Nuestro trabajo ha sido apoyado por becas de la American Heart Association (Gran Afiliado Sureste) 0765185B A partir de la concesión en la ayuda, la Fundación Elsa U. Pardee beca de investigación, y la Universidad Estatal de Wayne fondo de intramuros de inicio y de Investigación Cardiovascular del Instituto premio Iniciativa Isis. Este método de in vitro conjunto complejo macromolecular CFTR fue originalmente iniciado por el Dr. AP Naren (Universidad de Tennessee Health Science Center).

1. Expresión y purificación de proteínas recombinantes de fusión con la etiqueta en las bacterias <ol><li> Amplificar las regiones definidas de la C-colas (en los últimos 50-100 aminoácidos que contienen los motivos PDZ en C-terminal) de CFTR, LPA 2, MRP2, MRP4, β 2 AR, y NHERFs (de larga duración o dominios PDZ1 o PDZ2 ) por el método PCR. </li><li> Clonar los productos de PCR en pGEX4T-1 vector de fusión GST-proteínas (como GST-NHERFs, GST-MRP4 TC), pMAL-C2 vector de las proteínas de fusión MBP-(como MBP-β 2 AR CT, PAM-CFTR CT ), y pET30 para His-S-proteínas de fusión (tales como Su-S-CT CFTR, Su-S-PDZK1). </li><li> Transformación en una E. proteasa deficiente coli cepa (BL21-DE3) para minimizar la posible degradación de proteínas recombinantes. </li><li> Mantener el cultivo durante la noche a 37 ° C en medio Luria-Bertani (pH 7,0) que contenía los antibióticos adecuados (por ejemplo, ampicilina o kanamicina). Diluir el cultivo durante la noche a 1:10 y crecer durante otras 2 horas a 37 ° C. Enducir con 0,5-1 m M IPTG durante los próximos 4 horas a 30 ° C. Precipitar las células por centrifugación a 8.000 × g durante 10 min a 4 ° C. </li><li> Lisar las células en tampón de sacarosa (50 m M de Tris-HCl, pH 8,0, 1 mM EDTA, 1 mM PMSF, y 10% de sacarosa) que contenía lisozima (1 mg / ml), 0,2% de Triton X-100, y la proteasa inhibidores. Utilizar 20 ml de sedimento de células procedentes de 1 litro de cultivo. </li><li> Mezclar en un agitador rotatorio durante 30 min a 4 ° C. </li><li> Girar a 20.000 rpm durante 30 min a 4 ° C. Recoger el sobrenadante claro. </li><li> En el sobrenadante claro, añadir 1 ml de los siguientes resina / agarosa bolas (50% suspensión en tampón de sacarosa): <ul><li> Glutatión agarosa para las proteínas de fusión GST. </li><li> Talon cuentas de Sus-S proteínas de fusión. </li><li> La amilosa resina para las proteínas de fusión MBP. </li></ul></li><li> Mezclar durante 4 horas a 4 ° C en un agitador rotatorio. </li><li> Lavar las perlas por resuspensión en PBS 1x (15 ml), mezclando durante 2 millasn, girando a 800 xg durante 2 min, y desechar el sobrenadante. Repita este paso para seis veces. </li><li> Eluir la proteína de las perlas usando tampón de elución respectivo (2 tampón de elución ml por 1 ml de perlas). <ul><li> Tampón de elución de proteínas de fusión GST: 25 m M de Tris-HCl (pH 8,0), 140 m M de NaCl, y 20 m M glutatión reducido. </li><li> La elución de las proteínas tampón Sus fusión: 20 m M de Tris-HCl (pH 8,0), 500 m M de NaCl, y 200 m M imidazol. </li><li> La elución tampón para proteínas de fusión MBP: 20 m M de Tris-HCl (pH 8,0), 200 m M de NaCl, 1 mM EDTA, 1 mM DTT y 10 m M de maltosa. </li></ul></li><li> Dializar las proteínas eluidas contra 2 l de 1x PBS a 4 ° C (para evitar la degradación de proteínas es posible). Cambio de PBS cada 4 h durante cuatro veces. Concentrar la proteína utilizando un filtro Centricon (10.000 MW de corte, Millipore) y almacena como pequeñas alícuotas a -80 ° C. </li><li> Detarmiño la concentración de proteínas por el método de Bradford. Evaluación de la calidad de proteínas por SDS-PAGE utilizando BSA como estándares. Si la integridad de la proteína no es satisfactoria, los procedimientos secundarios de purificación tales como filtración en gel o intercambio iónico puede ser utilizado. (Por ejemplo, hemos utilizado una columna G-75 para purificar más Sepharosa fusión GST-proteína). </li></ol> 2. Cultivo de células y preparación de lisado celular <ol><li> Cultura de riñón de cría de hámster (BHK), células que sobre-expresan de forma estable CFTR-CFTR en peso y His10-o células BHK que transitoriamente sobre-expresan la bandera-LPA-2 en peso o la bandera de la LPA-2-ΔSTL, y de Madin-Darby de riñón canino (MDCK ) células que sobreexpresan establemente MRP2 en medio esencial mínimo de Eagle (MEM) que contienen 10% de suero fetal de ternero y penicilina / estreptomicina en matraces de poliestireno a 37 ° C con 5% de CO 2. </li><li> Cultura riñón embrionario humano 293 (HEK293) células que sobre-expresan de forma estable CFTR en peso y el CFTR His10 en Dulbecco&#39;S medio de Eagle modificado (DMEM) suplementado con fetal al 10% de suero bovino y penicilina / estreptomicina en matraces de poliestireno a 37 ° C con 5% de CO 2. </li><li> Lisar las células en tampón de lisis (PBS - 0,2% de Triton-X-100 suplementado con una mezcla de inhibidores de proteasas que contienen 1 m M de fluoruro de fenilmetilsulfonilo, 1 mg / ml de aprotinina, 1 mg / ml de leupeptina, 1 mg / ml de pepstatina); uso 500 l de tampón de lisis para cada plato 60-mm de Petri, y 1000 l de tampón de lisis para cada plato de Petri de 100 mm. </li><li> Rock the lisados ​​celulares durante 20 min a 4 ° C, y eliminar el material insoluble por centrifugación a 16.000 xg durante 15 min a 4 ° C. Determinar la concentración de proteína por el ensayo de Bradford. </li></ol> 3. En la Asamblea in vitro de un complejo macromolecular que contiene CFTR (CFTR PDZK1-MRP4) <ol><li> En 200 l de buffer de lisis (PBS - 0,2% de Triton X-100 + inhibidores de la proteasa), añadir 20 g purificado GST-MRP4-C-terminal50 bis (CT50) proteína de fusión. </li><li> Añadir varias cantidades (0-40 g) de su purificada-S-PDZK1 proteína de fusión. </li><li> Mezclar las dos proteínas en un mezclador rotatorio a 22 ° C durante 1-2 h. </li><li> Añadir 20 perlas de glutatión l (50% suspensión) en la mezcla de proteínas, y continuar mezclando durante otras 1. Este paso se conoce también como pares de enlace. </li><li> Durante este tiempo, preparar las HEK293 lisados ​​de células que sobreexpresan Bandera-CFTR (en peso) como se describió anteriormente en el Paso 2). </li><li> Lavar el complejo dos veces con tampón de lisis. Gira a 800 xg durante 1 minuto por cada lavado y cuidado aspirar el sobrenadante después de cada lavado. Tenga cuidado de no chupar las bolas en la parte inferior. </li><li> Añadir los anteriormente preparadas HEK293 lisados ​​celulares a las perlas, y mezcle suavemente a 4 ° C durante 3 horas (o durante la noche). </li><li> Lavar las perlas extensamente tres veces con tampón de lisis tal como se describe en el Paso 3,6). </li><li> Eluir las proteínas con 30 muestras de búfer l (5 x): 0,6 M<html> Tris-HCl (pH 6,8), 50% de glicerol, 2% de SDS, y 0,1% de azul de bromofenol; que contiene 5% β-mercaptoetanol. </li><li> Incubar en baño de agua 37 ° C durante 10-15 minutos, y giran a 5.000 rpm durante 30 s para precipitar las cuentas. </li><li> Cargar todo el eluato en 4-15% de gel. </li><li> Ejecutar SDS-PAGE durante aproximadamente 30-40 min. </li><li> Transferir bandas de proteína a la membrana de PVDF, durante 1,5 horas. </li><li> Bloque de la membrana en TBS-Tween que contenía 5% de leche sin grasa. </li><li> Incubar la membrana con el anticuerpo primario (tales como anti-IgG de CFTR, R1104) a 4 ° C, durante la noche. </li><li> Lavar la membrana con TBS-Tween durante 5 minutos, seis veces. </li><li> Incubar la membrana con el anticuerpo secundario (como cabra anti-ratón conjugado HRP-anticuerpo nd 2) durante 45 min a 22 ° C. </li><li> Lavar la membrana con TBS-Tween durante 5 minutos, seis veces. </li><li> Visualizar las bandas de proteínas a través de ECL. </li><li> Los datos representativos se muestran en la Figura 1. </li></ol>jove_title &quot;&gt; 4. Los resultados representativos Un ejemplo de complejo macromolecular que contiene CFTR de señalización que se montó en vitro se muestra en la Figura 1. Un complejo macromolecular se formó entre MRP4 C-terminal de 50 aminoácidos (MRP4-CT50), los PDZK1 y de larga duración CFTR (Figura 1, abajo). La formación del complejo mayor dosis-dependiente con cantidades crecientes de la proteína intermediario, PDZK1 (Figura 1, parte inferior) 18. <img alt="Figura 1" src="/files/ftp_upload/4091/4091fig1.jpg" /> Figura 1. Una representación pictórica del ensayo complejo macromolecular (arriba). Un complejo macromolecular fue detectado in vitro con tres proteínas (GST-CT50-MRP4, Su-S-PDZK1, y la bandera CFTRwt) de una manera dosis-dependiente (abajo) 18. <table border="1"><tbody><tr><td></td><td><strong&gt; CFTR NHERF1-β 2 AR (ref. 14) </td><td> CFTR NHERF2-LPA 2 (ref. 15) </td><td> CFTR proteínas PDZ-MRP 2 (ref. 17) </td><td> CFTR PDZK1-MRP4 (ref. 16) </td></tr><tr><td> Cuentas de afinidad </td><td> La amilosa de resina </td><td> S-proteína de agarosa </td><td> La amilosa de resina </td><td> El glutatión agarosa </td></tr><tr><td> Purificada proteína-1 </td><td> MBP-β 2 AR CT </td><td> Su-S-CFTR CT </td><td> MBP-CFTR CT </td><td> GST-MRP4 CT </td></tr><tr><td> Purificada proteína-2 </td><td> GST-NHERF1 </td><td> GST-NHERF2 </td><td> GST-proteínas PDZ </td><td> Su-S-PDZK1 </td></tr><tr><td> Purificada proteína-3 (o lisados ​​celulares) </td><td> CFTR en peso o CFTR His10 (BHK o lisados ​​de células HEK) </td><td> BanderaLPA-2-en peso o la bandera de la LPA-2-ΔSTL (lisados ​​de células BHK) </td><td> MRP2 (lisados ​​de células MDCK) </td><td> Purificada Bandera-CFTR en peso o CFTR His10-(o lisados ​​celulares) </td></tr><tr><td> Anticuerpo </td><td> IgG anti-CFTR </td><td> Anti-Flag HRP </td><td> Anti-IgG MRP2 </td><td> Anti-Flag HRP </td></tr></tbody></table> Tabla 1. Resumen de diversos complejos macromoleculares que contienen CFTR ensamblados in vitro.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+CFTR+Interactome+in+the+Macromolecular+Complexes.">Analysis of CFTR Interactome in the Macromolecular Complexes.</a> Met. Molecul. Med. 741, 255-270 (2011).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemokine+receptor+CXCR2+macromolecular+complex+regulates+neutrophil+functions+in+inflammatory+diseases.">A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.</a> J. Biol. Chem. , (2011).</li></ol>

No hay conflictos de interés declarado.

En este protocolo, se demostró un método para ensamblar in vitro y la detección de un complejo macromolecular que contiene CFTR señalización utilizando proteínas purificadas (o fragmentos de proteínas) y / o lisados ​​de células como se informó anteriormente 16-19,29,30. Para lograr los mejores resultados los siguientes puntos críticos durante el proceso de preparación requieren una atención especial: <ul><li> Es importante que el pH del tampón de elución se ajustó a 8,0 despu?...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> pGEX4T-1 vectores </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> anteriormente Amersham Biosciences </td></tr><tr><td> pMAL-C2 vector </td><td> New England BioLabs </td><td></td><td></td></tr><tr><td> pET30 vector </td><td> Merck Chemicals </td><td> 69077-3 </td><td> antes Novagen </td></tr><tr><td> Glutatión agarosa </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> La amilosa de resina </td><td> New England BioLabs </td><td> E8021S </td><td></td></tr><tr><td> Talon cuentas </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> glutatión reducido </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazol </td><td> Pescador </td><td> BP305-50 </td><td></td></tr><tr><td> maltosa </td><td> Pescador </td><td> BP684-500 </td><td></td></tr><tr><td> S-proteína de agarosa </td><td> Merck Chemicals </td><td> 69704-3 </td><td> antes Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> IgG anti-CFTR </td><td> Hechas a medida </td><td> R1104 </td><td> MAB reconociendo CFTR epítopo 722-734 de AA </td></tr><tr><td> Anti-IgG MRP2 </td><td> Chemicon International </td><td> MAB4148 </td><td> Ahora una parte de Millipore </td></tr></tbody></table> Tabla 2. Reactivos específicos y equipos.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Transmembrana de la fibrosis quística regulador de la conductancia (CFTR), un canal de cloruro encuentra principalmente en la membrana apical de las células epiteliales, juega un papel fundamental en la homeostasis de los fluidos 1-3 transepitelial. CFTR se ha implicado en dos enfermedades importantes: la fibrosis quística (FQ) 4 y la diarrea secretora 5. En CF, la síntesis o la actividad funcional de la CFTR Cl-canal se reduce. Este trastorno afecta a aproximadamente 1 de cada 2,500 caucásicos en los Estados Unidos 6. La actividad del CFTR excesiva también ha sido implicado en casos de toxina inducida diarrea secretora (por ejemplo, por la toxina del cólera y estable al calor enterotoxina de E. coli) que estimula la producción de AMPc o GMPc en el intestino 7. La acumulación de pruebas sugieren la existencia de interacciones físicas y funcionales entre CFTR y un número creciente de otras proteínas, incluyendo transportadores, canales iónicos, receptores, quinasas, fosfatasas, la señalIng. moléculas, y los elementos del citoesqueleto, y estas interacciones entre CFTR y sus proteínas de unión se ha demostrado que ser críticamente involucrada en la regulación mediada por CFTR transporte iónico transepitelial in vitro y también in vivo 8-19. En este protocolo, nos centraremos únicamente en los métodos que ayudan en el estudio de las interacciones entre la cola CFTR carboxilo terminal, que posee un motivo de unión a proteínas [conocido como PSD95/Dlg1/ZO-1 (PDZ) motivo], y un grupo de proteínas de andamiaje, que contienen un módulo de unión específica a que se refiere a dominios PDZ. Hasta ahora, varios diferentes PDZ proteínas de andamiaje se ha informado que se unen a la cola carboxilo terminal de CFTR con afinidades diferentes, tales como, las NHERF1 NHERF2, los PDZK1, los PDZK2, Cal (CFTR asociada a ligando), Shank2, y GRASP 20-27. El motivo PDZ en CFTR que es reconocido por PDZ proteínas de andamiaje son los últimos cuatro aminoácidos en el terminal C (es decir, 1477-DTRL-1480 en el ser humano CFTR) 20. Curiosamente,CFTR puede obligar a más de un dominio PDZ de ambos NHERFs y PDZK1, aunque con distintas afinidades 22. Esta multivalencia con respecto a CFTR unión ha demostrado ser de importancia funcional, lo que sugiere que las proteínas PDZ andamio puede facilitar la formación de complejos macromoleculares CFTR señalización para la señalización específicos / selectiva y eficiente en las células 16-18. Múltiples ensayos bioquímicos se han desarrollado para estudiar CFTR que implica interacciones de proteínas, como la co-inmunoprecipitación, ensayo de pull-down, por pares ensayo de unión, colorimétrico pares ensayo de unión, y el ensayo de ensamblaje complejo macromolecular 16-19,28,29 . Aquí nos centramos en las particularidades del procedimiento de montaje de un PDZ motivo dependiente de CFTR que contiene complejo macromolecular in vitro, que se utiliza ampliamente en nuestro laboratorio para estudiar proteína-proteína o de dominio de dominio-interacciones que implican CFTR 16-19,28,29.

Watch this Scientific Journal Video about In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes at JoVE.com

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Transmembrana de la fibrosis quística regulador de la conductancia (CFTR), un canal de cloro del epitelio, se ha informado de interactuar con varias proteínas y regular procesos celulares importantes, entre ellos los CFTR PDZ mediadas por motivo de las interacciones han sido bien documentados. Este protocolo describe los métodos que hemos desarrollado para montar un macromolecular CFTR PDZ-dependiente de señalización complejos<em&gt; In vitro</em&gt;.

Análisis in vitro de PDZ-dependientes de complejos macromoleculares de señalización CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

in-vitro-analysis-pdz-dependent-cftr-macromolecular-signaling

Research

JoVE Journal

Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

10.3K vistas.  Wayne State University School of Medicine. Transmembrana de la fibrosis quística regulador de la conductancia (CFTR), un canal de cloro del epitelio, se ha informado de interactuar con varias proteínas y regular procesos celulares importantes, entre ellos los CFTR PDZ mediadas por motivo de las interacciones han sido bien documentados. Este protocolo describe los métodos que hemos desarrollado para montar un macromolecular CFTR PDZ-dependiente de señalización complejos In vitro.

Video: In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Investigación

Biología

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

La detección de señalización de Efectos Complejos de aguas abajo de BMP4 uso In situ PLA, un ensayo de la ligadura de proximidad

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Un alto contenido de imágenes de flujo de trabajo para estudiar Grb2 complejos de señalización por clonación de expresión

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Mecánica estimulación inducida por calcio Propagación de Ondas en monocapas de células: el ejemplo de las especies bovina células endoteliales corneales

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólicas etiquetado y de membrana de fraccionamiento para Comparativo Proteómica Análisis de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensión celular

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Aislamiento y cultivo de ratón adulto cardiomiocitos de Señalización Celular y<em&gt; In vitro</em&gt; Hipertrofia Cardíaca

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

La medición de la tasa metabólica en<em&gt; Drosophila</em&gt; Mediante respirometría

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

En Vitro Reconstitución de complejos captadores de luz de Plantas y Algas

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.