Notre travail a été soutenu par des subventions de l&#39;American Heart Association (Grand Sud-affiliation) Début-grant-in-aid 0765185B, l&#39;Elsa U. Pardee Fondation une subvention de recherche, et la Wayne State University intra-muros de fonds de démarrage et de recherche cardiovasculaire de l&#39;Institut d&#39;attribution Initiative Isis. Cette méthode in vitro CFTR assemblage complexe macromoléculaire a été initialement mis au point par le Dr AP Naren (Université du Tennessee Health Science Center).

1. Expression et purification de protéines recombinantes de fusion associés à des bactéries <ol><li> Amplification des régions définies du C-queue (les 50-100 derniers acides aminés contenant des motifs PDZ à extrémité C-terminale) pour CFTR, APL 2, MRP2, MRP4, β 2 AR, et NHERFs (pleine longueur ou PDZ1 ou PDZ2 domaines ) par l&#39;approche de PCR. </li><li> Cloner les produits de PCR en pGEX4T-1 vecteur de la TPS-protéines de fusion (comme la TPS-NHERFs, TPS-MRP4 CT), pMAL-C2 vecteur de protéines de fusion MBP (comme MBP-β 2 AR CT, MBP-CFTR CT ), et pour ses pET30-S-protéines de fusion (tel que son-S-CFTR CT, Son-S-PDZK1). </li><li> Transformer dans un E. déficiente en protéase souche de E. coli (BL21-DE3) pour minimiser la dégradation possible de protéines recombinantes. </li><li> Cultiver la culture pendant une nuit à 37 ° C dans du milieu Luria-Bertani (pH 7,0) contenant des antibiotiques appropriés (tels que l&#39;ampicilline ou la kanamycine). Diluer la culture de nuit à 1:10 et se développer pendant encore 2 h à 37 ° C. Dansduire avec 0,5-1 m M IPTG pour le prochain 4 h à 30 ° C. Pellet les cellules par centrifugation à 8000 xg pendant 10 min à 4 ° C. </li><li> Lyse des cellules dans le tampon de saccharose (50 m M Tris-HCl, pH 8,0, 1 m M EDTA, 1 m M PMSF, et 10% de saccharose) contenant du lysozyme (1 mg / ml), 0,2% de Triton X-100, et de la protéase inhibiteurs. Utilisez 20 ml pour culot cellulaire provenant de 1 L de la culture. </li><li> Mélanger sur un agitateur rotatif pendant 30 min à 4 ° C. </li><li> Centrifuger à 20000 g pendant 30 min à 4 ° C. Recueillir le surnageant clair. </li><li> Dans le surnageant clair, ajouter 1 ml de résine les suivantes / agarose perles (coulis 50% dans le tampon de saccharose): <ul><li> Glutathion billes d&#39;agarose pour les protéines de fusion GST. </li><li> Talon perles pour Ses-S protéines de fusion. </li><li> Résine d&#39;amylose pour les protéines de fusion MBP. </li></ul></li><li> Mix pendant 4 h à 4 ° C sur un agitateur rotatif. </li><li> Laver les billes en remettant en suspension dans du PBS 1X (15 ml), mélanger pendant 2 min, tournant à 800 × g pendant 2 min, et élimination du surnageant. Répétez cette étape pour six fois. </li><li> Eluer la protéine à partir des billes en utilisant un tampon d&#39;élution respectifs (2 ml de tampon d&#39;élution pour 1 ml de billes). <ul><li> Tampon d&#39;élution des protéines de fusion GST: 25 m M Tris-HCl (pH 8,0), 140 m M de NaCl, et 20 m M glutathion réduit. </li><li> Tampon d&#39;élution pour ses protéines de fusion: 20 m M Tris-HCl (pH 8,0), à 500 m M de NaCl, et à 200 m M d&#39;imidazole. </li><li> Un tampon d&#39;élution pour des protéines de fusion MBP: 20 m M de Tris-HCl (pH 8,0), 200 m M de NaCl, 1 m M d&#39;EDTA, de 1 m M DTT, et 10 m M maltose. </li></ul></li><li> Dialyser les protéines éluées contre 2 L de PBS 1x à 4 ° C (pour éviter la dégradation des protéines du possible). Changer PBS toutes les 4 h, quatre fois. Concentrer la protéine à l&#39;aide d&#39;un filtre Centricon (10.000 MW de coupure, Millipore) et le magasin en tant que de petites aliquotes à -80 ° C. </li><li> Dethermine de la concentration en protéines par la méthode de Bradford. Évaluer la qualité des protéines par SDS-PAGE en utilisant la BSA comme normes. Si l&#39;intégrité de la protéine n&#39;est pas satisfaisante, les procédures de purification secondaires telles que la filtration sur gel ou d&#39;échange d&#39;ions peut être utilisé. (Par exemple, nous avons utilisé une colonne G-75 Sepharose pour purifier davantage la TPS-protéine de fusion). </li></ol> 2. Culture cellulaire et préparation lysat cellulaire <ol><li> Culture rein de bébé hamster (BHK) que de manière stable sur-expriment la protéine CFTR-poids et CFTR-his10 ou des cellules BHK que transitoirement sur-expriment le drapeau-APL 2-poids ou Drapeau-APL 2-ΔSTL, et Madin-Darby Canine Kidney (MDCK cellules) que de manière stable sur-express MRP2 à Eagle du milieu essentiel minimum (MEM) contenant 10% sérum de veau foetal et de la pénicilline / streptomycine dans des flacons en polystyrène à 37 ° C avec 5% de CO 2. </li><li> Culture embryonnaires humaines de rein 293 (HEK293) des cellules qui expriment de manière stable sur-CFTR-poids et CFTR-his10 dans Dulbecco«Moyen s de Eagle modifié (DMEM) supplémenté avec 10% de sérum bovin et de la pénicilline / streptomycine dans des flacons en polystyrène à 37 ° C avec 5% de CO 2. </li><li> Lyse des cellules dans le tampon de lyse (PBS - 0,2% de Triton-X-100 complété par un mélange d&#39;inhibiteurs de la protéase contenant 1 m M phénylméthylsulfonyle fluorure, 1 ug / ml d&#39;aprotinine, 1 ug / ml de leupeptine, 1 ug / ml de pepstatine); utilisation 500 ul de tampon de lyse pour chaque plat de 60 mm de Petri, et 1000 ul de tampon de lyse pour chaque plat de 100 mm de Petri. </li><li> Rock the lysats cellulaires pendant 20 min à 4 ° C, et de supprimer l&#39;insoluble par centrifugation à 16.000 g pendant 15 min à 4 ° C. Déterminer la concentration en protéines par la méthode de Bradford. </li></ol> 3. Assemblage in vitro d&#39;un complexe macromoléculaire CFTR contenant (CFTR-PDZK1-MRP4) <ol><li> Dans 200 ul de tampon de lyse (PBS - 0,2% de Triton X-100 + inhibiteurs de la protéase), ajouter 20 pg purifiée GST-MRP4-C-terminal50 bis (CT50) protéine de fusion. </li><li> Additionner les montants différents (0-40 mg) de purifier Son-S-PDZK1 protéine de fusion. </li><li> Mélanger les deux protéines sur un mélangeur rotatif à 22 ° C pendant 1-2 h. </li><li> Ajouter 20 billes de glutathion pi (50% suspension) dans le mélange de protéines, et continuer à mélanger pour un autre h 1. Cette étape est aussi appelée par paire de liaison. </li><li> Pendant ce temps, préparer les lysats cellulaires HEK293 qui surexpriment la protéine CFTR-Drapeau (en poids) tel que décrit ci-dessus à l&#39;étape 2). </li><li> Laver le complexe à deux reprises avec un tampon de lyse. Centrifuger à 800 xg pendant 1 min pour chaque lavage et aspirer délicatement le surnageant après chaque lavage. Faites preuve de prudence pour ne pas sucer les billes dans le fond. </li><li> Ajouter les HEK293 préparés ci-dessus lysats cellulaires aux billes, et mélanger délicatement à 4 ° C pendant 3 h (ou toute la nuit). </li><li> Laver les billes extensive trois fois avec un tampon de lyse telles que décrites à l&#39;étape 3.6). </li><li> Éluer les protéines en utilisant 30 uL tampon d&#39;échantillon (5 ×): 0,6 M<html> Tris-HCl (pH 6,8), glycérol 50%, 2% de SDS, et 0,1% de bleu de bromophénol, contenant 5% β-mercaptoéthanol. </li><li> Incuber à 37 ° C bain-marie pendant 10-15 min, et de spin à 5000 × g pendant 30 s pour précipiter les perles. </li><li> Chargez tous l&#39;éluat sur un gel de 4-15%. </li><li> Exécuter SDS-PAGE pour environ 30-40 min. </li><li> Transfert des bandes de protéines à la membrane PVDF, pendant 1,5 heures. </li><li> Bloquer la membrane dans du TBS-Tween contenant 5% de matière grasse du lait non. </li><li> Incuber la membrane avec l&#39;anticorps primaire (comme les anti-IgG CFTR, R1104) à 4 ° C, pendant la nuit. </li><li> Laver la membrane avec du TBS-Tween pendant 5 min, à six reprises. </li><li> Incuber la membrane avec un anticorps secondaire (par exemple la chèvre anti-souris conjugué à HRP 2 anticorps e) pendant 45 min à 22 ° C. </li><li> Laver la membrane avec du TBS-Tween pendant 5 min, à six reprises. </li><li> Visualisez les bandes de protéines par ECL. </li><li> Les données représentatives sont présentés dans la figure 1. </li></ol>jove_title &quot;&gt; 4. Les résultats représentatifs Un exemple de complexe macromoléculaire contenant du CFTR de signalisation qui a été assemblé in vitro est représenté sur la figure 1. Un complexe macromoléculaire a été formé entre MRP4 C-terminales de 50 acides aminés (MRP4-CT50), les PDZK1 et pleine longueur du gène CFTR (figure 1, en ​​bas). La formation d&#39;un complexe augmentation de la dose-dépendante avec des quantités croissantes de la protéine intermédiaire, PDZK1 (Figure 1, en ​​bas) 18. <img alt="Figure 1" src="/files/ftp_upload/4091/4091fig1.jpg" /> Figure 1. La représentation graphique de l&#39;analyse complexe macromoléculaire (en haut). Un complexe macromoléculaire a été détectée in vitro avec trois protéines (TPS-MRP4-CT50, Son-S-PDZK1, et Flag-CFTRwt) d&#39;une manière dose-dépendante (en bas) 18. <table border="1"><tbody><tr><td></td><td><strong&gt; CFTR-NHERF1-β 2 AR (réf. 14) </td><td> CFTR-NHERF2-APL 2 (réf. 15) </td><td> CFTR-PDZ protéines-MRP 2 (réf. 17) </td><td> CFTR-PDZK1-MRP4 (réf. 16) </td></tr><tr><td> Perles d&#39;affinité </td><td> Résine d&#39;amylose </td><td> S-protéine d&#39;agarose </td><td> Résine d&#39;amylose </td><td> Le glutathion agarose </td></tr><tr><td> Protéine purifiée-1 </td><td> MBP-β 2 AR CT </td><td> Son-S-CFTR CT </td><td> MBP-CFTR CT </td><td> TPS-MRP4 CT </td></tr><tr><td> Protéine purifiée-2 </td><td> TPS-NHERF1 </td><td> TPS-NHERF2 </td><td> TPS-PDZ protéines </td><td> Son-S-PDZK1 </td></tr><tr><td> Protéine purifiée-3 (ou lysats cellulaires) </td><td> CFTR-poids ou CFTR-his10 (BHK ou lysats cellulaires HEK) </td><td> DrapeauLPA-2-poids ou Drapeau-APL 2-ΔSTL (lysats cellulaires BHK) </td><td> MRP2 (lysats de cellules MDCK) </td><td> Purifiée Drapeau-CFTR-poids ou CFTR-his10 (ou lysats cellulaires) </td></tr><tr><td> Anticorps </td><td> Anti-IgG CFTR </td><td> Anti-Flag HRP </td><td> Anti-IgG MRP2 </td><td> Anti-Flag HRP </td></tr></tbody></table> Tableau 1. Résumé des différents complexes macromoléculaires CFTR contenant assemblés 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>

Pas de conflits d&#39;intérêt déclarés.

Dans ce protocole, nous avons démontré une méthode in vitro le montage et la détection d&#39;une protéine CFTR contenant un complexe macromoléculaire de signalisation utilisant des protéines purifiées (ou fragments de protéines) et / ou de lysats cellulaires comme indiqué précédemment 16-19,29,30. Pour obtenir les meilleurs résultats sur les points suivants critiques au cours du processus de préparation nécessitent une attention particulière: <ul><li> Il est important que le pH du...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> pGEX4T-1 vecteur </td><td> GE Healthcare </td><td> 28-9545-49 </td><td> anciennement Amersham Biosciences </td></tr><tr><td> pMAL-C2 vecteur </td><td> New England Biolabs </td><td></td><td></td></tr><tr><td> pET30 vecteur </td><td> EMD Chemicals </td><td> 69077-3 </td><td> anciennement Novagen </td></tr><tr><td> Glutathion billes d&#39;agarose </td><td> BD Biosciences </td><td> 554780 </td><td></td></tr><tr><td> Résine d&#39;amylose </td><td> New England Biolabs </td><td> E8021S </td><td></td></tr><tr><td> Perles Talon </td><td> Clontech </td><td> 635501 </td><td></td></tr><tr><td> glutathion réduit </td><td> BD Biosciences </td><td> 554782 </td><td></td&gt; </tr><tr><td> imidazole </td><td> Pêcheur </td><td> BP305-50 </td><td></td></tr><tr><td> maltose </td><td> Pêcheur </td><td> BP684-500 </td><td></td></tr><tr><td> S-protéine d&#39;agarose </td><td> EMD Chemicals </td><td> 69704-3 </td><td> anciennement 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> Fait sur mesure </td><td> R1104 </td><td> mAb reconnaissant CFTR épitope à aa 722-734 </td></tr><tr><td> Anti-IgG MRP2 </td><td> Chemicon International </td><td> MAB4148 </td><td> Maintenant une partie de Millipore </td></tr></tbody></table> Tableau 2. Réactifs et équipements spécifiques.

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

Kystique régulateur de la conductance transmembranaire de la fibrose (CFTR), un canal chlorure situés principalement au niveau des membranes apicales des cellules épithéliales, joue un rôle crucial dans l&#39;homéostasie du fluide transépithélial 1-3. CFTR a été impliqué dans deux maladies majeures: la fibrose kystique (CF) 4 et la diarrhée sécrétoire 5. Dans FC, la synthèse ou l&#39;activité fonctionnelle de la protéine CFTR Cl-canal est réduite. Ce trouble affecte environ 1 sur 2500 personnes de race blanche aux États-Unis 6. Activité de la CFTR excessive a également été impliquée dans des cas de toxine induit une diarrhée sécrétoire (par exemple, par la toxine du choléra et de la chaleur entérotoxine stable de E. coli) qui stimule la production de GMPc AMPc ou dans l&#39;intestin 7. Les preuves qui s&#39;accumulent suggèrent l&#39;existence d&#39;interactions physiques et fonctionnelles entre CFTR et un nombre croissant d&#39;autres protéines, y compris les transporteurs, canaux ioniques, récepteurs, les kinases, les phosphatases, de signauxmolécules ING et éléments du cytosquelette, et ces interactions entre la protéine CFTR et de ses protéines de liaison ont été montré à la critique impliquée dans la régulation de CFTR-médiée transport des ions transépithélial in vitro et également in vivo 8-19. Dans ce protocole, nous nous concentrons uniquement sur les méthodes que l&#39;aide dans l&#39;étude des interactions entre la queue CFTR carboxyle terminal, qui possède un motif liaison aux protéines [dénommé PSD95/Dlg1/ZO-1 (PDZ) motif], et une un groupe de protéines échafaudage, qui contiennent un module de liaison spécifique dénommé domaines PDZ. Jusqu&#39;à présent, plusieurs différentes protéines d&#39;échafaudage PDZ ont été signalés à se lier à la queue carboxyle terminal de la protéine CFTR avec des affinités différentes, telles que NHERF1, les NHERF2, les PDZK1, les PDZK2, CAL (CFTR associée à un ligand), Shank2, et le GRASP 20-27. Le motif PDZ dans CFTR qui est reconnu par PDZ protéines d&#39;échafaudage est les quatre derniers acides aminés à l&#39;extrémité C-terminale (c.-à-1477-DTRL-1480 chez l&#39;homme CFTR) 20. Il est intéressant,CFTR peut se lier plus d&#39;un domaine PDZ des deux NHERFs et PDZK1, quoique avec plus ou moins d&#39;affinités 22. Cette polyvalence à l&#39;égard de la protéine CFTR contraignant a été démontré que de l&#39;importance fonctionnelle, ce qui suggère que PDZ protéines d&#39;échafaudage peuvent faciliter la formation de complexes de signalisation macromoléculaire CFTR pour la signalisation spécifiques / sélective et efficace dans les cellules 16-18. Multiples dosages biochimiques ont été développées pour étudier la protéine CFTR-impliquant les interactions entre protéines, telles que la co-immunoprécipitation, pull-down assay, par paire test de liaison, colorimétrique paires test de liaison, et le dosage assemblage macromoléculaire complexe 16-19,28,29 . Nous nous concentrons ici sur les procédures détaillées d&#39;assemblage d&#39;une PDZ motif-dépendante CFTR contenant macromoléculaire complexe in vitro, qui est largement utilisé par notre laboratoire pour étudier protéine-protéine ou un domaine à domaine interactions impliquant la protéine 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

Kystique régulateur de la conductance transmembranaire de la fibrose (CFTR), un canal chlorure épithéliale, a été signalé à interagir avec des protéines et de réglementer divers processus cellulaires importants, parmi lesquels les CFTR PDZ motif-médiées par les interactions ont été bien documentés. Ce protocole décrit les méthodes que nous avons développés pour assembler un macromoléculaire CFTR PDZ-dépendante complexe de signalisation<em&gt; In vitro</em&gt;.

L&#39;analyse in vitro de PDZ-dépendants complexes macromoléculaires CFTR de signalisation

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

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JoVE Journal

Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

10.3K vues.  Wayne State University School of Medicine. Kystique régulateur de la conductance transmembranaire de la fibrose (CFTR), un canal chlorure épithéliale, a été signalé à interagir avec des protéines et de réglementer divers processus cellulaires importants, parmi lesquels les CFTR PDZ motif-médiées par les interactions ont été bien documentés. Ce protocole décrit les méthodes que nous avons développés pour assembler un macromoléculaire CFTR PDZ-dépendante complexe de signalisation In vitro

Vidéo: 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.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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

Recherche

Biologie

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.

Détection de signalisation Effector-Complexes en aval de BMP4 aide In situ PLA, un dosage Ligature Proximité

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.

Analyse rapide de la génétique des épithélio-mésenchymateuse signalisation lors de la régénération des cheveux

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 workflow d&#39;imagerie à haute teneur pour étudier Grb2 complexes de signalisation par clonage d&#39;expression

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.

La stimulation mécanique induite par le calcium propagation des ondes dans des monocouches de cellules: l&#39;exemple de bovins cellules endothéliales de la cornée

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.

Marquage métabolique et la membrane de fractionnement pour comparative Analyse protéomique de<em&gt; Arabidopsis thaliana</em&gt; Cultures Suspension cellulaires

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.

Isolement et culture de cardiomyocytes adultes de souris pour Signalisation cellulaire et<em&gt; In vitro</em&gt; Hypertrophie cardiaque

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.

Mesure des taux métabolique dans<em&gt; Drosophila</em&gt; En utilisant Respirométrie

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.

Reconstitution in vitro de complexes collecteurs de lumière de plantes et d&#39;algues vertes

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.