 ± 0,28 </td><td> 3,2 ± 0,55 × 10 7 </td></tr></tbody></table> Tabelle 2. Kinetische Parameter der Pre-SUMO1 die Reifung durch SENP1 durch quantitative FRET-Analysen. Die Standardabweichung kam aus den vier Proben in jeder Substratkonzentration.

<ol>
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FRET-Technologie verwendet wurde, um die Pre-SUMO1 SENP1 Reifung von 9 studieren. GFP-YFP wurde als FRET-Paar und ratiometrische Analyse, die das Verhältnis von Akzeptor zu Donor Emissionen verwendet, wurde verwendet, um die kinetischen Eigenschaften charakterisieren. Es besteht jedoch keine Berücksichtigung von Donor und Akzeptor Eigenfluoreszenz im herkömmlichen ratiometrische FRET-Analyse. Das Verhältnis nicht direkt mit der Menge an verdauten Substrat korrelieren. Hier ber...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> PCR II TOPO Kit </td><td> Invitrogen </td><td> K466040 </td></tr><tr><td> 2xYT </td><td> Research Products Corp Internationa.l </td><td> X15600 </td></tr><tr><td> Ni-NTA Agrose </td><td> Thermo Scientific </td><td> 88222 </td></tr><tr><td> Coomassie plus (Bradford) Assay Regent </td><td> Thermo Scientific </td><td> 23238 </td></tr><tr><td> 384-Well-Platte (Glasboden) </td><td> Greiner </td><td> 781892 </td></tr><tr><td> FlexStation II 384 Plattenlesegerät </td><td> Molecular Device </td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

Reversible posttranslationale Modifikationen von Proteinen mit Ubiquitin oder Ubiquitin-ähnliche Proteine ​​(UBLs) werden häufig verwendet, um dynamisch zu regulieren Protein-Aktivität und haben diverse Rollen in vielen biologischen Prozessen. Beispielsweise SUMO kovalent modifiziert eine große Anzahl oder Proteine ​​mit wichtige Rollen bei vielen zellulären Prozessen, einschließlich der Zellzyklus-Regulation, Zellüberleben und Tod, DNA-Schädigung Antwort und Stress-Response 1-5. SENP, wie SUMO-spezifische Protease, um Funktionen wie einer Endopeptidase bei der Reifung von SUMO Vorstufen oder als Isopeptidase SUMO von der Zielproteine ​​zu entfernen und Aktualisiere die SUMOylierung Zyklus 1,3,6,7. Die katalytische Effizienz oder Spezifität eines Enzyms wird am besten durch das Verhältnis der kinetischen Konstanten, k cat / K M gekennzeichnet. In mehreren Untersuchungen haben die kinetischen Parameter von SUMO-SENP paarweise durch verschiedene Verfahren, einschließlich Polyacrylamid Gel-basierten Western-Blot bestimmt worden ist, radIOActive-markiertes Substrat, eine fluoreszierende Verbindung oder Protein markierte Substrat 8-13. Doch die Polyacrylamid-Gel-Techniken, die die &quot;native&quot; Proteine ​​verwendet, aber aufwendig und technisch anspruchsvoll sind, haben die nicht leicht eignen sich für detaillierte quantitative Analyse. Das erhaltene k cat / K M aus Untersuchungen unter Verwendung Tetrapeptiden oder Proteine ​​mit einem ACC-(7-Amino-4-carbamoylmetylcoumarin) oder AMC (7-Amino-4-methylcumarin) Fluorophor waren entweder bis zu zwei Größenordnungen niedriger als die natürlichen Substrate oder kann nicht eine deutliche Differenzierung der iso-und Endopeptidase Aktivitäten SENPs. Kürzlich FRET-Protease-Tests wurden verwendet, um die Enzyme deubiquitinating (DUBs) oder SENPs mit der FRET-Paar von Cyan Fluorescent Protein (GFP) und gelb fluoreszierendes Protein (YFP) 9,10,14,15 studieren. Das Verhältnis von Akzeptor-Emission um Donoremission wurde als quantitative Parameter verwendet für Signal-Monitor für Protease ac FRETtivität Entschlossenheit. Allerdings ignoriert diese Methode Signal Kreuzkontaminationen bei der Akzeptor und Donor Emissionswellenlängen von Donor-und Akzeptor Eigenfluoreszenz und war somit nicht korrekt. Wir entwickelten einen neuartigen hochempfindliche quantitative und FRET-Protease Assay zur Bestimmung der kinetischen Parameter von Pre-SUMO1 SENP1 Reifung durch. Gentechnisch FRET-Paar und CyPet YPet mit deutlich verbesserten Wirkungsgrad und FRET Fluoreszenzquantenausbeute, wurden verwendet, um die CyPet-(pre-SUMO1)-YPet Substrat 16 zu erzeugen. Wir differenziert und quantifiziert absolute Fluoreszenz-Signale von der Donor-und Akzeptor beigetragen und FRET an den Akzeptor und Emissionswellenlängen sind. Der Wert von k cat / K M wurde als (3,2 ± 0,55) erhalten x10 7 M -1 s -1 von SENP1 Richtung Pre-SUMO1, die in Übereinstimmung mit den allgemeinen enzymatischen kinetischen Parameter ist. Daher ist diese Methode gültig und kann verwendet werden, einsa allgemeinen Ansatz auf andere Proteasen sowie charakterisieren.

Watch this Scientific Journal Video about Quantitative FRET FÃƒÂ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Eine neuartige Methode, die quantitative Analyse von FRET (Förster Resonance Energy Transfer)-Signale für das Studium Enzymkinetik beschrieben.<em&gt; K<sub&gt; M</sub</em&gt; Und<em&gt; K<sub&gt; Cat</sub</em&gt; Wurden zur Hydrolyse der katalytischen Domäne des SENP1 (SUMO / Sentrin spezifische Protease 1) vor SUMO1 (Small Ubiquitin-ähnlicher Modifikator) erhalten. Die allgemeinen Grundsätze dieses quantitative-FRET-basierten Protease kinetische Untersuchung auf andere Proteasen angewendet werden.

Quantitative FRET (Förster Resonance Energy Transfer) Analyse für SENP1 Protease Kinetics Bestimmung

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

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Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

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18.9K Aufrufe.  University of California, Riverside. Eine neuartige Methode, die quantitative Analyse von FRET (Förster Resonance Energy Transfer)-Signale für das Studium Enzymkinetik beschrieben. K M

Serie: Quantitative FRET Förster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Verbesserte Visualisierung und quantitative Analyse von Arzneimittelwirkungen mit mikrostrukturierten Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Forschung

Biotechnik

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Adhesion Frequency-Assay für In Situ Kinetics Analyse der Cross-Junktionaler Molecular Interactions an der Zell-Zell-Schnittstelle

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Mikrofluidik-basierten electrotaxis für On-Demand-Quantitative Analyse von<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Drug-induzierte Sensibilisierung der Adenylatzyklase: Assay Straffung und Miniaturisierung für Small Molecule-und siRNA-Screening-Anwendungen

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lumineszenz-Resonanzenergietransfer zu Konformationsänderungen in Membranproteinen in Säugerzellen exprimiert Studieren

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Mess TCR-pMHC Binding<em&gt; In-situ-</em&gt; Mit einem FRET-Mikroskopie Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Sensitive Detection von Proteopathic Seeding Aktivität mit FRET Durchflusszytometrie

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Mehrfarbenfluoreszenzdetektion für Droplet Mikrofluidik Verwendung von Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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.

Ein Protokoll für die Verwendung von Förster Resonance Energy Transfer (FRET) -force Biosensoren mechanische Kräfte über den Kern LINC-Komplex zu messen

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

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Eine höchste Durchsatz mikrofluidischen Plattform für einzellige Genom-Sequenzierung

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Quantitative FRET (Förster Resonance Energy Transfer) Analyse für SENP1 Protease Kinetics Bestimmung

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