我々は、国立衛生研究所を（1R21CA138373感謝し、この作品の金融支援のためのがん（SU2C）にスタンドアップ。HYのためのシドニーキンメル財団からのがん研究、キンメルScholarの賞の協会から2009年エリオン賞に感謝です。がん研究（SKF - 08 - 101）、および国立科学財団の学部初期のキャリア賞（NSF0954819）。

1。クローニングの考慮事項<ol><li> NheIとBamHIの制限部位と5&#39; -リン酸化によって挟ま関心のTMDを表す商業的に作製したオリゴヌクレオチドを順次消化pTox7（直接BamHI制限部位14日以降1塩基対の挿入によって私たちの研究室で修正された）（図3）に連結することができるBamHIおよびNheIで。例のオリゴヌクレオチドを​​以下に示します。 5&#39;ctagcTMDSEQUENCEg3&quot; 3&#39;gTMDSEQUENCEcctag5&quot; </li></ol> TMDシーケンスは、12から24残基（より短い配列はおそらくベクトル符号化された疎水性残基により伸長される）でなければなりません。ここで、逐次残渣の挿入とToxRに対するTMDの相対的な回転に付随する残基の欠失の結果TMD設計4つのバリエーションを作成する必要がある、インタフェースを調査するために。最後に15,16は 、アラビノース濃度は0.001と0.01％の間で変化させてください（w / v）の異なるTMD配列の間のβ-ガラクトシダーゼの信号の最大の違いが観察される濃度を識別するために、異なる発現レベルをテストすること、異なる親和性が最高の区別することができる条件を特定することをお勧めします。アラビノースと抗生物質、0.4mMのIPTGに加えて別のTMDSとの間の親和性の違いを高めるために使用することができます。 ToxRの測定は、少なくとも4連で実行する必要があります。全体の手順が異なるプラスミドの変換で少なくとも3回繰り返す必要があります。 2。細菌培養の成長<ol><li>静かに15mlの培養チューブに氷と転送に関するFHK12テントセル（200μl）を解凍します。プラスミドDNA（200 ng）を追加し、30分間氷上で細胞をインキュベートする。 </li><li> 42℃で90秒のためのインキュベーションにより、熱ショック細胞℃で2分間氷上でインキュベートしたC、。 </li><li> SOCメディア（800μl）を添加し、37℃でサンプルをインキュベート℃で1時間インキュベート振とうしながらC（300 rpm）を</li><li>三連で15 mlの培養管の形質転換混合物の50μlとクロラムフェニコール（30μg/ mlの）とアラビノース（0.0025％w / v）を、5 mlのLB培地に接種する。 37サンプルをインキュベート℃で20時間攪拌（300 rpm）し振とうしながら（文化の代わりに5μlを96ウェルプレートの培地100μlを接種するために使用することができます。このメソッドは、大量のサンプルを扱うときにエラーが若干高くなるものの、便利です。蒸発を避けるために、つながるとなるメディアとの最も外側の井戸を埋める、エラーが、サンプルのためにそれらを使用しないでください。最後に、蓋およびパラフィルムでプレート間のダブルラップジョイント）。 </li></ol> 3。 β-ガラクトシダーゼ活性の測定<ol><li>予熱プレートリーダー〜28℃の</li><li>上段（水層）をのみ取ることを確認しながら、大規模なピペットの先端でリザーバにZ -バッファを転送します。 96ウェルプレートのウェルに調製したばかりのZ-buffer/chloroformの100μlを移す。 4部プレートのウェルに各培養液5μlを移す。ブランクとなる4つのウェルから培養を省略します。 </li><li>細胞密度を決定するためにプレートのOD 595を測定します。 </li><li>プレートの全ウェルにZ-buffer/SDSの50μlを追加します。細胞をlyzeに10分間プレートリーダーでプレートを振る。細胞懸濁液は、溶解後に明確であり、必要に応じて揺れのステップを繰り返すことを確認してください。不完全な溶解はZ-buffer/chloroformが新しく調製されていない示唆している。 </li><li>たてのすべてのウェルにZ-buffer/ONPG準備50μlを加え20分間毎に30秒プレートリーダーと測定OD 405にプレートを返します。 </li><li>次式を（空白を減算することに注意してください）​​を使用してβ-ガラクトシダーゼ活性を算出する。 OD 405 /分の比率は、線形モデルのフィットを用いてOD 405の範囲0.0から1.0までのすべてのデータポイントを使用して計算する必要があります。 <img src="files/ftp_upload/2721/2721eq1.jpg" alt="式（1）" /> 別の日に記録したときにミラーユニットは異なることがあります。したがって、GPAなどの基準の構造は、各試験で測定する必要があります。その値はToxR値の正常化に使用することができます。 </li></ol> 4。タンパク質発現のための制御<ol><li>西洋では構成要素間でも、タンパク質の発現を確認するためにブロッティング行います。微量遠心機で三連の文化や遠心50μlの（2000回転、4分）を組み合わせる。ピペッティングにより上清を除去し、2 ×サンプルローディングバッファーに残留ペレットを再懸濁します。 </li><li>標準的な8％のゲルは7.5μLをロードし、1時間5分間125 Vで電気泳動を行う。転送後、抗MBP HRP標識抗体とインキュベートし、可視化、キメラタンパク質は時々48 kDaの周りに見られるいくつかの分解生成物と約70 kDaので観察される。内因性のMBPは、45 kDaの（図5を参照）で観察される。 </li></ol>5。適切な膜挿入のための制御マルトース結合タンパク質の欠損細胞株は、キメラTMD構築物の適切な膜の挿入を評価するために使用されます。唯一の炭素源としてマルトースと最少培地で増殖させた場合、正常にペリプラズムにあるマルトース結合タンパク質と膜積分発現産物を発現する細胞のみが成長することができます。 <ol><li> PD28のセルを（FHK12セルの説明と同様に）変換し、LB培地2 mlを接種する。 37細胞を成長° Cを一晩（300 rpm）し振とうしながら。 </li><li>ペレットは、3500 rpmで遠心分離により細胞を、10分、4℃と大きい先端または穏やかにボルテックスで穏やかにピペッティングによりPBS（2ml）に再懸濁により洗浄。ペレットは、細胞（上記のような）、二度目のPBSで洗浄、ペレット、最後にPBS（1 ml）に懸濁します。 </li><li> 37三連とインキュベートで5ミリリットル最少培地に接種するために再懸濁した細胞を25μlを使用して° C（300 rpm）し振とうしながら。プレートリーダーを用いて96ウェルプレートや読書に各サンプルの200μlを転送することにより、15〜25時間の間にOD 595の測定値、約2時間ごとにかかります。 </li></ol> 6。代表的な結果： 図4に示すように膜貫通ドメインのオリゴマー化の傾向を分析するToxR転写レポーターアッセイの使用の例。以前に我々はToxRを含む様々な技術によってmultispanning膜積分タンパク質の潜在的な膜タンパク質- 1（LMP - 1）からの膜貫通ドメインのオリゴマー化を検討した14の膜貫通ドメイン5つが（TM5）オリゴマー化する強い傾向を示すことが示された。これは、陽性対照に匹敵する高いミラーユニット、、GPA、十分に確立された二量化シーケンスで示されています。 TM5の有害な突然変異、D150Aは、オリゴマー化するシーケンスの能力を低下させる。 LMP - 1 TM1は大幅にオリゴマー化せず、ただブランク、非形質転換FHK12細胞のための信号の上に、非常に低いミラーユニットの信号を示す。 <img src="/files/ftp_upload/2721/2721fig1.jpg" alt="図1" /> ToxRのレポーターアッセイを描いた図は、1。漫画。膜貫通ドメイン（TMD）はToxR および LacZ の転写の活性化の二量体でオリゴマー化の結果を駆動する。 LacZの遺伝子産物は、β-ガラクトシダーゼは、オリゴマー化するTMDの傾向の尺度として定量することができる。 <img src="/files/ftp_upload/2721/2721fig2.jpg" alt="図2" /> 図2。光吸収種のo - nitrophenolate（ONP）の製造におけるβ-ガラクトシダーゼの結果によるONPGの加水分解。 <img src="/files/ftp_upload/2721/2721fig3.jpg" alt="図3" /> 図3。pToxR7のプラスミドマップ。 <img src="/files/ftp_upload/2721/2721fig4.jpg" alt="図4" /> 図4代表ToxR転写レポーターアッセイは、潜在的な膜蛋白質A - 1の膜貫通ドメインのオリゴマー化の傾向を分析する。膜貫通領域1（TM1）は唯一の弱い相互作用を示すながら貫通ドメイン5（TM5）は、強くoligomerizes。 TM5の突然変異のD150Aは大幅にオリゴマー化する能力が減少します。 GPAは、強い二量化のための正の制御シーケンスとして含まれています。空白は非形質転換FHK12セルを表します。 <img src="/files/ftp_upload/2721/2721fig5.jpg" alt="図5" /> 図5。タンパク質発現のウエスタンブロット。 <img src="/files/ftp_upload/2721/2721fig6.jpg" alt="図6" /> 図6。ペリプラズムへの適切な膜の挿入のために制御するPD28相補アッセイ。ネガティブコントロールは、マルトース結合タンパク質の欠損構造を表します。

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利害の衝突は宣言されません。

ToxR転写レポーターアッセイでは、オリゴマー化する可能性と膜貫通配列を同定するために手軽な方法です。相互作用が細菌の内膜内に発生しているので、このアッセイは、細胞膜模倣環境でシステムを研究の妥当性に関連する問題を回避することができます。単一のプラスミドに複数のTMDSのクローニングを容易に並列に行うことができ、全体のアッセイは96ウェルプレートフォーマットで?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> BamHI制限酵素</td><td>インビトロジェン</td><td> 15201023 </td><td>インビトロジェンの酵素は、代替のサプライヤーよりも効率的であることがわかった</td></tr><tr><td> NheI制限酵素</td><td>インビトロジェン</td><td> 15444011 </td><td>インビトロジェンの酵素は、代替のサプライヤーよりも効率的であることがわかった</td></tr><tr><td> 15 mLの培養管</td><td>フィッシャーサイエンティフィック</td><td> 14から956 - 1J </td><td></td></tr><tr><td> SOCメディア</td><td> Teknova </td><td> S0225 </td><td>適切なボリュームに構成し、オートクレーブにより滅菌。 </td></tr><tr><td> LB培地</td><td>シグマアルドリッチ</td><td> L7275 </td><td>適切なボリュームに構成し、オートクレーブにより滅菌。 </td></tr><tr><td>クロラムフェニコール</td><td>シグマアルドリッチ</td><td> CO 3位 78 </td><td>冷凍庫に保存されているエタノール30 mg / mLの原液</td></tr><tr><td>アラビノース</td><td>フルカ</td><td> 10839 </td><td>水中の2.5％ストック溶液（w / v）を冷凍庫に保存され</td></tr><tr><td>のNa 2 HPO 4 </td><td>シグマアルドリッチ</td><td> S9390 </td><td></td></tr><tr><td>のNaH 2 PO 4 </td><td>シグマアルドリッチ</td><td> S9638 </td><td></td></tr><tr><td>塩化カリウム</td><td> Mallinckrodtケミカルズ</td><td> 6858〜06 </td><td></td></tr><tr><td>のMgSO 4 · 7H 2 O </td><td>シグマアルドリッチ</td><td> 63138 </td><td></td></tr><tr><td>ドデシル硫酸ナトリウム（SDS） </td><td>シグマアルドリッチ</td><td> L6026 </td><td></td></tr><tr><td> 2 - ニトロフェニルβ- D -ガラクトピラノシド（ONPG） </td><td>シグマアルドリッチ</td><td> 73660 </td><td></td></tr><tr><td> Z -バッファ</td><td></td><td></td><td> 16.1グラムのNa 2 HPO 4 5.5グラムのNaH 2 PO 4 0.75グラムのKCl 0.246グラムのMgSO 4 1リットル、pH7.0にメイクアップ</td></tr><tr><td> Z-buffer/chloroform </td><td></td><td></td><td> 200mLのβ-メルカプトエタノール、2 mLのクロロホルム、Z -バッファ、20 mLに構成しています。 1分間、800 rpmで1分間遠心するための渦。各プレートのための新鮮ください。 </td></tr><tr><td> Z-buffer/SDS </td><td></td><td></td><td> 10mLのZ -バッファに溶解した160 mgのSDS </td></tr><tr><td> Z-buffer/ONPG </td><td></td><td></td><td> 10mLのZ -バッファの40 mgをONPG。各プレートのための新鮮な確認</td></tr><tr><td> β-メルカプトエタノール</td><td>カルビオケム</td><td> 444203 </td><td></td></tr><tr><td>抗MBP抗体（HRP標識） </td><td> NEB </td><td> E8038S </td><td></td></tr><tr><td>マルトースと最少培地</td><td></td><td></td><td> 1 × M9塩、0.4％マルトース、1 mg / mLのチアミン、2mMのMgSO 4を </td></tr><tr><td> 96ウェル平底プレート</td><td>ザルスタット</td><td> 83.1835.300 </td><td></td></tr><tr><td>プレートリーダー</td><td>ベックマンコールター</td><td> DTX880マルチモード検出器</td><td></td></tr><tr><td>ウォーターバス</td><td> VWRI </td><td> 89032-204 </td><td></td></tr><tr><td>振とう培養器</td><td> FormaScientific </td><td></td><td></td></tr></tbody></table>

transmembrane domain oligomerization propensity determined by toxr assay

リン脂質二重層で単にアンカーとしてのタンパク質の膜貫通ドメインの極端に簡略化ビューには、誤りであるので、長く持っています。多くの場合、膜貫通タンパク質は、アクションの非常に高度なメカニズムを進化させてきた。膜タンパク質はその構造と機能を調節することができる1〜3つの方法は、構造化された膜貫通のオリゴマーを形成する、疎水性ヘリックスの直接および特定の連絡先があります。4,5多くの最近の作業は6,7は 、それにもかかわらず。優先的に水溶液とドライブの蛋白質の関連付け、異なる分子間力と比較して膜の環境で見つかったアミノ酸の分布に焦点を当てているタンパク質の膜貫通ドメインにおける分子認識の研 ​​究は、まだのものに遅れている水溶性の領域。主要なハードルが残っている：貫通オリゴマー化が達成できる驚くべき特異性と親和性にもかかわらず、それらの関連の8の直接測定が困難です。内在性膜蛋白質機能の研究に適用される伝統的な方法論が検討中のシーケンスの固有の不溶性によって妨げられることができる。膜貫通ドメインを表す合成ペプチドを研究から得られた生物物理学的な洞察は、有用な構造的洞察を提供することができます。しかし、細胞膜を模倣するためにこれらの研究で使用される洗剤ミセルまたはリポソーム系の生物学的関連性はしばしば疑問視され、ペプチドは、これらの条件の下で天然様の構造を採用していないと各機能の動作は、真のネイティブ膜内で作用機序を反映していない？天然のリン脂質二重層の膜貫通配列の相互作用を研究するためには、LangoschラボはToxR転写レポーターアッセイを開発した。関心の9膜貫通ドメインがペリプラズムとToxRの位置については、マルトース結合タンパク質とのキメラタンパク質として発現されるレポートを提供するオリゴマー化のレベル（図1）の。 過去10年間で、いくつかの他のグループ（例：エンゲルマン、DeGrado、シャイ）さらにこのToxRのレポーターアッセイを最適化し適用した。10-13、様々なToxRアッセイは、細胞膜のタンパク質間相互作用をテストするためのゴールドスタンダードとなっています。我々はここに主にLangoschによって開発されたプロトコルを、次の我々の研究室で行われた代表的な実験操作を示す。この一般的に適用できる方法では、E.の膜貫通ドメインの自己会合の解析に有用です。 β-ガラクトシダーゼの生産はTMDオリゴマー化の傾向を評価するために使用される大腸菌 、。 TMD誘発性二量体化すると、ToxRはβ-ガラクトシダーゼのLacZ遺伝子のアップレギュレーションを引き起こすCTXのプロモーターに結合する。比色読み出しはlyzed細胞へのONPGを添加することによって得られる。光吸収の種O - nitrophenolate（ONP）（図2）の産生におけるβ-ガラクトシダーゼの結果によるONPGの加水分解。

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

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

一回膜貫通ドメイン（TMDS）のオリゴマー化の傾向を評価するための効率的な手順が説明されています。 ToxRに融合されたTMDからなるキメラタンパク質は、大腸菌のレポーター株に発現されています。 TMD誘発性オリゴマーはToxR、転写およびレポータータンパク質の生産、-ガラクトシダーゼの活性化の二量体化を引き起こす。

ToxRアッセイによって決定される膜貫通ドメインのオリゴマー化の傾向

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

transmembrane-domain-oligomerization-propensity-determined-toxr

Research

JoVE Journal

Biology

15.1K ビュー.  University of Colorado at Boulder. 一回膜貫通ドメイン（TMDS）のオリゴマー化の傾向を評価するための効率的な手順が説明されています。 ToxRに融合されたTMDからなるキメラタンパク質は、大腸菌のレポーター株に発現されています。 TMD誘発性オリゴマーはToxR、転写およびレポータータンパク質の生産、-ガラクトシダーゼの活性化の二量体化を引き起こす。

ビデオ: Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

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

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

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

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

で嚢胞性線維症膜コンダクタンス制御因子タンパク質の発現および精製サッカロマイセスセレビシエ

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

生物学

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

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

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

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

で表現する嚢胞性線維症膜コンダクタンス制御因子タンパク質の精製<em&gt;サッカロマイセス·セレビシエ</em

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

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

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

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

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

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

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

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

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

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

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

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

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

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

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

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

生細胞内のタンパク質断片相補アッセイ（PCA）によるゲノムワイドなタンパク質間相互作用のスクリーニング

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

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

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

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

膜貫通タンパク質の再構成、顕微鏡およびパッチクランプ研究のための巨大単膜小胞への電位依存性イオンチャネル、KvAP、

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

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

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

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

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

イントラ小胞体のCaの光学マッピング<sup&gt; 2+</sup&gt;とランゲンドルフ灌流ウサギの心臓における膜電位

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

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

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

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

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

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

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

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

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

以下のための遺伝学的および生化学的アプローチ<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;タンパク質のオリゴマーの評価：リアノジン受容体のケーススタディ

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

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

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

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

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

小胞体とミトコンドリアの相互作用の研究によって、<em&gt;その場で</em固定された細胞における&gt;近接ライゲーションアッセイ

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