CGは、欧州連合（EU）の地平線の下で、欧州連合（EU）セブンス枠組み計画（マリー・キュリーのキャリアの統合n˚8304162）からと欧州研究評議会（ERC）から、アジャンス国立デラRECHERCHE（ANR-13-JSV1-0008）からの助成金によってサポートされています2020年の研究と技術革新プログラム（ERC開始グラントn˚639300）。

磁気ビーズに1リガンド抱合注：リガンド結合は、2.8μmの直径（原液濃度が10 8ビーズ/ mLで、30mgのビーズ/ mL）で超常磁性トシル活性化ビーズを使用して実行されます。以下のプロトコルを、60 mm組織培養プレートにおいて80％コンフルエンシーまで増殖させたMRC-5細胞に相当する約2×10 5細胞のサンプルに基づいています。それに応じて異なる合流で異なるサイズまたは細胞のプレートを使用している場合は、ビーズおよび試薬の音量を調整します。セル当たり2ビーズを持っているために、超常磁性ビーズの量を使用してください。したがって、4×10 5ビーズを60mmプレートに必要とされます。 <ol><li>徹底的に少なくとも30秒をボルテックスすることにより、元のバイアル中のビーズを再懸濁します。 1.5 mLのマイクロチューブに小分けし再懸濁し、超常磁性ビーズ（4×10 6ビーズに相当）の40μLを。 </li><li> 磁気separatiにチューブを置きます溶液から磁気ビーズを分離するためにスタンドに。 、上清を捨て磁気分離スタンドからチューブを取り外し、1 mLの0.1 Mリン酸ナトリウムpH7.4の中でビーズを再懸濁します。 <ol><li> 1 mLの0.1 Mリン酸ナトリウム（pH7.4）で二回繰り返して洗浄ステップ。 </li></ol></li><li>最終洗浄後、1 mLの0.1 Mリン酸ナトリウムpH7.4の中でビーズを再懸濁します。 </li><li>ビーズを含有する1 mLの0.1 Mリン酸ナトリウムpH7.4に100μgのウシフィブロネクチン（FN）、または任意の他の細胞接着受容体リガンドを結合し、ピペッティングにより混合します。 </li><li> FNまたは陰性対照のためのBSA、ポリ-D-リジンまたはアポトランスフェリンと他の特異的リガンドを置換することによって、前の手順を繰り返します。 </li><li>必要に応じて、ゲル分析のために、架橋効率の分析のためのリガンド溶液の10μLのアリコートを取り、濃縮Laemmliサンプルバッファーの適切な量と混合。 </li><li>ロータに37℃で12〜24時間のためのソリューションを含むリガンドとビーズをインキュベートします。 </li><li>反応はせいぜい10から20秒間、超音波処理（連続39 Wの電力）を発生した後、ビーズ凝集体が現れた場合。 </li><li>磁気分離スタンドを用いてビーズを分離し、残りの溶液を吸引し、1mLのPBS / 0.2％BSAのpHは7.6ソリューションを追加します。 37℃でローター上で1時間インキュベートします。 </li><li> 1mLのPBS / 0.2％BSAをpHが7.6で二回磁石を用いた洗浄ビーズ。 1 mLのPBS / 0.2％BSAのpHは7.6で再懸濁ビーズ。 </li><li>超音波処理ビーズは、凝集物はせいぜい20〜30秒間表示されます。 </li><li>必要に応じて、（濃縮されたLaemmliサンプルバッファーの適切な量と混合）ウエスタンブロットによって結合効率を分析するために、10μLのアリコートを削除します。 </li><li> 1ヶ月まで4℃で細胞アッセイまたはストアビーズに​​進みます。 </li><li>必要に応じて、磁気ビーズにFN架橋を分析するためにクーマシーブルーでSDS-PAGEゲルおよび染色を実行します。 </li></ol>背に接着受容体に結合したリガンドでコーティングされたビーズ上の張力軍の適用に関する事項細胞の表面<ol><li>適切な成長培地（10％FCSを補充したDMEM通常4.5グラム/ LのD-グルコース）、60ミリメートルの組織培養皿上で培養接着細胞が80％コンフルエントに達するまで。 </li><li>調製非変性非イオン性溶解緩衝液（20mMのトリスHCl pH7.6の、150mMのNaCl、2mMのMgCl 2を、0.1％NP-40）及びチルマイクロチューブ。 </li><li> 1 mLのPBS / 0.2％BSAのpHは7.6でセクション1.10を再懸濁からリガンドでコーティングしたビーズをペレット化。暖かい培地と渦の5ミリリットルへのリガンドでコーティングしたビーズ溶液100μLを加えます。 </li><li> 60mm培養皿中の培地を吸引し、5mLにビーズを補充した暖かい増殖培地を追加します。 </li><li>ビーズが沈降し、細胞に接着することを可能にする細胞培養条件（37℃、5％CO 2）下で20分間インキュベートします。ビーズは、食作用によって内在化することができるので、20分を超えないことが重要です。 </li><li>必要に応じて、10倍または20倍オブジェを使用して光学顕微鏡でビーズを観察ctiveと少し取り付け、フローティングビーズとを区別するための皿を振とうすることにより、ビーズの付着を確認してください。 </li><li>インキュベーター内で培養皿を維持しながら、上面に取り付けられたラウンド38ミリメートルのネオジム磁石と蓋のための通常の皿のふたを交換します。磁石は、蓋の下面に配置された2つの小さい13ミリメートルネオジム磁石によって適所に保持されます。磁石は非常に強力であるため、慎重に操作します。 </li><li>細胞培養条件で所望の時間ポイントの張力（37℃、5％CO 2）に供した細胞をインキュベートします。 </li><li>処理後、氷の上に皿を置き、慎重に皿から媒体全体を吸引。 </li><li>細胞溶解緩衝液の300μLを追加し、氷上で10分間インキュベートします。予め冷却し1.5 mLのマイクロ遠心チューブに細胞スクレーパーと転送を使用して、溶解物を収集します。 </li><li>磁気分離スタンドを用いた磁気ビーズをペレット化し、新しいプレチルに、全細胞溶解物を転送1.5 mLのマイクロ遠心チューブを編。さらなる分析のために-20℃でこの画分を保管してください。 </li><li> 1mLの氷冷溶解緩衝液でビーズを3回洗浄。 、ビーズペレットに50μLのLaemmliサンプルバッファーを追加し、乾燥ブロックヒーターを使用して、95℃で5分間ピペッティングし、沸騰によって混ぜます。この画分は、単離された接着複合体が含まれています。 -20℃での生化学的解析や店舗に進みます。 </li><li>ビーズ分離（約300μL）後に得られた全細胞溶解物から、ウェスタンブロット分析のために、50μLのアリコートを削除します。 注：そのようなタンパク質 - タンパク質相互作用の研究（免疫沈降、GSTプルダウンアッセイ）またはGTPアーゼ活性実験などのさらなる生化学的研究のために使用することができる左250μL（溶解緩衝液を変更は、関心のGTPアーゼに応じて必要であり得ます）。 </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+molecular+processes+mediate+cellular+mechanotransduction.">Dynamic molecular processes mediate cellular mechanotransduction.</a> Nature. 475 (7356), 316-323 (2011).</li><li>Seo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mechanogenetic+Toolkit+for+Interrogating+Cell+Signaling+in+Space+and+Time.">A Mechanogenetic Toolkit for Interrogating Cell Signaling in Space and Time.</a> Cell. 165 (6), 1507-1518 (2016).</li></ol>

著者には、競合する金融利益を宣言しません。

ここで説明する方法は、細胞表面接着受容体に張力を適用し、それらのその後の精製を可能にする簡単な方法を構成しています。しかし、いくつかのステップは、効率的な接着精製および潜在的な最適化を行うことが重要である標的接着受容体に依存して行うことができます。私たちは、ユーザーが以下の発生する可能性のある潜在的な問題を提示します。 我々は、2.8μ...

後生動物では、機械的張力は、増殖、分化および生存1、2などの細胞プロセスの無数の調節を介して組織発達および恒常性を指示します。機械的張力は、細胞外マトリックスから生じ得るか、接着性細胞、サンプル細胞外マトリックス上に引っ張ると緊張に敏感な分子を介してその剛性をプローブアクトミオシン収縮機構を介して、それらの細胞外環境を生成することができます。張力に応じて、機械受容接着タンパク質は、複雑なシグナル伝達カスケードを誘発する立体配座の変化を受けます。次に、これらのシグナル伝達経路は、細胞外環境への細胞の挙動を調節、増殖、分化および生存を包含するmechanoresponseを調整します。このようなプロセスはすぐにメカのループにフィードバックするために短期的な期間（数秒から数分）で決済することができます<html>機械受容の構造を変更することによってnotransduction。例えば、インテグリンベースの接着は、Rho GTPアーゼによって媒介される細胞骨格リモデリング3、4、5を介して張力に応じて強化します。並行して、他のシグナル伝達経路は、最終的に細胞の運命6に影響を与える遺伝的プログラムを制御するための時間と日にわたって活性化されます。 、多くの研究は、細胞の決定論と病気の発症1、2上にマトリックス剛性の効果を強調しているのに対し、接着媒介メカノの正確な分子メカニズムはまだとらえどころのないまま。 様々なアプローチは、蛍光共鳴エネルギー移動（FRET）-tensionセンサ7、流れシステムを含む細胞挙動に対する細胞発生力又は外力の影響を研究するために開発されていますLASS = &quot;外部参照&quot;&gt; 8、コンプライアント基板9、磁気ピンセット、光ピンセット10と、原子間力顕微鏡（AFM）11。ここでは、特定の接着レセプターに適用引張力に応答してメカノ経路を特徴付けるために超常磁性ビーズを使用してプロトコルを提示します。超常磁性ビーズを磁場中に置かれたときに可逆的に磁化粒子です。いったん特定の受容体のリガンドでコーティングされ、これらのビーズは、細胞外加力の影響を研究するための強力なツールを提供します。 17、大部分は接着細胞の生化学的解析を容易にするための利点を提示する-この方法は、いくつかの研究3、5、12により確認されています。生化学分析に続いて同様のコラーゲンでコーティングされた磁気ビーズを使用して、初期の作品は、の増加を報告しましたテンション5、18、19に応答して、タンパク質チロシンリン酸化およびRhoAの活性化。以下に記載される方法はまた、インテグリン3の張力から下流のシグナル伝達経路を特徴づけるために、フィブロネクチン（FN）でコーティングされたビーズを使用されています。本研究では、Guilluyら。テンションがインテグリン接着複合体への2つのグアニンヌクレオチド交換因子（GEF）、LARGとGEF-H1の募集を通じてのRhoAを活性化することを示しました。それ以来、他の研究は、GEF-H1は、ここに記載の方法のロバスト性を実証し、異なる方法20,21を用いて、細胞生じる張力に応じて接着複合体に動員されることを示しました。その結果、活性化されたRhoAは、細胞骨格の再構築を介して、接着強化を促進することが示されました。このシステムはまた、Tの張力を探索するために使用されました細胞/細胞接着レセプターO。インテグリン関連する接着複合体12と同様にビンキュリン募集の増加を誘導したE-カドヘリンの細胞外ドメインで被覆した磁気ビーズ上に力を加えます。コリンズらは、PECAM-1に張力のアプリケーションは、インテグリンとRhoAの活性化13を促進することを観察しました。磁気ビーズを使用して、別の実験的なアプローチは、単離された核の張力の研究です。 nesprin-1核エンベロープタンパク質に対する抗体でコーティングされたビーズを使用して、核エンベロープ複合体は、それらが動的機械的張力22に応答して調節されることを示すために精製しました。これらの結果は、メカノ経路の研究では、このメソッドの力強さをサポートしています。フローまたは牽引力システムは、一般的な細胞プロセスを刺激しながら、さらに、磁気ビーズは、具体的には、受容体リガンドのいずれかを使用して、細胞接着受容体を標的と<suPクラス= &quot;外部参照&quot;&gt; 3、または細胞表面受容体13、15に対するモノクローナル抗体。 この方法の別の利点は、簡単なリガンドアフィニティ精製手順を介して接着複合体の単離であります。よく、細胞に対するリガンドでコーティングされたビーズの添加は接着受容体に結合し、いくつかの接着タンパク質23の動員を誘発することが知られています。リガンドでコーティングされた磁気ビーズへの力のさらなる適用は、さまざまなテンション依存性シグナル伝達経路4、24を仲介する巨大分子のプラットフォームにこれらの接着複合体をオンにします。磁石を用いてビーズ濃度に続く細胞溶解は、接着プラットフォームの単離を可能にします。接着複合体を精製するために使用される他の方法は、既に付着細胞に使用されてきました。それらは、タンパク質 - タンパク質相互作用を節約するために化学的架橋を組み合わせます洗剤および剪断流または超音波処理20、21、25、26、27、28による細胞溶解ステップ。最後のステップは、接着複合体を含む得られた腹側形質膜のコレクションです。これらの方法とは異なり、磁気ビーズを選択的接着受容体の特定のファミリーを標的とすることにより、細胞接着複合体の高い精製度を可能にします。磁気ビーズは、既にリガンドでコーティングされたマイクロビーズ29、30に取り付けられた非付着性細胞内接着複合体を精製するために使用されてきました。力は、短い持続時間（分秒）に適用される模倣生物学的状況下に記載の方法。したがって、精製された接着複合体の分子組成の両方を調査するための強力なツールを提供し下流の機械受容シグナル伝達経路。 ここでは、接着表面タンパク質に引張力を適用するために磁気ビーズを使用するための詳細な実験プロトコルを提示します。永久ネオジム磁石は、培養皿の表面の上に配置されます。シングル2.8μmの磁気ビーズ上の力は一定（約30〜40 PN）31となるように、磁石の磁極面は6ミリメートルの高さに配置されています。張力刺激の持続時間は、対象の分子と活性化のその時間スケールに応じてオペレータによって決定されます。細胞は、最終的に接着複合体は、磁石を用いてビーズ分離によって精製され、生化学分析が処理され、溶解されます。このプロトコルは、リガンドでコーティングされた超常磁性ビーズの準備、および生化学的分析に続いて、磁石を通して緊張のアプリケーションが含まれています。さらに、当社は、張力がインテグリンベースのADHに適用されることを実証するデータの代表的なサンプルを提供しますesions接着リモデリングを誘導し、タンパク質チロシンリン酸化を変化させます。

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the upper face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">DX88-N52</td>
			<td style="width:375px;">grade N52 dimension: 1 1/2&#34; dia. x 1/2&#34; thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the lower face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">D84PC-BLK</td>
			<td>grade N42 dimension: 1/2&#34; dia. x 1/4&#34; thick Black Plastic Coated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dynabeads M280 Tosylactivated</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">14203</td>
			<td>superparamagnetic beads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DynaMag-2 Magnet</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fibronectin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">F1141-5MG</td>
			<td>Fibronectin from bovine plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Poly-D-Lysine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">P7280-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Apo-Transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">T1428-50MG</td>
			<td>Bovine Apo-Transferrin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">A7906-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">DMEM high glucose, GlutaMAX supplement, pyruvate&#160;</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:103px;">31966-021</td>
			<td>DMEM+GlutaMAX-I 500 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">60*15 mm culture dish</td>
			<td style="width:115px;">Falcon</td>
			<td style="width:103px;">353004</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

機械受容細胞表面接着複合体は、細胞がその周囲の機械的特性を感知することを可能にします。最近の研究では、接着部位での力感知分子、および系統特異的遺伝子の発現を調節し、表現型出力を駆動力依存性転写因子の両方を同定しました。しかし、生化学的経路に機械的張力を変換するシグナル伝達ネットワークは、とらえどころのないままです。細胞表面受容体に適用される機械的張力の際に係合したシグナル伝達経路を探索するために、超常磁性マイクロビーズを使用することができます。ここでは、細胞表面接着タンパク質に力を適用するために磁気ビーズを使用するためのプロトコルを提示します。このアプローチを使用して、様々な生化学的アプローチによってのみならず力依存性細胞質のシグナル伝達経路を調査することが可能であるが、リガンドでコーティングされたビーズに付着した接着複合体の磁気分離によってリモデリングを接着します。このプロトコルは、リガンド - コの準備を含んでいます超常磁性ビーズをated、と定義する引張力の適用は、生化学的分析が続きます。さらに、我々はインテグリンベースの接着に適用し、そのテンションを実証するデータの代表的なサンプルを提供する接着改造をトリガし、タンパク質チロシンリン酸化を変化させます。

技術の概略は 、 図1Aに示されています。リガンド結合後、磁気ビーズを20分間、細胞と共にインキュベートされ、その後、永久磁石は、時間の様々な量を約30〜40 pNでの引張力を適用するために使用されます。 図1bは、MRC5細胞接着受容体に結合した2.8μmのFNで被覆した磁気ビーズを示します。 細胞溶解後の超常磁性ビーズの洗浄ステ?...

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

彼らは機械的張力を送信し、組織の恒常性と開発に関与するシグナル伝達経路を開始するように、細胞表面の癒着は、メカノの中心です。ここでは、接着受容体へのリガンドで被覆された磁性マイクロビーズと力のアプリケーションを使用して、張力に応答して活性化される生化学的経路を切開するためのプロトコルを提示します。

磁気ビーズを使用した機械的張力に応答して細胞表面接着リフォームの分析

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

analyzing-cell-surface-adhesion-remodeling-response-to-mechanical

Research

JoVE Journal

Biology

8.4K ビュー.  Centre de recherche UGA - INSERM U1209 - CNRS UMR. 彼らは機械的張力を送信し、組織の恒常性と開発に関与するシグナル伝達経路を開始するように、細胞表面の癒着は、メカノの中心です。ここでは、接着受容体へのリガンドで被覆された磁性マイクロビーズと力のアプリケーションを使用して、張力に応答して活性化される生化学的経路を切開するためのプロトコルを提示します。 

ビデオ: Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

診断ツールを開発するマイクロエレクト​​ロメカニカルシステム（MEMS）の使用

生物学

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

集積光と原子間力顕微鏡による研究機械的刺激に対する細胞応答を生きる

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

タンパク質 - タンパク質相互作用を研究するために磁気ビーズを用いて液相アフィニティキャプチャーアッセイ：ポリオ-nanobodyの例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D共焦点顕微鏡を用いたゼブラフィッシュにおける顔面形態形成を分析する

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

ATP依存性クロマチンリモデリング酵素の活性を分析するための生化学的アッセイ

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

キイロタマホコリカビ急性の機械的刺激に対する応答の評価

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

MCF7細胞単層における細胞基質接着面積と細胞形状分布の定量化

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...