Damos las gracias a los Institutos Nacionales de Salud (1R21CA138373 y Stand Up to Cancer (SU2C) para apoyos financieros de este trabajo. HY está agradecido por el premio Elion 2009 de la Asociación Americana de Investigación del Cáncer, el Premio Académico Kimmel de la Fundación Sidney Kimmel para Investigación sobre el Cáncer (SKF-08-101), y el Premio Nacional de Ciencias Facultad de Carrera Temprana Fundación (NSF0954819).

1. Consideraciones sobre la clonación <ol><li> Oligonucleótidos preparados comercialmente, en representación de la TMD de interés flanqueado por NheI y sitios de restricción BamHI y 5 &#39;fosforilados se pueden ligar en pTox7 (modificado en nuestro laboratorio mediante la inserción de un par de bases directamente después de que el sitio de restricción BamHI 14) (Figura 3) digerido secuencialmente con BamHI y NheI. Un oligonucleótido ejemplo se muestra a continuación: 5&#39;ctagcTMDSEQUENCEg3 &#39; 3 &#39;gTMDSEQUENCEcctag5&#39; </li></ol> La secuencia de TMD debe ser 12-24 residuos (las secuencias más cortas, presumiblemente, se prolongarán por residuos hidrofóbicos vector codificado). Con el fin de investigar la interfaz, cuatro variantes del diseño TMD se debe crear en inserciones secuenciales de residuos y residuos concomitante resultado deleciones en la rotación de la relativa a la TMD toxR 15,16. Por último, la concentración de arabinosa se ​​debe variar entre 0,001 y 0,01% (w / v) para identificar la concentración máxima en que las diferencias en las señales de galactosidasa β-entre las diferentes secuencias de TMD se observan, pruebas de diferentes niveles de expresión, se recomienda identificar las condiciones bajo las cuales las afinidades se pueden distinguir mejor. Además de arabinosa y antibióticos, 0,4 mM IPTG se puede utilizar para resaltar las diferencias de afinidades entre TTM diferentes. La medición toxR debe realizar por lo menos en cuatro ejemplares. Todo el procedimiento se debe repetir por lo menos tres veces con diferentes transformaciones plásmido. 2. El crecimiento de los cultivos bacterianos <ol><li> Suavemente deshielo FHK12 células competentes (200 l) en el hielo y la transferencia en un tubo de cultivo de 15 ml. Añadir ADN plásmido (200 ng) e incubar las células en hielo durante 30 min. </li><li> Choque térmico de las células mediante la incubación durante 90 s en 42 ° C, seguido de incubación en hielo durante 2 min. </li><li> Añadir SOC medios de comunicación (800 l) y se incuban las muestras a 37 ° C con agitación (300 rpm) durante una h. </li><li> Inocular 5 ml de LB medios con cloranfenicol (30 mg / ml) y arabinosa (0,0025% w / v) con 50 l de la mezcla de transformación en tubos de 15 ml de cultivo por triplicado. Incubar las muestras a 37 ° C con agitación (300 rpm) durante 20 h. (También 5 l de la cultura se puede utilizar para inocular 100 l de medio en una placa de 96 pocillos. Este método es útil cuando se trata de un gran número de muestras, a pesar de los errores será ligeramente superior. Con el fin de evitar la evaporación, lo que llevaría de error, llenar los pozos ultraperiféricas con los medios de comunicación, pero no las utilizan para las muestras. Por último, haga doble envoltura de la articulación entre la tapa y la placa con parafilm). </li></ol> 3. Medición de la actividad β-galactosidasa <ol><li> Precaliente la placa-lector a 28 ° C. </li><li> Transferir el Z-buffer en un depósito con una gran punta de la pipeta, asegurándose de que sólo ocupan la parte superior (acuosa) de la capa. Transferencia de 100 l de Z-buffer/chloroform recién preparada a los pocillos de una placa de 96 pocillos. La transferencia de 5 l de cada cultura en los pocillos de la placa en cuatro ejemplares. Omitir la cultura de los cuatro pozos que servirá como blanco. </li><li> Mida el diámetro exterior 595 de la placa para determinar la densidad celular. </li><li> Añadir 50 l de Z-buffer/SDS a todos los pocillos de la placa. Agitar la placa en la placa-lector durante 10 minutos para Lyze las células. Asegúrese de que las suspensiones celulares son claras después de la lisis y repita el paso agitando si es necesario. Lisis incompleta sugiere la Z-buffer/chloroform no estaba recién preparado. </li><li> Añadir 50 l recién preparada Z-buffer/ONPG a todos los pozos y devolver el plato para el lector de placas y medir OD 405 cada 30 s durante 20 min. </li><li> Calcular β-galactosidasa actividad mediante la siguiente ecuación (recordando que restar el espacio en blanco). La relación de OD 405 / min, deberá calcularse utilizando todos los puntos de datos en el OD 405 entre 0.0 y 1.0 usando un modelo de ajuste lineal. <img src="files/ftp_upload/2721/2721eq1.jpg" alt="La ecuación 1" /> Unidades Miller difieren a veces cuando se graba en diferentes días. Por lo tanto, una construcción de referencia como GpA se debe medir en cada prueba. Sus valores pueden ser utilizados para la normalización de los valores toxR. </li></ol> 4. Control de la expresión de proteínas <ol><li> Realizar Western Blot para comprobar incluso la expresión de proteínas entre los constructos. Combine 50 l de las culturas por triplicado y se centrifuga (2000 rpm, 4 min) en una microcentrífuga. Eliminar el sobrenadante con pipeta y resuspender el pellet residual en 2 x tampón de carga de la muestra. </li><li> 7,5 l de carga en un gel estándar de 8% y llevar a cabo la electroforesis a 125 V durante 1 hora 5 min. Después de la transferencia, se incuba con anticuerpos anti-MBP HRP-anticuerpo conjugado y visualizar, la proteína quimérica se observa en aproximadamente 70 kDa con algunos productos de degradación a veces visto alrededor de 48 kDa. MBP endógena también se observa a 45 kDa (ver Figura 5). </li></ol>5. De control para la inserción de la membrana adecuada Una línea de células deficientes en la proteína de unión a maltosa se utiliza para evaluar la inserción adecuada de la membrana de la construcción de TMD quimérico. Cuando se cultiva en medio mínimo con maltosa como única fuente de carbono, sólo las células que expresan una membrana integral producto de expresión de proteínas de unión a maltosa correctamente ubicado al periplasma son capaces de crecer. <ol><li> Transformar las células PD28 (como se describe para FHK12 células) e inocular 2 ml de medio LB. Crecer las células a 37 ° C con agitación (300 rpm) durante la noche. </li><li> Precipitar las células por centrifugación a 3500 rpm, 10 min, 4 ° C y se lavan mediante resuspensión en PBS (2 ml) por pipeteo suave con una buena propina o agitación suave. Que sedimenten las células (como antes), lavar con PBS por segunda vez, pellets y, finalmente, volver a suspender en PBS (1 ml). </li><li> Utilice 25 l de células resuspendidas para inocular 5 ml de medio mínimo por triplicado y se incuba a 37 ° C con agitación (300 rpm). Tomar lecturas de OD 595 entre 15-25 h, aproximadamente cada 2 horas por la transferencia de 200 l de cada muestra en una placa de 96 pozos y la lectura utilizando la placa-lector. </li></ol> 6. Los resultados representativos: Un ejemplo de la utilización del ensayo de reportero toxR transcripción para analizar la propensión de oligomerización dominios transmembrana en la muestra en la Figura 4. Anteriormente hemos investigado la oligomerización de los dominios transmembrana de la multispanning membrana de proteínas de membrana integral latente proteína-1 (LMP-1) por varias técnicas, incluyendo toxR 14 dominio transmembrana cinco (TM5) se demostró que presentan una fuerte tendencia a oligomerize.; Esto se demuestra por las altas unidades Miller, comparable a la del control positivo, el PAM, una secuencia bien establecida dimerizing. Una mutación deletérea en TM5, D150A, reduce la capacidad de la secuencia de oligomerize. LMP-1 TM1 no significativamente oligomerize y presenta una señal de la Unidad de Miller muy baja, justo por encima de la señal en blanco, no transformadas FHK12 células. <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="Figura 1" /> Dibujos Figura 1. Representa el reportero de ensayo toxR. Dominio transmembrana (TMD), impulsados ​​resultados oligomerización en la dimerización de toxR y la activación de la transcripción de lacZ. El producto del gen de LacZ, β-galactosidasa se ​​puede cuantificar como una medida de la propensión de un TMD de oligomerize. <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="Figura 2" /> Figura 2. La escisión hidrolítica de ONPG por los resultados de β-galactosidasa en la producción de la especie que absorbe la luz o-nitrofenolato (ONP). <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="Figura 3" /> Figura 3. Mapa del plásmido de pToxR7. <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="Figura 4" /> Figura 4. Representante ensayo toxR reportero transcripcional analizar la propensión de oligomerización de la proteína latente de membrana-1 dominios transmembrana. Dominio transmembrana 5 (TM5) oligomeriza fuertemente, mientras que un dominio transmembrana (TM1) exhibe sólo una interacción débil. D150A mutación en TM5 reduce de forma significativa su capacidad para oligomerize. GpA se incluye como una secuencia de control positivo para la dimerización fuerte. Blanco representa sin transformar FHK12 células. <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="Figura 5" /> Figura 5. Western blot para la expresión de la proteína. <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="Figura 6" /> Figura 6. PD28 ensayo de complementación para el control de la inserción correcta de la membrana del periplasma. Control negativo representa una construcción deficiente en proteínas de unión a maltosa.

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El ensayo toxR reportero de la transcripción es una forma fácil para identificar las secuencias transmembrana con el potencial de oligomerize. Dado que las interacciones se producen dentro de la membrana interna bacteriana, este ensayo se eludan las cuestiones relacionadas con la validez de los sistemas que estudian en la membrana mimética-ambientes. Teniendo en cuenta que la clonación de TTM múltiples en un solo plásmido fácilmente se puede hacer en paralelo y el ensayo completo se puede realizar en formato de 9...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Enzima de restricción BamHI </td><td> Invitrogen </td><td> 15201023 </td><td> Invitrogen enzimas resultaron ser más eficientes que los proveedores alternativos </td></tr><tr><td> NheI enzima de restricción </td><td> Invitrogen </td><td> 15444011 </td><td> Invitrogen enzimas resultaron ser más eficientes que los proveedores alternativos </td></tr><tr><td> Tubos de 15 ml de cultivo </td><td> Fisher Scientific </td><td> 14-956-1J </td><td></td></tr><tr><td> SOC medios de comunicación </td><td> Teknova </td><td> S0225 </td><td> Hecho hasta el volumen adecuado y esterilizado en autoclave. </td></tr><tr><td> Medio LB </td><td> Sigma-Aldrich </td><td> L7275 </td><td> Hecho hasta el volumen adecuado y esterilizado en autoclave. </td></tr><tr><td> Cloranfenicol </td><td> Sigma-Aldrich </td><td> CO 3 78 </td><td> Solución stock de 30 mg / ml en etanol almacenados en el congelador </td></tr><tr><td> Arabinosa </td><td> Fluka </td><td> 10839 </td><td> Solución madre de 2,5% (w / v) en el agua almacenada en el congelador </td></tr><tr><td> Na 2 HPO 4 </td><td> Sigma-Aldrich </td><td> S9390 </td><td></td></tr><tr><td> NaH 2 PO 4 </td><td> Sigma-Aldrich </td><td> S9638 </td><td></td></tr><tr><td> KCl </td><td> Mallinckrodt productos químicos </td><td> 6858-06 </td><td></td></tr><tr><td> MgSO 4 .7 H 2 O </td><td> Sigma-Aldrich </td><td> 63138 </td><td></td></tr><tr><td> Dodecilsulfato sódico (SDS) </td><td> Sigma-Aldrich </td><td> L6026 </td><td></td></tr><tr><td> 2-nitrofenil β-D-galactopiranósido (ONPG) </td><td> Sigma-Aldrich </td><td> 73660 </td><td></td></tr><tr><td> Z-buffer </td><td></td><td></td><td> 16,1 g de Na 2 HPO 4 5,5 g de NaH 2 PO 4 0,75 g de KCl 0.246g MgSO4 Completar hasta 1 l, pH 7,0 </td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200 ml de β-mercaptoetanol, 2 ml de cloroformo, que hasta 20 ml con Z-buffer. Vortex durante 1 min, centrifugar durante 1 min a 800 rpm. Hacer de nuevo para cada plato. </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 160 mg SDS disuelto en 10 ml de Z-buffer </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 40 mg en 10 ml de ONPG Z-buffer. Hacer de nuevo para cada plato </td></tr><tr><td> β-mercaptoetanol </td><td> Calbiochem </td><td> 444203 </td><td></td></tr><tr><td> Anti-MBP anticuerpo monoclonal (HRP conjugado) </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td> Los medios de comunicación con un mínimo de maltosa </td><td></td><td></td><td> 1 x M9 sales, el 0,4% de maltosa, 1 mg / ml de tiamina, 2 mM MgSO 4 </td></tr><tr><td> Placa de 96 pocillos de fondo plano </td><td> Sarstedt </td><td> 83.1835.300 </td><td></td></tr><tr><td> Plate-lector </td><td> Beckman Coulter </td><td> DTX880 Detector multimodo </td><td></td></tr><tr><td> Baño de agua </td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td> Incubadora de agitación </td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

El punto de vista simplista de los dominios transmembrana de la proteína como un mero anclas en bicapas de fosfolípidos desde hace mucho tiempo que ya fueron desbancados. En muchos casos, que atraviesa la membrana las proteínas han desarrollado mecanismos muy sofisticados de acción. 3.1 Una forma en que las proteínas de membrana son capaces de modular sus estructuras y funciones es por contacto directo y específico de las hélices hidrofóbicas, formando oligómeros estructura transmembrana. 4,5 Mucho recientes el trabajo se ha centrado en la distribución de los aminoácidos preferentemente en el medio ambiente de la membrana en comparación con una solución acuosa y las fuerzas intermoleculares diferentes que la asociación de proteínas en coche. 6,7 Sin embargo, los estudios de reconocimiento molecular en el dominio transmembrana de las proteínas aún por detrás de los de soluble en agua las regiones. El principal obstáculo sigue siendo: a pesar de la especificidad y afinidad notable que oligomerización transmembrana puede lograr, 8 medición directa de su asociación es un reto. Las metodologías tradicionales aplicadas al estudio de la función integral de proteína de la membrana puede ser obstaculizada por la insolubilidad inherente de las secuencias en estudio. Puntos de vista biofísico adquiridos al estudiar péptidos sintéticos que representan los dominios transmembrana puede proporcionar una visión estructural de utilidad. Sin embargo, la importancia biológica de las micelas de detergente o de los sistemas de liposomas utilizados en estos estudios para imitar las membranas celulares con frecuencia se cuestiona, no adoptar una estructura de péptidos nativos-como en estas condiciones y que su comportamiento funcional, reflejen verdaderamente el modo de acción dentro de una membrana nativa ? Con el fin de estudiar las interacciones de las secuencias transmembrana en bicapas de fosfolípidos naturales, el laboratorio Langosch desarrollado ensayos toxR transcripcional reportero. 9 El dominio transmembrana de interés se expresa como una proteína quimérica con la proteína de unión a maltosa para la ubicación de la periplasma y toxR que presente un informe del nivel de oligomerización (Figura 1). En la última década, varios otros grupos (por ejemplo, Engelman, DeGrado, Shai) optimizado y aplicado este reportero de ensayo toxR. 10-13 Los ensayos toxR varios se han convertido en un estándar de oro para probar interacciones proteína-proteína en las membranas celulares. Nosotros en este documento demuestran una operación típica experimental llevado a cabo en nuestro laboratorio que todo sigue los protocolos desarrollados por Langosch. Este método de aplicación general es útil para el análisis de dominio de transmembrana la libre asociación de E. coli, donde se utiliza β-galactosidasa de producción para evaluar la propensión oligomerización TMD. Al TMD inducida por dimerización, toxR se une al promotor ctx causando la regulación del gen lacZ de β-galactosidasa. Una lectura colorimétrica se obtiene mediante la adición de ONPG a las células lisadas. Escisión hidrolítica de ONPG por los resultados de β-galactosidasa en la producción de la especie que absorbe la luz o-nitrofenolato (ONP) (Figura 2).

Watch this Scientific Journal Video about Transmembrane Domain Oligomerization Propensity determined by ToxR Assay at JoVE.com

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Un procedimiento eficaz para evaluar la propensión de oligomerización dominios transmembrana de un solo paso (TTM) se describe. Proteínas quiméricas que consiste en la DTM fundido a toxR se expresan en una cepa de E. coli reportero. TMD inducida por oligomerización causas dimerización de toxR, activación de la transcripción y la producción de la proteína reportera,-galactosidasa.

Dominio transmembrana Oligomerización propensión determinada por el ensayo toxR

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

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15.1K vistas.  University of Colorado at Boulder. Un procedimiento eficaz para evaluar la propensión de oligomerización dominios transmembrana de un solo paso (TTM) se describe. Proteínas quiméricas que consiste en la DTM fundido a toxR se expresan en una cepa de E. coli reportero. TMD inducida por oligomerización causas dimerización de toxR, activación de la transcripción y la producción de la proteína reportera,-galactosidasa.

Video: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expresión y purificación de la proteína transmembrana de la fibrosis quística regulador de la conductancia en la Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Investigación

Biología

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

La purificación de la fibrosis quística regulador de conductancia transmembrana de proteínas expresadas en<em&gt; Saccharomyces cerevisiae</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

Caracterización de G receptores acoplados a proteína por un ensayo de movilización de calcio basado en fluorescencia

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Genoma toda la Proteína-proteína interacción Screening por Complementación de ensayo de proteínas-fragmento (PCA) en las células vivas

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstitución de una proteína transmembrana, el canal iónico dependiente de voltaje, KvAP, en Gigante vesículas unilamelares para Microscopía y Patch Clamp Estudios

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Mapeo óptico de Intra-retículo sarcoplásmico Ca<sup&gt; 2 +</sup&gt; Y potencial transmembrana en el corazón de conejo perfundidos Langendorff

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Los enfoques genéticos y bioquímicos para<em&gt; En Vivo</em&gt; y<em&gt; In Vitro</em&gt; Evaluación de la oligomerización de proteínas el receptor de rianodina Estudio de caso

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Estudio del retículo endoplasmático y las mitocondrias por Interacciones<em&gt; In Situ</em&gt; Proximidad ligadura de ensayo en células fijadas

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...