Nosso trabalho foi suportado por concessões do American Heart Association (Grande Afiliado Sudeste) A partir de doações em ajuda-0765185B, a Elsa U. Pardee Foundation bolsa de investigação, e Wayne State University fundo de inicialização intramural e Cardiovascular Research Institute prêmio Iniciativa de Isis. Este método de in vitro conjunto complexo macromolecular CFTR foi originalmente pioneira pelo Dr. AP Naren (University of Tennessee Health Science Center).

1. Expressão e Purificação de Proteínas de Fusão recombinantes Tagged em bactérias <ol><li> Amplificar regiões definidas do C-caudas (os últimos 50-100 aminoácidos contendo os motivos PDZ em C-terminal) para CFTR, LPA 2, MRP2, MRP4, β 2 AR, e NHERFs (comprimento completo ou PDZ1 ou PDZ2 domínios ) por metodologia de PCR. </li><li> Clonar os produtos de PCR em pGEX4T-1 vector para GST-proteínas de fusão (tal como GST-NHERFs, GST-MRP4 CT), pMAL-C2 vector para MBP de fusão de proteínas (tais como MBP-β 2 AR CT, MBP-CFTR CT ), e pET30 para His-S-proteínas de fusão (como o seu-S-CFTR CT, His-S-PDZK1). </li><li> Transformar num E. protease deficiente coli estirpe (BL21-DE3) para minimizar a degradação da proteína recombinante possível. </li><li> Crescer a cultura durante a noite a 37 ° C em meio Luria-Bertani meio (pH 7,0) contendo antibióticos apropriados (tais como a ampicilina ou canamicina). Dilui-se a cultura durante a noite a 1:10 e crescer durante mais 2 h, a 37 ° C. Emduzir com 0,5-1 m M IPTG para a próxima 4 horas a 30 ° C. Sedimentar as células por centrifugação a 8000 × g durante 10 min a 4 ° C. </li><li> Lisar as células em tampão de sacarose (50 m M de Tris-HCl, pH 8,0, 1 m M de EDTA, 1 m M de PMSF, e 10% de sacarose) contendo lisozima (1 mg / mL), Triton 0,2% X-100, e protease inibidores. Use 20 mL de sedimento de células proveniente de 1 L de cultura. </li><li> Misturar num agitador rotativo durante 30 min a 4 ° C. </li><li> Girar a 20.000 xg durante 30 min a 4 ° C. Recolher o sobrenadante claro. </li><li> Para o sobrenadante claro, adicionar 1 mL de resina nas seguintes / agarose (suspensão de 50% em tampão de sacarose): <ul><li> Pérolas de glutationa agarose para proteínas de fusão GST. </li><li> Contas Talon para His-S proteínas de fusão. </li><li> Amilose resina para proteínas de fusão MBP. </li></ul></li><li> Mistura-se durante 4 horas a 4 ° C em um agitador rotativo. </li><li> Lavar os grânulos por ressuspensão em PBS 1x (15 mL), misturando durante 2 min, girando a 800 × g durante 2 min, e descartar o sobrenadante. Repita este passo para seis vezes. </li><li> Eluir a proteína a partir das pérolas usando tampão de eluição respectivo (2 mL de tampão de eluição por 1 ml de esferas). <ul><li> Tampão de eluição para as proteínas de fusão GST: 25 m M de Tris-HCl (pH 8,0), 140 m M de NaCl, e 20 m M glutationa reduzida. </li><li> Tampão de eluição de proteínas sua fusão: 20 m M de Tris-HCl (pH 8,0), 500 m M de NaCl, e 200 m M imidazol. </li><li> Tampão de eluição para proteínas de fusão MBP: 20 m M de Tris-HCl (pH 8,0), 200 m M de NaCl, 1 m M de EDTA, 1 m M de DTT, e 10 m M maltose. </li></ul></li><li> Dialisar as proteínas eluídas contra 2 L de 1x PBS a 4 ° C (para evitar a degradação da proteína possível). Alterar PBS a cada 4 horas, durante quatro vezes. Concentra-se a proteína utilizando um filtro de Centricon (10.000 cutoff MW, Millipore) e armazenada como pequenas alíquotas a -80 ° C. </li><li> Detarminho concentração da proteína pelo método de Bradford. Avaliar a qualidade de proteína por SDS-PAGE utilizando BSA como padrão. Se a integridade da proteína não for satisfatório, os processos de purificação secundários, tais como filtração em gel ou de permuta iónica podem ser utilizados. (Por exemplo, utilizou-se um G-75 coluna de Sepharose para purificar ainda mais GST-proteína de fusão). </li></ol> 2. Cultura de Células e Preparação lisado celular <ol><li> Cultura rim de hamster bebé (BHK) que de forma estável sobre-expresso CFTR-wt e CFTR-his10 ou células BHK que transitoriamente sobre-express-Flag LPA 2-peso ou Bandeira-LPA 2-ΔSTL e Madin-Darby rim canino (MDCK ) as células que expressam de forma estável sobre-MRP2 em meio essencial mínimo de Eagle (MEM) contendo 10% de soro fetal de vitelo e penicilina / estreptomicina em frascos de poliestireno a 37 ° C com 5% de CO 2. </li><li> Cultura de rim embrionário humano 293 (HEK293) células que de forma estável sobre-expresso CFTR-wt e CFTR-his10 em Dulbecco&#39;Média s modificado de Eagle (DMEM) suplementado com 10% de soro fetal bovino e penicilina / estreptomicina, em frascos de poliestireno a 37 ° C com 5% de CO 2. </li><li> Lisar as células em tampão de lise (PBS - 0,2% de Triton-X-100 suplementado com uma mistura de inibidores de protease contendo 1 M de fluoreto de fenilmetilsulfonil m, 1 ug / ml de aprotinina, 1 ug / mL de leupeptina, 1 ug / mL de pepstatina); utilização 500 uL de tampão de lise para cada placa de Petri de 60 mm, e 1000 uL de tampão de lise para cada placa de Petri de 100 mm. </li><li> Balançar a célula lisados ​​durante 20 min a 4 ° C, e remover o material insolúvel por centrifugação a 16.000 xg durante 15 min a 4 ° C. Determinar a concentração de proteína pelo ensaio de Bradford. </li></ol> 3. Em Assembléia Vitro de um complexo macromolecular contendo CFTR (CFTR-PDZK1-MRP4) <ol><li> Em 200 uL de tampão de lise (PBS - 0,2% de Triton-X 100 + inibidores da protease), adicionar 20 ug de GST-purificada MRP4-C-terminal50 aa (CT50) proteína de fusão. </li><li> Adicionar várias quantidades (0-40 ug) de Purificou His-S-PDZK1 proteína de fusão. </li><li> Misturar as duas proteínas num misturador rotativo a 22 ° C durante 1-2 h. </li><li> Adicionar 20 pérolas de glutationa microlitro (50% de lama) na mistura de proteínas, e continuar a misturar por mais 1 h. Este passo é também referido como pares de ligação. </li><li> Durante este tempo, preparar os lisados ​​de células HEK293 que superexpressam a bandeira-CFTR (em peso) como acima descrito no Passo 2). </li><li> Lavar o complexo duas vezes com tampão de lise. Girar a 800 × g durante 1 min para cada lavagem e aspirar cuidadosamente o sobrenadante após cada lavagem. Tenha cuidado para não sugar as contas no fundo. </li><li> Adicionar os preparados acima HEK293 lisados ​​de células para os grânulos, e misturar suavemente a 4 ° C durante 3 h (ou durante a noite). </li><li> Lavar os grânulos extensivamente três vezes com tampão de lise, conforme descrito no Passo 3,6). </li><li> Eluir as proteínas utilizando 30 uL Tampão de amostra (5 ×): 0,6 M<html> Tris-HCl (pH 6,8), glicerol a 50%, SDS a 2%, e 0,1% de azul de bromofenol; contendo 5% de β-mercaptoetanol. </li><li> Incubar a 37 ° C banho de água durante 10-15 min, e giram a 5000 × g durante 30 s para precipitar os grânulos. </li><li> Carregar todo o eluído em gel de 4-15%. </li><li> Executar SDS-PAGE para cerca de 30-40 min. </li><li> Transferir bandas de proteína para a membrana PVDF, durante 1,5 horas. </li><li> Bloquear a membrana em TBS-Tween contendo gordura do leite a 5% não. </li><li> Incubar a membrana com o anticorpo primário (tais como anti-CFTR IgG, R1104) a 4 ° C, durante a noite. </li><li> Lava-se a membrana com TBS-Tween durante 5 min, seis vezes. </li><li> Incubar a membrana com anticorpo secundário (tal como o anticorpo de cabra anti-rato conjugado com HRP nd 2) durante 45 min a 22 ° C. </li><li> Lava-se a membrana com TBS-Tween durante 5 min, seis vezes. </li><li> Visualizar as bandas de proteínas através de ECL. </li><li> Os dados representativos são mostrados na Figura 1. </li></ol>jove_title &quot;&gt; 4. resultados representativos Um exemplo de CFTR contendo complexo macromolecular de sinalização que foi montado in vitro é mostrado na Figura 1. Um complexo macromolecular foi formado entre MRP4 C-terminal 50 aminoácidos (MRP4-CT50) e PDZK1, e de comprimento completo CFTR (Figura 1, parte inferior). A formação do complexo aumentou dose-dependente com quantidades crescentes de proteína do intermediário, PDZK1 (Figura 1, parte inferior) 18. <img alt="A Figura 1" src="/files/ftp_upload/4091/4091fig1.jpg" /> Figura 1. Uma representação pictórica do ensaio complexo macromolecular (topo). Um complexo macromolecular foi detectada in vitro com três proteínas (GST-MRP4-CT50, His-S-PDZK1, e-Flag CFTRwt) de uma forma dependente da dose (inferior) 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-PDZ proteínas-MRP 2 (ref. 17) </td><td> CFTR-PDZK1-MRP4 (ref. 16) </td></tr><tr><td> Contas de afinidade </td><td> Resina de amilose </td><td> S-proteína de agarose </td><td> Resina de amilose </td><td> Glutationa agarose </td></tr><tr><td> Purificou-proteína-1 </td><td> MBP β-2 AR CT </td><td> His-S-CFTR CT </td><td> MBP-CFTR CT </td><td> GST-MRP4 CT </td></tr><tr><td> Purificou-2 de proteína </td><td> GST-NHERF1 </td><td> GST-NHERF2 </td><td> GST-PDZ proteínas </td><td> His-S-PDZK1 </td></tr><tr><td> Proteína purificada-3 (ou lisados ​​de células) </td><td> CFTR-wt ou CFTR-his10 (BHK ou lisados ​​de células HEK) </td><td> BandeiraLPA-2-em peso ou bandeira-LPA 2-ΔSTL (lisados ​​de células BHK) </td><td> MRP2 (lisados ​​de células MDCK) </td><td> Purificou-Flag CFTR-wt ou CFTR-his10 (ou lisados ​​de células) </td></tr><tr><td> Anticorpo </td><td> Anti-IgG CFTR </td><td> Anti-Flag HRP </td><td> Anti-IgG MRP2 </td><td> Anti-Flag HRP </td></tr></tbody></table> Resumo Tabela 1. De vários complexos de CFTR contendo macromoleculares montadas in vitro.

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Neste protocolo que demonstraram um método para a montagem in vitro e detecção de um complexo macromolecular CFTR contendo sinalização utilizando proteínas purificadas (ou fragmentos de proteínas) e / ou lisados ​​celulares como relatado anteriormente 16-19,29,30. Para alcançar melhores resultados os seguintes pontos críticos durante o processo de preparação requerem especial atenção: <ul><li> É importante que o pH do tampão de eluição ser ajustado para 8,0 após a adição d...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> pGEX4T-1 vector </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 Química </td><td> 69077-3 </td><td> anteriormente Novagen </td></tr><tr><td> Esferas de agarose de glutationa </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Resina de amilose </td><td> New England Biolabs </td><td> E8021S </td><td></td></tr><tr><td> Contas Talon </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> glutationa reduzida </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> maltose </td><td> Pescador </td><td> BP684-500 </td><td></td></tr><tr><td> S-proteína de agarose </td><td> Merck Química </td><td> 69704-3 </td><td> anteriormente Novagen </td></tr><tr><td> Anti-Flag HRP </td><td> Sigma </td><td> A8592 </td><td></td></tr><tr><td> Anti-IgG CFTR </td><td> Feito sob encomenda </td><td> R1104 </td><td> mAb reconhecendo CFTR epítopo em aa 722-734 </td></tr><tr><td> Anti-IgG MRP2 </td><td> Chemicon International </td><td> MAB4148 </td><td> Agora uma parte da Millipore </td></tr></tbody></table> Tabela 2. Reagentes específicos e equipamentos.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

A fibrose cística reguladora da condutância transmembrana (CFTR), um canal de cloreto localizados principalmente nas membranas apicais das células epiteliais, desempenha um papel crucial na homeostasia de fluidos transepitelial 1-3. CFTR tem sido implicado em duas doenças principais: fibrose cística 4 (CF) e secretora diarreia 5. Na FC, a síntese ou a actividade funcional do CFTR Cl-canal é reduzida. Esta doença afecta cerca de 1 em 2500 caucasianos nos Estados Unidos 6. Actividade CFTR excessiva também tem sido implicado em casos de toxina induzida por diarreia secretória (por exemplo, por toxina da cólera e enterotoxina calor coli estável E.) que estimula a produção de cAMP ou cGMP no intestino 7. Evidências sugerem a existência de interações físicas e funcionais entre CFTR e um número crescente de outras proteínas, incluindo os transportadores, canais iônicos, receptores, quinases, fosfatases, sinalmoléculas ing, e os elementos do citoesqueleto, e estas interacções entre CFTR e as suas proteínas de ligação têm sido mostrados para ser criticamente envolvidos na regulação mediada por CFTR transporte de iões transepitelial in vitro e também in vivo 8-19. Neste protocolo, vamos nos concentrar apenas sobre os métodos que auxiliam no estudo das interações entre CFTR cauda carboxilo terminal, que possui um motivo de ligação às proteínas [referido como PSD95/Dlg1/ZO-1 (PDZ) motivo], e um grupo de andaime proteínas, que contêm um módulo de ligação específica referida como domínios PDZ. Até agora, várias proteínas diferentes PDZ andaime têm sido relatados para se ligar ao terminal carboxilo da cauda de CFTR, com afinidades diferentes, tais como NHERF1 e NHERF2 e PDZK1 e PDZK2, CAL (CFTR-associated ligando), Shank2, e agarrar 20-27. O motivo PDZ dentro CFTR que é reconhecido pelo PDZ andaime proteínas são os últimos quatro aminoácidos no terminal C (ie, 1477-DTRL-1480 em CFTR humano) 20. Curiosamente,CFTR pode ligar mais do que um domínio PDZ de ambos os NHERFs e PDZK1, embora com diferentes afinidades 22. Este multivalência com respeito à ligação CFTR foi mostrado para ser de importância funcional, sugerindo que as proteínas PDZ andaime pode facilitar a formação de complexos macromoleculares CFTR sinalização para a sinalização específicas / selectiva e eficaz em células 16-18. Vários ensaios bioquímicos têm sido desenvolvidos para estudar CFTR-envolvendo interacções proteína, tais como co-imunoprecipitação, suspenso ensaio, pares ensaio de ligação, por pares de ensaio colorimétrico de ligação, e doseamento macromolecular montagem complexa 16-19,28,29 . Aqui vamos nos concentrar sobre os procedimentos detalhados de montagem de um PDZ motivo dependente da CFTR macromolecular contendo complexo in vitro, que é usado amplamente por nosso laboratório para estudar proteína-proteína ou domínio domínio interações envolvendo 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

A fibrose cística regulador da condutância transmembranar (CFTR), um canal de cloreto epitelial, tem sido relatado para interagir com várias proteínas e regulam importantes processos celulares, entre eles os CFTR PDZ motivo mediadas-interacções têm sido bem documentados. Este protocolo descreve métodos desenvolvidos para montar um macromolecular CFTR PDZ-dependente sinalização complexo<em&gt; In vitro</em&gt;.

Análise in vitro de PDZ-dependentes Complexos Macromoleculares CFTR Sinalização

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 visualizações.  Wayne State University School of Medicine. A fibrose cística regulador da condutância transmembranar (CFTR), um canal de cloreto epitelial, tem sido relatado para interagir com várias proteínas e regulam importantes processos celulares, entre eles os CFTR PDZ motivo mediadas-interacções têm sido bem documentados. Este protocolo descreve métodos desenvolvidos para montar um macromolecular CFTR PDZ-dependente sinalização complexo In vitro.

Vídeo: 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.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

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

Biologia

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.

Detecção de Sinalização de Efeitos Complexos Downstream de BMP4 Usando In situ PLA, um Ensaio Ligadura Proximidade

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

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Colheita e Cryo-resfriamento cristais de proteínas de membrana crescida no mesofases lipídicas para a determinação da estrutura de Cristalografia Macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

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.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

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.

Um fluxo de trabalho de imagem de alta de conteúdo para estudar Grb2 Complexos de Sinalização por clonagem de expressão

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.

A estimulação mecânica induzida por cálcio Propagação de Ondas em monocamadas de células: O Exemplo de Bovinos endotélio corneano

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.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

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.

Isolamento e Cultura de rato adulto Cardiomyocytes de sinalização celular e<em&gt; In vitro</em&gt; A 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.

A medição da taxa metabólica em<em&gt; Drosophila</em&gt; Usando respirometria

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.

In Vitro Reconstituição de Complexos de Light-colheita de plantas e algas verdes

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