我々は、資金調達のために化学と生化学のウースター工科大学部に感謝の意を表します。

1。細胞株のトランスフェクショントランスフェクションの方法1 （増加の主細胞のトランスフェクション効率のためのロンザのNucleofection） <ol><li>細胞は、最近の経過とトランスフェクションの前に、対数増殖期にである必要があります。 phophateで細胞を洗浄するトリプシン処理により生理食塩水（PBS）と収穫のセルをバッファ。 </li><li> 5分間1500 × gで遠心分離により細胞を収集する。 </li><li> 6ウェルプレートに滅菌カバースリップを追加。カバースリップは、強化された細胞接着のために被覆されてもよい。場所1.5培地の添加（この場合はダルベッコ改変イーグル培地（DMEM）、10％ウシ胎児血清（FBS））各ウェルに、インキュベーター内のメディアを平衡。 </li><li> （PBS）リン酸緩衝生理食塩水の最小限の量でトリプシン処理し、再懸して細胞を収集する。 </li><li>細胞をカウントし、サンプルあたり〜5 × 10 5細胞を提供するために再懸濁の正しい量を遠心してください。 </li><li>サンプルあたり100μl室温のNucleofectorソリューションで慎重に細胞を再懸濁します。 </li><li> 5μgのプラスミドDNAを細胞懸濁液100μLを組み合わせると、気泡を含まないように注意しながらNucleofectorキュベットにサンプルを移す。 </li><li>適切なNucleofectorプログラムを選択し（これは最低限の死亡率との最適なトランスフェクション効率を確立する前のテストが必要な場合があります）。 </li><li> Nucleofectorキュベットホルダに細胞/ DNA懸濁液を含むキュベットを挿入し、選択したプログラムを開始。 </li><li>完了すると、キュベットを取り出し、（6 - ウェルプレートから撮影）に予め平衡化培地の〜500μLを加える。 </li><li>すぐにウェルあたり1.5mlの最終容量で6ウェルプレートに試料を移す。細胞はコントロールのために、混合集団または個別にどちらに播種してください。 </li></ol>トランスフェクションの方法2 （細胞株に対してキアゲンEffecteneトランスフェクション） <ol><li>最初の200μLECのバッファへの各プラスミドDNA試料の0.8μgを添加することによりキアゲンEffectene試薬を用いてトランスフェクション複合体を準備します。 </li><li>各サンプルに6.4μLエンハンサーの試薬を追加、チューブをフリックすることで簡単にミックス、そして室温で2分間インキュベートする。 </li><li>チューブをフリックすることにより、各サンプル、ミックスに20μLEffectene試薬を追加し、室温で15分間反応させます。 </li><li>このインキュベーションの間、トランスフェクションのために関心のある2つの細胞株を準備する。最近継代細胞（&lt;80％コンフルエント）リン酸で一度洗浄は生理食塩水（PBS）を。バック1.5mLの新鮮な暖めたと平衡化した増殖培地（ダルベッコ変法イーグル培地（DMEM）この場合、10％ウシ胎児血清（FBS））を追加します。 </li><li>トランスフェクション複合体は、15分間インキュベートした後、各サンプルにメディアを1.2 mLを転送する。ピペッティング、そしてすぐに6 - ウェルプレートの二つのウェルの各々に複合体の〜700μLを転送して混合。滴下を追加し、プレートを旋回して静かに混和。細胞は少なくとも3時間トランスフェクションすることができます（セルのタイプによって異なる場合があります）。 </li><li>新しい6ウェルプレートに滅菌カバースリップを追加。カバースリップは、強化された細胞接着のために被覆されてもよい。 </li><li>細胞はトランスフェクションした後、各ウェルに約100μLトリプシン溶液を加える簡単に完全にコートの各ウェルをするためにプレートを攪拌し、そして即座にトリプシンをオフに吸引し、PBSで細胞を2回洗浄し、最小限のtrysinizationにより細胞を収穫。細胞が解除したら、1.5 mLの新鮮な培地を加​​える。 </li><li>この時点で、細胞を滅菌カバーグラスの上に混在するか、別の集団（コントロール用）のいずれかでプレーティングする必要があります。 </li><li>細胞は12時間で回復することができます。 </li></ol> 2。細胞融合<ol><li>細胞から培地を取り出して、PBSで2回洗浄する。 </li><li>この時点で、それはタンパク質合成を阻害するシクロヘキシミド（100μg/ mL）を持つ細胞を処置することをお勧めします。細胞は2時間インキュベートし、PBSで細胞を2回洗浄することができます。 </li><li>室温で125秒のために無血清DMEMに50％ポリエチレングリコール1000（PEG 1000）のソリューションに公開することにより細胞を融合させる。 </li><li> PEG 1000ソリューションを削除し、そして2mLの新鮮暖めと平衡メディアを追加するには、PBSで細胞を洗浄する。 </li><li> 24時間 - 細胞が18のためにインキュベートすることができます。 </li></ol>蛍光顕微鏡<ol><li>文化メディアを取り出し、そしてPBSで細胞を2回洗浄する。 </li><li>各ウェルに4％パラホルムアルデヒド溶液（PBS中）1 mLを加えることによって細胞を固定する。ゆっくりと15分間振とうする。 </li><li> PBSで2回固定された細胞を洗う。 </li><li>慎重にプレートからカバースリップを削除する、余分なPBSをオフに放出する。に顕微鏡封入剤の一滴（90％グリセロール、100mMのトリスpH7.5、2％DABCO（1,4 - diazabicoyclo -オクタン）を含むDAPI（4&#39;,6 -ジアミジノ-2 - フェニル）を搭載したスライド。反転</li><li>スライド上の過剰なマウンティングメディウムを外し、シールCOverは、マニキュアでスリップ。 </li><li>共焦点または落射蛍光顕微鏡を介して細胞を視覚化する。 </li></ol> 3。代表的な結果図2は、一次線維芽細胞およびH1299非小細胞肺癌細胞を用いて例のヘテロカリオンの実験を示しています。この実験の目的のタンパク質（チキン貧血ウイルス- VP3は）落射蛍光顕微鏡により可視化として、形質転換細胞の種類の一次細胞型および核局在化に主に細胞質局在が表示されます。タンパク質は、H1299細胞における主要な包皮繊維芽細胞（PFF）またはDsRedのGFPのどちらかへの融合（pEGFPを- N1ベクターを、Clontech社）（DsRedを- N1ベクターを、Clontech社）として表されます。自己融合を含むコントロールサンプル（上の2行）は、定常状態の局在パターンの変化なしで多核細胞が表示されます。このような変更は、特に核融合の条件またはsyncitiaの形成に感受性のために発生する可能性があります。ヘテロカリオンの融合（一番下の行）は、GFP融合主細胞由来の形質転換細胞の材料の導入に応じて、DsRedを融合蛋白質と蛋白質と共局在の核エントリを示しています。示した例は、両方fluoroprotein信号の存在によって示される2つだけのセル、各タイプの1つ、に起因する比較的まれなヘテロカリオンの融合です。局在遷移のこれらのタイプの検出に加えて、核 - 細胞質シャトル活動は簡単に両方の核内の単一の蛍光物質の存在によって検出することができます。午前1時01細胞融合を調べて、翻訳の抑制に依存するであろうときに決定のこのタイプは明らかである。 <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="図1" /> 図1。主な線維芽細胞とH1299の非小細胞癌細胞を用いたヘテロカリオンの方法のフローチャート。関心の遺伝子は、二つの蛍光タンパク質遺伝子のいずれかにインフレーム融合を生成するために複製されます。細胞株は、一過性のどちらかコンストラクトをトランスフェクトし、回復するために許可されています。ヘテロカリオンの形成は、さらにインキュベートしたポリエチレングリコール（PEG）による簡単な処理により誘導される。最後に、目的のタンパク質のサブ細胞内局在を蛍光顕微鏡の様々な形態を用いて観察される。 <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="図2" /> 図2。核細胞質人身売買とチキンの貧血ウイルスVP3タンパク質の活性を往復には、ヘテロカリオンの融合により視覚化した。一番上の行：DsRedタンパク質- VP3を発現している自己融合H1299細胞の代表画像中段：。。PEG融合後にGFP - VP3を発現している主要な包皮線維芽細胞（PFF）の代表落射蛍光画像下段：PFFとH1299細胞のヘテロカリオンの融合。黄色は、GFPとDsRedの信号の共局在を示している。細胞は、ライカAF6000E蛍光顕微鏡とライカのイメージングソフトウェアを使用して画像化した。

<ol>
	<li>Belaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPing+heterokaryons+to+analyze+nucleo-cytoplasmic+shuttling+of+yeast+proteins.">FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.</a> RNA. 12, 921-930 (2006).</li><li>Bhaumik, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+target+of+a+transcriptional+activator+revealed+by+fluorescence+resonance+energy+transfer.">In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.</a> Genes Dev. 18, 333-343 (2004).</li><li>Cohen, H. R., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+exhibits+nuclear+retention+and+exhibits+reduced+association+with+the+export+factor+TAP/NXF1.">XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.</a> Chromosoma. 116, 373-383 (2007).</li><li>Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper1+is+a+nucleoplasmic+shuttling+protein+that+negatively+modulates+Wnt+signaling+in+the+nucleus.">Dapper1 is a nucleoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus.</a> J. Biol. Chem. 51, 35679-3588 (2008).</li><li>Grespin, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+Hormone+Receptor+&#945;1+Follows+a+Cooperative+CRM1/Calreticulin-mediated+Nuclear+Export+Pathway.">Thyroid Hormone Receptor &#945;1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway.</a> J. Biol. Chem. 283, 25576-25588 (2008).</li><li>Heilman, D. W., Teodoro, J. G., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptin+Nucleocytoplasmic+Shuttling+Is+Required+for+Cell+Type-Specific+Localization,+Apoptosis,+and+Recruitment+of+the+Anaphase-Promoting+Complex/Cyclosome+to+PML+Bodies.">Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies.</a> J. Virology. 80, 7535-7545 (2006).</li><li>Jeffries, S., Capobianco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+by+Notch+requires+nuclear+localization.">Neoplastic transformation by Notch requires nuclear localization.</a> Mol Cell Biol. 20, 3928-3941 (2000).</li><li>Keaton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+trafficking+of+G2/M+regulators+in+yeast.">Nucleocytoplasmic trafficking of G2/M regulators in yeast.</a> Mol. Biol. Cell. 19, 4006-4018 (2008).</li><li>Ling, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-Cytoplasmic+Shuttling+Is+Not+Required+for+the+Epstein-Barr+Virus+EBNA-LP+Transcriptional+Coactivation+Function.">Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function.</a> J. Virology. 83, 7109-7116 (2009).</li><li>Lin, X. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RXR&#945;+acts+as+a+carrier+for+TR3+nuclear+export+in+a+9-cis+retinoic+acid-dependent+manner+in+gastric+cancer+cells.">RXR&#945; acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.</a> J. Cell Science. 117, 5609-5621 (2004).</li><li>Vissinga, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Export+of+NBN+Is+Required+for+Normal+Cellular+Responses+to+Radiation.">Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation.</a> Mol Cell Biol. 29, 1000-1006 (2009).</li><li>Zimmerman, R. S., Rosenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+p19Arf+localization+accompany+apoptotic+crisis+during+pre-B-cell+transformation+by+Abelson+murine+leukemia+virus.">Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.</a> J. Virol. 82, 8383-8391 (2008).</li></ol>

利害の衝突は宣言されません。

ヘテロカリオンのプロトコルは、細胞型特異的局在の挙動だけでなく、核 - 細胞質シャトル活動の特性評価のための効率的かつ容易に解釈する方法を提供するここに紹介。このメソッドは、蛍光分析の感度だけでなく、画像の生きている細胞の能力を活用し、タンパク質のダイナミクスの研究を行う。技術は簡単に多くの異なる細胞タイプに適応し、ライブや固定細胞のイメージングの両方の影響を受けやすいです。例えば、倒立蛍光顕微鏡は、ライブイメージングやタイムラプスビデオキャプチャに使用することができます。これとは対照的に、マウントされている細胞は、定常状態の局在の決定または離散ローカリゼーションの詳細な共焦点イメージングのための落射蛍光顕微鏡のいずれかで使用することができます。また、このような光退色後の蛍光回復（FRAP）、または退色で蛍光損失（FLIP）などの技法は、ローカリゼーション速度1の判定に使用することができます。プロトコルの代替蛍光色素の取り込みは、コンサート2で実施される蛍光共鳴エネルギー移動（FRET）などのタンパク質相互作用の実験のためにできるようになります。このようなレプトマイシンBまたはドミナントネガティブとして細胞輸送の様々な阻害剤は、GTPアーゼの構造が特定のインポートまたはエクスポートの経路5,6,9への依存を確立するために使用することができる蘭。 <ul><li>往復の活性が関与する特定の実験では、ケアが新たなタンパク質の合成に起因する局在の影響を避けるようにしなければならない。これらのケースでは、翻訳の阻害剤または期間限定ポイントを使用する必要があります。しかし、翻訳阻害に起因するアーティファクトは、可能性は残っている。各セルのタイプのいずれかを含む細胞融合は、ほとんどの往復とローカライゼーションの行動の明確な解釈のために望まれている。細胞数と融合条件（タイミングや濃度）の最適化は、良好な1:1の融合イベントを促進するために必要な場合があります。 </li><li>プロトコルの特定のステップの最適化が使用される特定の細胞タイプに応じて必要となることに注意することは重要です。このような融合効率、トランスフェクション効率、および融合後の生存率などの実験手順の側面は、細胞型との間で大きく異なることがあります。特に、主要な細胞型は、複雑な培養条件と低いトランスフェクション効率のために管理するために挑戦することができます。このようなロンザNucleofector装置のような技術の使用は、細胞だけでなく、劇的に増加したトランスフェクション効率の迅速な処理を可能にすることができる。さらに、エレクトロポレーション法は続く核融合のステップへの懸濁液中の細胞を簡単に転送が可能になります。 </li></ul>

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P5493</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3023602</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s modified Eagle&#x2019;s medium (DMEM)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH30243FS</td>
<td>High glucose</td>
</tr>
<tr>
<td>paraformaldehyde</td>
<td>&nbsp;</td>
<td>FisherChemical</td>
<td>O4042-500</td>
<td>&nbsp;</td>
<tr>
<td>4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D9542-10MG</td>
<td>&nbsp;</td>
<tr>
<td>Hyclone Serum (fetal bovine)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH3008803HI</td>
<td>&nbsp;</td>
<tr>
<td>Falcon 6-well plates</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-772-1B</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene glycol 1000</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>528875-25GM</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545-85</td>
<td>&nbsp;</td>
<tr>
<td>DABCO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2522-25G</td>
<td>&nbsp;</td>
<tr>
<td>Nucleofector HDF kit</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPD-1001</td>
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heterokaryon technique for analysis of cell type-specific localization

タンパク質のかなりの数は、細胞内輸送やシャトルnucleocytolasmicによって規制されている。これらのタンパク質は、核のインポート/ RNAとタンパク質の輸出、転写調節、およびアポトーシスを含む細胞機能の多様な配列を表示します。興味深いことに、細胞分裂、分化および変換を含む、主要な携帯電話再編は、多くの場合、3,4,8,10などの活動を含む。これらのタンパク質と、それぞれの調節機構の詳細な研究は、これらの局在の変化のための刺激は、とらえどころのないことができるよう挑戦し、そして自分自身が非常にダイナミックで追跡することが困難な場合も動作することができます。携帯電話発癌を対象とした研究は、例えば、理解の経路と6,7,11,12正常な初代培養細胞や形質転換細胞間で異なるタンパク質の活動の恩恵を受け続けることでしょう。として多くのタンパク質は、方法が効率的にこれらのタンパク質と彼らは薬剤が標的とするため発癌とのオープン新たな分野への理解を向上させるために立って参加する経路を特徴づけるために、変換中または変換の結果として局在変化を示す。 ここでは、タンパク質輸送の解析方法を提示し、プライマリおよび形質転換哺乳動物の細胞間のアクティビティを往復。このメソッドは、定常状態または動的なタンパク質の局在を検出するために使用できる柔軟なプロトコルを提供するために、蛍光顕微鏡によるヘテロカリオンの融合の生成を兼ね備えています。図1に示すように、2つの別個の細胞型は、一時的に目的の遺伝子に接続されているfluoroproteinの遺伝子を有するプラスミド構築物でトランスフェクトされています。発現後、細胞をポリエチレングリコールを用いて融合させ、及びタンパク質の局在は、その後の様々な方法を用いて画像化することができます。ここで紹介するプロトコールは、特別なテクニックが追加される可能性があるために基本的なアプローチです。

Watch this Scientific Journal Video about Heterokaryon Technique for Analysis of Cell Type-specific Localization at JoVE.com

Heterokaryon Technique for Analysis of Cell Type-specific Localization

細胞タイプ特異的タンパク質の局在化の特性評価と核細胞質間輸送のための柔軟かつ効率的な方法が記載されている。このヘテロカリオンのアプローチは、細胞融合後の画像の蛋白質のローカリゼーションへの蛍光標識融合タンパク質を使用しています。プロトコルは、定常状態の局在やライブセルイメージングに基づいて、より動的な決定に適している。

細胞種特異的局在の解析のためのヘテロカリオンのテクニック

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

heterokaryon-technique-for-analysis-of-cell-type-specific-localization

Research

JoVE Journal

Biology

16.3K ビュー.  Worcester Polytechnic Institute- WPI. 細胞タイプ特異的タンパク質の局在化の特性評価と核細胞質間輸送のための柔軟かつ効率的な方法が記載されている。このヘテロカリオンのアプローチは、細胞融合後の画像の蛋白質のローカリゼーションへの蛍光標識融合タンパク質を使用しています。プロトコルは、定常状態の局在やライブセルイメージングに基づいて、より動的な決定に適してい?...

ビデオ: Heterokaryon Technique for Analysis of Cell Type-specific Localization

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可視化RNAの局在にアフリカツメガエル卵母細胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

セル通信解析のための単一細胞のマイクロインジェクション

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

マイクロマニピュレーション技術形成ダイナミクスの解析と細胞骨格のレギュレータの売り上げ高

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 細胞型特異エピゲノムのプロファイリング

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

ゼブラフィッシュレンズ核局在化と Sutural 整合性の評価

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

マウス肝臓における細胞型特異的遺伝子発現プロファイリング

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

超構造保存細胞における膜蛋白質局在化の電子顕微鏡分析のためのクライオAPEX法

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

一分子局在顕微鏡を用いたヒトiPSC由来心筋細胞におけるαアクチニンネットワークの解析

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...