私たちの仕事は、米国心臓協会（グレーター東南アジアアフィリエイト）昭和·助成金の0765185B、エルザU.パーディー財団研究助成金、ウェイン州立大学の学内のスタートアップ資金と血管研究所イシスイニシアティブ賞からの助成金によってサポートされています。 in vitroで CFTRの高分子複合体のアセンブリ内のこのメソッドは、もともと博士AP Naren（テネシー大学ヘルスサイエンスセンター）によって開拓された。

1。細菌における組換えHisタグ融合タンパク質の発現および精製<ol><li> CFTR、LPA 2、MRP2、MRP4、β2 AR、そしてNHERFs（フルレングスまたはPDZ1またはPDZ2ドメインのC-テイル（C末端にPDZモチーフを含む最後の50から100アミノ酸）の定義された領域を増幅するPCR法による）。 </li><li>そのようなMBP-β2 AR CT、MBP-CFTR CTとしてMBP融合タンパク質（詳細については、GST-融合タンパク質のpGEX4T-1ベクター（例えば、GST-NHERFsとして、GST-MRP4 CT）やpMal-C2ベクターにPCR産物をクローニングし）、および彼の-S-融合タンパク質（例えば、彼の-S-CFTR CT、彼の-S-PDZK1など）のpET30。 </li><li>プロテアーゼ欠損E.に変換大腸菌株（BL21-DE3）可能な組換えタンパク質の分解を最小限に抑えます。 </li><li>適切な抗生物質（例えばアンピシリンまたはカナマイシンなど）を含むルリア - ベルターニ培地（pH7.0）に37°Cで一晩培養する。 1:10に一晩培養物を希釈し、37℃でさらに2時間のために育つ℃、で0.5〜1メートル、Mは 30℃で、次の4時間IPTGでduce 8000遠心分離によってペレットは、細胞×gで10分間4℃で</li><li> （50メートルMトリス-HCl、pH8.0で、1メートルM EDTA、1メートルM PMSF、10％ショ糖）がリゾチーム（1 mg / mL）は、0.2％トリトンX-100、およびプロテアーゼを含むショ糖バッファーで細胞を溶解阻害剤。細胞ペレットは、培養液1 Lに由来するための20mLを使用しています。 </li><li> 4℃で30分間ロータリーシェーカーに混ぜる</li><li> 4℃で30分間20,000×gで遠心透明な上清を収集します。 </li><li>透明な上清中には、以下の樹脂/アガロースビーズ（50％ショ糖バッファ内のスラリー）を1 mLを追加します。 <ul><li> GST融合タンパク質のグルタチオンアガロースビーズ。 </li><li>彼の-Sの融合タンパク質のタロンビーズ。 </li><li> MBP融合タンパク質のアミロース樹​​脂。 </li></ul></li><li> 4℃で4時間混合°回転シェーカー上でC。 </li><li> 2マイルのために混合し、1×PBS（15mL）に再懸濁することによりビーズを洗浄2分、および廃棄上清を800×gで回転するN、。 6回のためにこの手順を繰り返します。 </li><li>それぞれの溶出バッファー（ビーズを1mLあたり2 mLの溶出バッファー）を用いて、ビーズからタンパク質を溶出します。 <ul><li> GST融合タンパク質の溶出バッファー：25メートルMのTris-HCl（pH 8.0）で、140メートルM NaCl、および20メートルMは、グルタチオンを減少させた。 </li><li>彼の融合タンパク質の溶出バッファー：20メートルMのTris-HCl（pH 8.0）で、500メートルM NaCl、および200メートルMイミダゾール。 </li><li> MBP融合タンパク質の溶出バッファー：20メートルMのTris-HCl（pH 8.0）で、200メートルMのNaCl、1メートルM EDTA、1メートルM DTT、10 M Mマルトース。 </li></ul></li><li> 4℃（可能なタンパク質の分解を避けるため）での1X PBSを2 Lに対して溶出したタンパク質を透析。 PBSを4回4時間毎に変更します。 -80℃で小分けとしてセントリコンフィルタ（10,000 MWカットオフ、ミリポア）とストアを使用してタンパク質を濃縮℃に</li><li> DETブラッドフォード法によるタンパク質濃度オコジョ。 SDS-PAGEは、標準としてBSAを用いてタンパク質の品質を評価します。タンパク質の整合性が十分でない場合は、そのようなゲルろ過やイオン交換などの二次精製手順を使用することができます。 （例えば、我々はさらにGST-融合タンパク質を精製するためにG-75セファロースカラムを使用している）。 </li></ol> 2。細胞培養と細胞ライセートの調製<ol><li>安定的に過剰発現CFTR-WTおよびCFTR-his10またはBHK細胞の一過性過剰発現フラグ-LPA 2重量またはフラグ-LPA 2ΔSTL、およびMadin-Darbyイヌ腎その文化ベビーハムスター腎臓（BHK）細胞（MDCK 37℃でポリスチレンフラスコ内で10％ウシ胎児血清およびペニシリン/ストレプトマイシンを含むイーグル最小必須培地（MEM）°5％CO 2とCの安定過剰発現MRP2た）細胞である。 </li><li>文化、ヒト胚性腎臓293（HEK293）細胞が安定的に過剰発現CFTR-WT及びダルベッコのCFTR-his10&#39;、5％CO 2で37℃、10％ウシ胎児血清およびペニシリン/ポリスチレンフラスコ中のストレプトマイシンを補充したの改変イーグル培地（DMEM）。 </li><li> Lysis Bufferで細胞を溶解（PBS -トリトン-X-100は1メートルMのフェニルメチルスルホニルフルオライドを含むプロテアーゼ阻害剤の混合物を添加した0.2％、1μg/ mLのアプロチニン、1μg/ mLのロイペプチン、1μg/ mLのペプスタチン）の使用各60 mmのペトリ皿、各100 mmのペトリ皿1000μL溶解バッファー500μL溶解バッファー。 </li><li>細胞は℃、4℃で15分間16,000遠心により不溶性物質×gを削除し4℃で20分間ライセートロックブラッドフォードアッセイによりタンパク質濃度を決定します。 </li></ol> 3 CFTR含有高分子複合体の in vitro総会で （CFTR-PDZK1-MRP4） <ol><li>溶解バッファー200μLのPBS（ - 0.2％トリトンX-100 +プロテアーゼ阻害剤）に、20μgの精製したGST-MRP4-C端子を追加する50 AA（CT50）の融合タンパク質。 </li><li>彼の-S-PDZK1融合タンパク質を精製した種々の量（0-40μg）を追加します。 </li><li> 22 1-2°C hのロータリーミキサーの2つのタンパク質を混ぜ合わせます。 </li><li>タンパク質混合物に20μLグルタチオンビーズ（50％スラリー）を追加し、別の1時間ミックスを続けています。この手順は、ペアワイズ結合と呼ばれています。 </li><li>この間、HEK293細胞ライセートを調製する、上記ステップ2で説明したように過剰発現したFlag-CFTR（WT））。 </li><li>溶解緩衝液で2回の複合体を洗ってください。各洗浄のために1分間800×gで遠心し、慎重にそれぞれの洗浄後の上清を吸引除去する。底にビーズを吸うしないように細心の注意を払ってください。 </li><li>ビーズに上記調製したHEK293細胞ライセートを追加し、軽く3時間（または一晩）を4℃で混合します。 </li><li>ステップ3.6）で説明したように溶解緩衝液で広範囲に三回ビーズを洗浄する。 </li><li> 30μLサンプルバッファー（5×）を使用してタンパク質を溶出：0.6 M<html>トリス-HCl（pH6.8）を、50％グリセロール、2％SDS、および青ブロモフェノール0.1パーセント、5％β-メルカプトエタノールを含む。 </li><li> 5000 10〜15分、スピン℃の水浴×gで30秒間ビーズを沈殿させた37にインキュベートします。 </li><li> 4から15までパーセントゲル上にすべての溶出液をロードします。 </li><li>約30〜40分間SDS-PAGEを実行します。 </li><li> 1.5時間、PVDF膜にタンパク質のバンドを転送します。 </li><li> 5％無脂肪牛乳を含むTBS-Tweenでの膜をブロックします。 </li><li> 4の一次抗体（例えば抗CFTR抗体、R1104など）の膜℃、一晩インキュベートします。 </li><li> 5分、6回TBS-Tweenで膜を洗浄します。 </li><li> 22℃で45分間二次抗体（例えば、ヤギ抗マウスHRP標識第2抗体など）で膜をインキュベート</li><li> 5分、6回TBS-Tweenで膜を洗浄します。 </li><li> ECLを介してタンパク質バンドを可視化することができます。 </li><li>代表的なデータは、 図1に示されています。 </li></ol>jove_title &quot;&gt; 4。代表的な結果 in vitroで組み立てられたCFTR含有高分子のシグナル伝達複合体の例を図1に示されています。高分子複合体は、MRP4 C末端50アミノ酸（MRP4-CT50）PDZK1と、フルレングスCFTR（ 図1 下 ）の間に形成されました。複合体形成は、仲介蛋白質PDZK1（ 図1、 下 ）18の増加量の用量依存的に増加した。 <img alt="図1" src="/files/ftp_upload/4091/4091fig1.jpg" /> 図1：高分子複合体アッセイの絵画表現（ 上 ）。高分子複合体は、用量依存的に三つのタンパク質（GST-MRP4-CT50、彼の-S-PDZK1と、フラグ-CFTRwt）（ 底部 ）18を用いてin vitro で検出されました。 <table border="1"><tbody><tr><td></td><td><strong&gt; CFTR-NHERF1-β2 AR（文献14） </td><td> CFTR-NHERF2-LPA 2（参考文献15） </td><td> CFTR-PDZタンパク質-MRP 2（参考文献17） </td><td> CFTR-PDZK1-MRP4（文献16） </td></tr><tr><td> アフィニティービーズ </td><td>アミロース樹​​脂</td><td> S-タンパク質アガロース</td><td>アミロース樹​​脂</td><td>グルタチオンアガロース</td></tr><tr><td> タンパク質-1を精製した </td><td> MBP-β2 AR CT </td><td>彼の-S-CFTR CT </td><td> MBP-CFTR CT </td><td> GST-MRP4 CT </td></tr><tr><td> タンパク質-2を精製した </td><td> GST-NHERF1 </td><td> GST-NHERF2 </td><td> GST-PDZタンパク質</td><td>彼の-S-PDZK1 </td></tr><tr><td> （または細胞溶解液）タンパク質A-3を精製 </td><td> CFTR-WTまたはCFTR-his10（BHKまたはHEK細胞溶解物） </td><td>フラグ-LPA 2重量またはフラグ-LPA 2ΔSTL（BHK細胞ライセート） </td><td> MRP2（MDCK細胞溶解液） </td><td>フラグ-CFTR-WTまたはCFTR-his10（または細胞ライセート）を精製した</td></tr><tr><td> 抗体 </td><td>抗CFTR抗体</td><td>抗Flag HRP </td><td>抗IgG抗体MRP2 </td><td>抗Flag HRP </td></tr></tbody></table> in vitroで組み立てられた様々なCFTR含有高分子複合体の表1にまとめ。

<ol>
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B., Li, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessory+protein+facilitated+CFTR-CFTR+interaction,+a+molecular+mechanism+to+potentiate+the+chloride+channel+activity.">Accessory protein facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity.</a> Cell. 103, 169-179 (2000).</li><li>Sun, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3KARP+mediates+the+association+of+ezrin+and+protein+kinase+A+with+the+cystic+fibrosis+transmembrane+conductance+regulator+in+airway+cells.">E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells.</a> J. Biol. Chem. 275, 29539-29546 (2000).</li><li>Cheng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Golgi-associated+PDZ+domain+protein+modulates+cystic+fibrosis+transmembrane+regulator+plasma+membrane+expression.">A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.</a> J. Biol. Chem. 277, 3520-3529 (1074).</li><li>Scott, R. O., Thelin, W. R., Milgram, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+PDZ+protein+regulates+the+activity+of+guanylyl+cyclase+C,+the+heat-stable+enterotoxin+receptor.">A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor.</a> The Journal of biological chemistry. 277, 22934-22941 (1074).</li><li>Lee, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+cystic+fibrosis+transmembrane+conductance+regulator+by+competitive+interactions+of+molecular+adaptors.">Dynamic regulation of cystic fibrosis transmembrane conductance regulator by competitive interactions of molecular adaptors.</a> The Journal of biological chemistry. 282, 10414-10422 (2007).</li><li>Gee, H. Y., Noh, S. H., Tang, B. L., Kim, K. H., Lee, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+DeltaF508-CFTR+trafficking+via+a+GRASP-dependent+unconventional+secretion+pathway.">Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway.</a> Cell. 146, 746-760 (2011).</li><li>Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+study+of+intermolecular+and+intramolecular+interactions+regulating+CFTR+function.">Methods for the study of intermolecular and intramolecular interactions regulating CFTR function.</a> Met. Molecul. Med. 70, 175-186 (2002).</li><li>Li, C., Roy, K., Dandridge, K., Naren, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+assembly+of+cystic+fibrosis+transmembrane+conductance+regulator+in+plasma+membrane.">Molecular assembly of cystic fibrosis transmembrane conductance regulator in plasma membrane.</a> The Journal of biological chemistry. 279, 24673-24684 (2004).</li><li>Li, C., Naren, A. 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>

利害の衝突が宣言されません。

このプロトコルでは、前述の16-19,29,30を報告した in vitroで組み立て、精製タンパク質（またはタンパク質断片）および/ ​​または細胞ライセートを用いて高分子シグナル伝達複合体を含むCFTRの検出方法を示した。最良の結果を達成するための準備プロセス中に次の重要なポイントは、特別な注意が必要です。 <ul><li>これは、溶出緩衝液のpHは、ステップ1）で説明し...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> pGEX4T-1ベクトル</td><td> GEヘルスケア</td><td> 28-9545-49 </td><td>旧アマシャムバイオサイエンス</td></tr><tr><td>やpMal-C2ベクトル</td><td>ニューイングランドバイオラボ</td><td></td><td></td></tr><tr><td> pET30ベクトル</td><td> EMDケミカルズ</td><td> 69077から3 </td><td>以前はNovagen社</td></tr><tr><td>グルタチオンアガロースビーズ</td><td> BDバイオサイエンス</td><td> 554780 </td><td></td></tr><tr><td>アミロース樹​​脂</td><td>ニューイングランドバイオラボ</td><td> E8021S </td><td></td></tr><tr><td>タロンビーズ</td><td>クロンテック</td><td> 635501 </td><td></td></tr><tr><td>還元グルタチオン</td><td> BDバイオサイエンス</td><td> 554782 </td><td></tD&gt; </tr><tr><td>イミダゾール</td><td>フィッシャー</td><td> BP305-50 </td><td></td></tr><tr><td>マルトース</td><td>フィッシャー</td><td> BP684-500 </td><td></td></tr><tr><td> S-タンパク質アガロース</td><td> EMDケミカルズ</td><td> 69704から3 </td><td>以前はNovagen社</td></tr><tr><td>抗Flag HRP </td><td>シグマ</td><td> A8592 </td><td></td></tr><tr><td>抗CFTR抗体</td><td>カスタムメード</td><td> R1104 </td><td> AA 722から734でCFTRのエピトープを認識するモノクローナル抗体</td></tr><tr><td>抗IgG抗体MRP2 </td><td>ケミコンインターナショナル</td><td> MAB4148 </td><td>ミリポアの今一部</td></tr></tbody></table> 表2特異的試薬および装置。

in vitro analysis of pdz-dependent cftr macromolecular signaling complexes

嚢胞性線維症膜コンダクタンス制御因子（CFTR）、主に上皮細胞の頂端膜に位置するクロライドチャネルは、1〜3上皮液の恒常性に重要な役割を果たしている。嚢胞性線維症（CF 4）及び分泌性下痢5：CFTRは、二つの主要な疾患に関与している。 CFでは、CFTRのCl-チャネルの合成または機能活性が低下します。この疾患は、米国6の約1 2500の白人に影響を与えます。過度のCFTR活性はまた、腸内7でcAMPまたはcGMP産生を刺激する毒素によって誘導される分泌性下痢（例えば、コレラ毒素と熱安定性大腸菌エンテロトキシンによる）の症例に関与している。 証拠を蓄積すると、CFTRとトランスポーター、イオンチャネル、受容体、キナーゼ、ホスファターゼ、信号を含む他のタンパク質の増加との間の物理的および機能的相互作用の存在を示唆る分子および細胞骨格要素、およびCFTRとその結合蛋白質との間でこれらの相互作用が非常にin vitroおよびin vivoでも8-19に CFTR媒介上皮イオン輸送の調節に関与していることが示されている。このプロトコルでは、メソッドだけに焦点を当て、そのタンパク質結合モチーフを持っているCFTRカルボキシル末端尾部の間の相互作用の研究を支援し、[モチーフPSD95/Dlg1/ZO-1（PDZ）と呼ばれる] PDZドメインと呼ばれる特定の結合モジュールが含まれている足場タンパク質のグループ。これまでのところ、いくつかの異なるPDZ足場タンパク質は、そのようなNHERF1などの様々な親和性、NHERF2、PDZK1、PDZK2、CAL（CFTR関連リガンド）、Shank2とCFTRのカルボキシル末端尾部に結合し、20から27を把握することが報告されている。 PDZ足場タンパク質によって認識されているCFTR内PDZモチーフはC末端（すなわち、ヒトCFTRの1477-DTRL-1480）20最後の4つのアミノ酸である。興味深いことに、CFTRは、様々な親和性を22とはいえ、NHERFsとPDZK1両方の複数のPDZドメインをバインドすることができます。 CFTRの結合に関しては、この多価は、PDZ足場タンパク質は、細胞16から18の特定/選択的かつ効率的なシグナリングのためのシグナル伝達複合体CFTRの高分子の形成を促進する可能性が示唆された、機能的意義のあることが示されている。 複数の生化学的なアッセイは、CFTR-含むそのような共免疫沈降、プルダウンアッセイでは、ペアワイズ結合アッセイ、ペアワイズ結合アッセイ比色し、高分子の複雑なアセンブリアッセイ16-19,28,29としてタンパク質相互作用を研究するために開発されている。ここでは、CFTR 16-19,28,29を含むタンパク質-タンパク質またはドメインドメインの相互作用を研究する我々の研究室で広く使用され、in vitroでのPDZモチーフに依存したCFTR含有高分子複合体を組み立てるの詳細な手順に焦点を当てています。

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

嚢胞性線維症膜コンダクタンス制御因子（CFTR）、上皮クロライドチャネルは、様々なタンパク質と相互作用し、重要な細胞プロセスを調節することが報告されているそれらの間でCFTR PDZモチーフを介する相互作用は十分に文書化されています。このプロトコルは、我々は複雑なシグナル伝達PDZ-依存性のCFTRの高分子を組み立てるために開発された方法について説明します。<em&gt; in vitroで</em&gt;。

PDZ-依存性のCFTRの高分子シグナル伝達複合体のin vitroでの解析で

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 ビュー.  Wayne State University School of Medicine. 嚢胞性線維症膜コンダクタンス制御因子（CFTR）、上皮クロライドチャネルは、様々なタンパク質と相互作用し、重要な細胞プロセスを調節することが報告されているそれらの間でCFTR PDZモチーフを介する相互作用は十分に文書化されています。このプロトコルは、我々は複雑なシグナル伝達PDZ-依存性のCFTRの高分子を組み立てるために開発された方法につ...

ビデオ: 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.

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

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

生物学

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.

使用BMP4のエフェクター-錯体のダウンストリームシグナリングの検出その場で PLA、近接ライゲーションアッセイ

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.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

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.

発現クローニングによりGrb2のシグナル伝達複合体を研究するために高コンテンツイメージングワークフロー

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.

細胞単層に機械的刺激によって誘発されるカルシウム波の伝播：ウシ角膜内皮細胞の例

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.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

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.

細胞シグナリングのための成体マウスの心筋細胞の単離と文化<em&gt; in vitroで</em&gt;心肥大

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.

代謝率の測定に<em&gt;ショウジョウバエ</em&gt;呼吸計測を使用した

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.

植物や緑藻の光捕集複合体のin vitro再構成では、

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.