Estamos muy agradecidos a Victor GJ Rodgers por sus valiosos consejos. Agradecemos a todos los miembros del grupo Liao para el trabajo en colaboración muy estrecha y ayuda con este estudio. Este estudio fue financiado por los Institutos Nacionales de Salud (Grant AI076504 a JL).

1. Construcciones de plásmidos <ol><li> Amplificar los marcos de lectura abiertos de los genes por PCR, y clonar los productos de PCR en pCRII-TOPO vector. </li><li> Confirmar los productos mediante secuenciación, y clonar el ADNc que codifica CyPet-(pre-SUMO1)-YPet, CyPet SUMO1-, YPet y dominios catalíticos de SENP1 en la pET28 (b) vector con una etiqueta de hexahistidina N-terminal. </li></ol> 2. Expresión y purificación de proteínas <ol><li> Transformar células de Escherichia coli de la cepa BL21 (DE3) con pET28 (b) vectores que codifican CyPet-(pre-SUMO1)-YPet, CyPet SUMO1-, YPet y los dominios catalíticos de SENP1. </li><li> Crecido las bacterias transformadas en medio 2xYT durante 3 horas a 37 ° C (con agitación a 250 rpm) hasta alcanzar una densidad óptica de 0,5 a 600 nm. </li><li> Añadir 100 mM isopropil-β-D-tiogalactósido (IPTG) (concentración final) para inducir la expresión de la proteína y agitar durante 16 horas a 25 ° C a 200 rpm. </li><li> Coseche las bacterias por centrifugación a 10.000 rpm a 4 ° C durante 5 min y se resuspenden en un tampón de 20 mM Tris-HCl (pH 7,4), NaCl 50 mM, y 5 mM de imidazol. </li><li> Sonicar las células durante 10 min en 5-sec intervalos a una potencia de 25 W utilizando MiSonics 4.000 sonicador y recogerlas por centrifugación a 35.000 xg a 4 ° C durante 30 min. </li><li> Configurar la columna con 500 ml de Ni-NTA perlas para el cultivo de L 1 y transferir el sobrenadante a la columna. </li><li> La resina se lava con tampón de 20 mM Tris-HCl (pH 7,4), NaCl 500 mM, y 10 mM imidazol dos veces. </li><li> Proteína se eluye con tampón de 20 mM Tris-HCl (pH 7,4), NaCl 500 mM, y 500 mM de imidazol y la diálisis en un tampón de 20 mM Tris-HCl (pH 7,4), NaCl 50 mM y 1 mM de ditiotreitol (DTT) a 4 ° C durante la noche. </li><li> Determinar las concentraciones de las proteínas purificadas mediante el ensayo de Bradford. Método alternativo para las mediciones de concentración de proteínas será SDS-PAGE electroforesis en gel y después se tiñeron con azul de Coomassie seguido con imágenescuantitativo. </li></ol> 3. Cuantitativo FRET análisis de espectro <ol><li> La estrategia general para el ensayo FRET se basa en la señalización (Figura 1). El par de FRET, CyPet y YPet, fue etiquetada para la N-y C-terminales, respectivamente, de pre-SUMO1. SENP1 escinde la proteína de fusión CyPet-(pre-SUMO1)-YPet en el lugar de Gly-Gly en SUMO1 del C-terminal y, por lo tanto, libera la cola con SUMO YPet. La señal de FRET se interrumpe, lo que resulta en un aumento de la emisión de CyPet y una dramática disminución de la emisión de YPet en la longitud de onda de excitación CyPet. </li><li> Cuando es excitado por la luz de longitud de onda de 414 nm, la emisión total de fluorescencia de CyPet-(pre-SUMO1)-YPet a 530 nm se puede derivar de tres fuentes: la emisión del absoluto FRET inducida YPet, el CyPet directa de emisiones y YPet directa de emisiones (Figura 2). </li></ol><img alt="Ecuación 1" src="/files/ftp_upload/4430/4430eq1.jpg" /> donde FL 530/414 &lt;/ Sub&gt; es el total de las emisiones de fluorescencia a 530 nm cuando se excita a 414 nm, FL FRET es el absoluto señal de FRET, FL CyPet (cont) es la emisión CyPet directa cuando se excita a 414 nm, y FL YPet (cont) es la YPet directas de las emisiones cuando se excita a 414 nm. El subíndice de (cont) representa la contribución. <ol start="3"><li> La emisión directa de CyPet a 530 nm era proporcional a su emisión a 475 nm cuando se excita a 414 nm con una relación constante de α. CyPet-SUMO1 se preparó a concentraciones de 50, 100, 200, 500, y 750 nM y 1 mM y las emisiones a 475 y 530 nm se midió después de excitación a 414 nm para determinar α (Figura 3-1). </li><li> La emisión directa de YPet a 530 nm con excitación a 414 nm era proporcional a su emisión a 530 nm cuando se excita a 475 nm con una relación constante de β. YPet se preparó a concentraciones de 50, 100, 200, 500, 750 and 1.000 nm, y la emisión a 530 nm se midió cuando las muestras fueron excitados por longitudes de onda de 414 y 475 nm para determinar β (Figura 3-2). </li><li> Cuando CyPet-(pre-SUMO1)-YPet fue digerido por SENP1, la escisión libera CyPet-SUMO1 y la cola SUMO1 con YPet. Cuando el compuesto se excita a 414 nm, la emisión de fluorescencia a 530 nm (FL &#39;530/414) se redujo, pero aún se pueden dividir en tres partes como: </li></ol><img alt="Ecuación 2" src="/files/ftp_upload/4430/4430eq2.jpg" /> donde FL &#39;530/414 es la emisión total de fluorescencia a 530 nm después de la digestión cuando se excita a 414 nm, FL&#39; FRET es el restante absoluto señal de FRET, CyPet FL &#39;(475/414) es la emisión CyPet a 475 nm después de la digestión cuando excita a 414 nm (en este caso la emisión CyPet es de dos partes: sin digerir CyPet-(pre-SUMO1)-YPet y digerido CyPet-SUMO1), y YPet FL(530/475) es la emisión YPet cuando se excita a 475 nm, que es constante si CyPet-(pre-SUMO1)-YPet se digiere o no. <ol start="6"><li> Después de la digestión por SENP1, el restante FRET emisión (FL-FRET) es: </li></ol><img alt="Ecuación 3" src="/files/ftp_upload/4430/4430eq3.jpg" /> donde C es la concentración total de CyPet-(pre-SUMO1)-YPet y x es la concentración de digerido CyPet-(pre-SUMO1)-YPet. <ol start="7"><li> Mediante la combinación de todos los elementos, la emisión de fluorescencia detectada a 530 nm bajo excitación de 414 nm (530 FL &#39;/ 414) es: </li></ol><img alt="Ecuación 4" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4. FRET basada en el Ensayo de la Proteasa Estudio de cinética enzimática <ol><li> CyPet-(pre-SUMO1)-YPet se incubó con el dominio catalítico de SENP1 a 37 ° C en un tampón de 20 mM Tris-HCl (pH 7,4), 50 mM NaCl, 0,1% (v /v) de Tween-20 y 1 mM de DTT a un volumen total de 80 ml y se transfirió a 384-así placa. </li><li> Carreras se llevaron a cabo mediante la medición de la emisión de fluorescencia a 475 y 530 nm después de una excitación a 414 nm en un lector de placas de fluorescencia de múltiples pocillos durante 5 min con 15-sec intervalos. </li><li> La velocidad de reacción (v) se correlacionó con el cambio en la cantidad de sustrato (S) como: </li></ol><img alt="Ecuación 5" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li> La concentración de producto aumentado exponencialmente desde 0 como [S] 0 (concentración de sustrato original) cuando t = 0: </li></ol><img alt="Ecuación 6" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> Cuando t = 0, la velocidad inicial (V 0) es: </li></ol><img alt="Ecuación 7" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li> La concentration de SENP1 se fijó, y la concentración de CyPet-(pre-SUMO1)-YPet se aumentó de 100 veces más que la concentración de la enzima, que es requerido por la ecuación de Michaelis-Menten. </li><li> Todas las lecturas fluorescentes fueron analizadas por el método de análisis cuantitativo FRET y se representan en GraphPad Prism Software V para adaptarse a la ecuación de Michaelis-Menten. La regresión no lineal también se puede realizar con la ayuda de paquetes de software más comunes, como los programas de hojas de cálculo (por ejemplo, Microsoft Excel). </li></ol> 5. Los resultados representativos La maduración de pre-SUMO1 por SENP1 puede determinarse controlando los cambios en la señal de fluorescencia a 475 y 530 nm durante el proceso. El resultado mostró que la velocidad de la pre-SUMO1 digestión por SENP1 en una manera dependiente de la dosis-sustrato (Figura 4). Esto sugiere que el dominio catalítico de SENP1 exhibe una excelente actividad para la maduración de los pre-SUMO1. La reacción inicialvelocidades ción se calcularon por el análisis anterior con diferentes concentraciones de sustrato (Tabla 1). El k cat / K M se utiliza generalmente para comparar las eficiencias de los diferentes enzimas con un sustrato o una enzima en particular con diferentes sustratos. K M y V max se puede obtener de la ecuación de Michaelis-Menten por el trazado de las diversas velocidades iniciales, correspondientes a las diferentes concentraciones de CyPet-(pre-SUMO1)-YPet (Figura 5) k gato se obtuvo en forma.: <img alt="Ecuación 8" src="/files/ftp_upload/4430/4430eq8.jpg" /> De acuerdo con el análisis anterior, se calculó un K M fue de 0,21 ± 0,04 mM, el gato k fue 6,90 ± 0,28 s -1, y la k cat / K M proporción fue de (3,2 ± 0,55) x 10 7 M -1 </sup<html> &gt; S -1. <img alt="Figura 1" src="/files/ftp_upload/4430/4430fig1.jpg" /> Figura 1. Gráfico de ensayo de la proteasa FRET basado en un pre-sumos SENP de maduración. <img alt="Figura 2" src="/files/ftp_upload/4430/4430fig2.jpg" /> Figura 2. Análisis cuantitativo de la señal fluorescente como contribuciones por el donante, aceptor y FRET. La disección de los espectros de emisión de CyPet-(pre-SUMO1)-YPet bajo excitación a 414 nm. CyPet FL (530/414) Se emisión CyPet de a 530 nm bajo excitación de 414 nm, FL FRET es la emisión FRET inducida YPet a 530 nm bajo excitación de 414 nm, y FL YPet (530/414) es la emisión YPet de a 530 nm bajo excitación de 414 nm. load/4430/4430fig3.jpg &quot;/&gt; Figura 3. Cálculo del factor de α y β dirección de emisión. <img alt="Figura 4" src="/files/ftp_upload/4430/4430fig4.jpg" /> Figura 4. Análisis cuantitativo de CyPet-(pre-SUMO1)-YPet digerido por diferentes proporciones de al dominio catalítico de SENP1. Las reacciones se controlaron dentro de los primeros 5 min. <img alt="Figura 5" src="/files/ftp_upload/4430/4430fig5.jpg" /> Figura 5. Michaelis-Menten análisis gráfico de digestión CyPet-(pre-SUMO1)-YPet de por SENP1. Los datos se representaron gráficamente y se analizaron por GraphPad Prism V y regresión no lineal. <table border="1"><tbody><tr><td> [S] (mM) </td><td> V 0 (mM / 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> Tabla 1. Determinación de las velocidades iniciales de la maduración de los pre-SUMO1 por SENP1. En cada concentración de sustrato, cuatro muestras se utilizaron para medir la digestión. La desviación estándar de vino de las variaciones de estas cuatro muestras. <table border="1"><tbody><tr><td> K M (M) </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> Tabla 2. Parámetros cinéticos de la maduración del pre-SUMO1 por SENP1 cuantitativos de análisis FRET. La desviación estándar de vino de las cuatro muestras en cada concentración de sustrato.

<ol>
	<li>Johnson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+modification+By+SUMO.">Protein modification By SUMO.</a> Annual Review of Biochemistry. 73, 355-382 (2004).</li><li>Gill, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO+and+ubiquitin+in+the+nucleus:+different+functions,+similar+mechanisms.">SUMO and ubiquitin in the nucleus: different functions, similar mechanisms.</a> Genes &amp; Development. 18, 2046-2059 (2004).</li><li>Hay, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO:+a+history+of+modification.">SUMO: a history of modification.</a> Molecular Cell. 18, 1-12 (2005).</li><li>M&#252;ller, S., Hoege, C., Pyrowolakis, G., Jentsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO,+ubiquitin's+mysterious+cousin.">SUMO, ubiquitin's mysterious cousin.</a> Nature. , 202-210 (2001).</li><li>Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SP-RING+for+SUMO:+new+functions+bloom+for+a+ubiquitin-like+protein.">SP-RING for SUMO: new functions bloom for a ubiquitin-like protein.</a> Cell. , 5-8 (2001).</li><li>Drag, M., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeSUMOylating+enzymes-SENPs.">DeSUMOylating enzymes-SENPs.</a> IUBMB Life. 60, 734-742 (2008).</li><li>Mukhopadhyay, D., Dasso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+in+reverse:+the+SUMO+proteases.">Modification in reverse: the SUMO proteases.</a> Trends in Biochemical Sciences. 32, 286-295 (2007).</li><li>Reverter, D., Lima, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+optimization+of+fluorescent+proteins+for+intracellular+FRET.">Evolutionary optimization of fluorescent proteins for intracellular FRET.</a> Nature Biotechnology. 23, 355-360 (2005).</li></ol>

No hay conflictos de interés declarado.

Tecnología FRET ha sido utilizada para estudiar la maduración pre-SUMO1 de por SENP1 9. CFP-YFP se utilizó como el par de FRET y el análisis radiométrico, que es la relación de aceptor a las emisiones de los donantes, se utilizó para caracterizar las propiedades cinéticas. Sin embargo, no existe ninguna consideración de donante y aceptor de auto-fluorescencia en la ratiométrica tradicional análisis FRET. La relación no se correlacionan directamente con la cantidad de sustrato digerido. <p clas...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> PCR II TOPO Kit </td><td> Invitrogen </td><td> K466040 </td></tr><tr><td> 2xYT </td><td> Investigación de Productos Internationa.l Corp. </td><td> X15600 </td></tr><tr><td> Ni-NTA agarosa </td><td> Thermo Scientific </td><td> 88222 </td></tr><tr><td> Más Coomassie (Bradford) Regent Ensayo </td><td> Thermo Scientific </td><td> 23238 </td></tr><tr><td> 384-así placa (con fondo de cristal) </td><td> Greiner </td><td> 781892 </td></tr><tr><td> FlexStation II 384 lector de placas </td><td> Dispositivo Molecular </td><td></td></tr></tbody></table>

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

Reversibles modificaciones postraduccionales de proteínas con proteínas de ubiquitina o ubiquitina como-(Ubls) son ampliamente utilizados para regular dinámicamente actividad de la proteína y tiene diversas funciones en muchos procesos biológicos. Por ejemplo, SUMO modifica covalentemente un gran número o proteínas con papeles importantes en muchos procesos celulares, incluyendo la regulación del ciclo celular, la supervivencia celular y la muerte, la respuesta de daño del ADN, y la respuesta de estrés 1-5. SENP, como SUMO-proteasa específica, funciona como una endopeptidasa en la maduración de los precursores SUMO o como un isopeptidase para eliminar SUMO de sus proteínas diana y refrescar el ciclo SUMOylation 1,3,6,7. La eficiencia catalítica o la especificidad de una enzima se caracteriza mejor por la relación de las constantes cinéticas, k cat / K M. En varios estudios, los parámetros cinéticos de SUMO-SENP pares han sido determinados por varios métodos, incluyendo en gel de poliacrilamida basado en western-blot, radIOActive sustrato marcado, compuesto fluorescente o proteína sustrato marcado 8-13. Sin embargo, las técnicas de poliacrilamida-gel a base, que utilizaron los &quot;nativos&quot; de proteínas, pero son laboriosos y técnicamente exigente, que no se prestan a un análisis cuantitativo detallado. La obtenido k cat / K M de estudios usando tetrapéptidos o proteínas con un ACC (7-amino-4-carbamoylmetylcoumarin) o AMC (7-amino-4-metilcumarina) fluoróforo eran o bien hasta dos órdenes de magnitud menor que los sustratos naturales o no puede diferenciar claramente las iso-y las actividades de endopeptidasa SENPs. Ensayos de la proteasa Recientemente, basados ​​en FRET se utilizan para estudiar las enzimas deubiquitinating (DUBs) o SENPs con el par de FRET cian proteína fluorescente (PPC) y la proteína amarilla fluorescente (YFP) 9,10,14,15. La relación de emisión de aceptor de emisión del donante se utiliza como el parámetro cuantitativo para FRET monitor de señal de proteasa acdad determinación. Sin embargo, este método ignora la señal contaminaciones cruzadas en el aceptor y longitudes de onda de emisión por donantes aceptor y donante de auto-fluorescencia y por lo tanto no era correcta. Hemos desarrollado un novedoso altamente sensible y cuantitativo FRET basada en el ensayo de la proteasa para la determinación de los parámetros cinéticos de pre-SUMO1 maduración por SENP1. Un par de ingeniería FRET CyPet y YPet con mejorado significativamente la eficiencia de FRET y rendimiento cuántico de fluorescencia, se usaron para generar el CyPet-(pre-SUMO1)-YPet sustrato 16. Hemos diferenciado y cuantificados absolutos señales de fluorescencia aportados por el donante y el aceptor y FRET en las longitudes de onda de emisión de aceptor y, respectivamente. El valor de k cat / K M se obtuvo como (3,2 ± 0,55) x 10 7 M -1 s -1 de SENP1 hacia pre-SUMO1, que está de acuerdo con los parámetros generales de cinéticas enzimáticas. Por lo tanto, esta metodología es válida y se puede utilizar unsa Enfoque general para caracterizar otras proteasas también.

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

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

Un nuevo método que implica el análisis cuantitativo de FRET (transferencia de energía por resonancia de Förster) señales se describe para el estudio de la cinética de enzimas.<em&gt; K<sub&gt; M</sub</em&gt; Y<em&gt; K<sub&gt; Cat</sub</em&gt; Fueron obtenidos para la hidrólisis del dominio catalítico de SENP1 (SUMO / sentrin proteasa específica 1) para pre-SUMO1 (Small ubiquitina-como modificador). Los principios generales de este estudio cuantitativo proteasa-FRET basada cinética se puede aplicar a otras proteasas.

Cuantitativo FRET (transferencia de energía de resonancia de Förster) Análisis para SENP1 Proteasa Determinación Kinetics

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 vistas.  University of California, Riverside. Un nuevo método que implica el análisis cuantitativo de FRET (transferencia de energía por resonancia de Förster) señales se describe para el estudio de la cinética de enzimas. K M

Video: Quantitative FRET F&#246;rster 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.

Una mejor visualización y análisis cuantitativo de los efectos de drogas Uso de células micropatterned

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

Investigación

Bioingeniería

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.

Ensayo de adherencia de frecuencia para In Situ El análisis de la cinética de unión de la Cruz-Las interacciones moleculares en la interfaz célula-célula

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&#039; 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.

Electrotaxis microfluidos basados ​​en On-Demand Análisis cuantitativo de<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.

Inducido por fármacos Sensibilización de adenilciclasa: Racionalización Ensayo y miniaturización de moléculas pequeñas y siRNA aplicaciones de cribado

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.

La luminiscencia de energía de resonancia de transferencia para estudiar cambios conformacionales en proteínas de membrana expresado en células de mamífero

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.

Medición de TCR-pMHC Encuadernación<em&gt; In Situ</em&gt; Usando un ensayo de microscopía basada en FRET

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.

Detección Sensible de Proteopathic Siembra Actividad con FRET Citometría de Flujo

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.

Multicolor detección por fluorescencia de la gotita de microfluídica el uso de fibras ópticas

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

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.

Un protocolo para el uso de Transferencia de energía de resonancia de Förster (FRET) y forzar Biosensores para medir fuerzas mecánicas en todo el complejo nuclear de LINC

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

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Ingeniería Molecular Recognition con polímeros biomiméticos en una sola pared nanotubos de carbono

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...