Ubp1を発現するプラスミドpCG001は、親切に医学研究のジョン·カーティン学校でロハンベイカーによって提供されました。この作品は、ダイム研究助成1〜2011年度の3月ダイムバジルオコナースターター客員研究賞＃5-FY20とNSFキャリアアワード0746796の行進によって賄われていた。

1。ひずみエンジニアリングワークフロー<ol><li>シーケンスのエンコード（）の膜局在化配列、（b）の高速成熟蛍光タンパク質とすると、目的のタンパク質（ 例えば転写因子）とフレーム内の（c）にプロテアーゼ認識配列のN末端 ​​。を挿入我々は、TSR-金星レポーター2標的膜を使用し、発現バクテリオファージλリプレッサーCIのタンパク質分子の数をカウントするユビキチンプロテアーゼ認識配列（UB）に融合した。我々は、野生型CIはコーディング領域とEにコンストラクトを組み込ん交換、融合タンパク質TSR-金星-UB-CIの構築方法についての詳細λ赤組3を用いて 、 大腸菌の染色体は、refに詳細に記載されている。 1。 </li><li>配列決定することによって、すべての変更を確認します。 </li><li>をコードするプラスミドを、目的の蛍光レポーターとタンパク質を発現している株に上記で使用した認識配列に特異的プロテアーゼを変換翻訳融合インチ我々は、C-末端ユビキチン残4の直後に切断するプロテアーゼUbp1を使用していました。 </li></ol> 2。培養細胞とサンプルを準備<ol><li> 1 Eを選ぶ1X MEMアミノ酸と適切な抗生物質を補充した1ミリリットルM9最少培地5にたて縞寒天プレートからの関心の大腸菌コロニー。十分な長さのOD 600が &gt; 1.0（600nmでの光学密度）に到達するために、所望の温度でシェーカーでインキュベートする。 </li><li> OD 600 = 0.02に適切な抗生物質で1ミリリットル新鮮M9培地に培養を希釈します。適切な温度でシェーカーでインキュベートする。低温成長のために必要であれば初期細胞密度を増加させることができる。 </li><li> OD 600 = 0.2から0.3（37℃、OD 600での初期培養細胞を使用したC = 0.02、これは3〜4時間かかります）場合には、アガロースゲルパッド（ステップ3）を製造開始。 </li><li>時OD細胞はイメージングのための準備が整いました<sub&gt;の600 = 0.3から0.4。 </li><li>卓上遠心機で1分間10,000 xgで1.5 mlマイクロチューブ、遠心分離機に1ミリリットルインキュベート文化を転送します。 </li><li>上清を捨て、1 mlの新鮮なM9培地を加え、穏やかにペレットを再懸濁します。 </li><li> 1分間、10,000×gで遠心分離します。 </li><li> 1.6と1.7を繰り返します。上清を捨てる。 </li><li>穏やかに1ミリリットルM9培地に再懸濁します。タイムラプスイメージングのための低細胞密度を確保するために100倍に - 細胞を直接顕微鏡イメージングまたは希釈10に使用することができます。低細胞密度、拡張タイムラプスイメージングのために重要であることに注意してください。イメージング室（ステップ4）は実験中に封入されている。細胞の指数関数的成長のために、高い初期細胞濃度は有意に蛍光タンパク質の成熟を削減し、細胞増殖に影響を与え、ゲルパッド、長引く成長後にチャンバー内の酸素を使い果たすことができます。 </li></ol> 3。アガロース溶液を準備<ol><li> 10から20ミリグラムの重量を量る1.5 mlのマイクロチューブに低融点温度アガロース。 </li><li> 3％アガロース溶液を作製するために抗生物質を含まない適切な音量M9最少培地を追加します。 </li><li> 70℃で30分間アガロース溶液を加熱℃、溶液が完全に溶融し、均一であることを保証するためにチューブを反転を溶解した。ゲルパッドは、この時点で注ぐことができる（ステップ4）、または温度が50℃まで低下し、後で使用するために数時間保持することができる。 </li></ol> 4。商工会議所にジェルパッドを作製<ol><li>イメージングの前に約50分は、水とMicroaqueductスライドをすすぎます。 </li><li> 1は、カバーガラスを掃除（6で説明したような厳しい洗浄方法を使用して）圧縮空気を吹き付けることにより滅菌水と乾いたスライドをMicroaqueductすすいでください。 </li><li>それが、これは内のアプリケーション用に変更することができMicroaqueductスライド（のガラス側の入口と出口を覆うようにMicroaqueductスライド上にゴム製のガスケットを配置どのメディア）は、サンプルを通して灌流される。 Microaqueductスライドの中央に50μlをアガロース溶液（ステップ3）を適用します。 </li><li>洗浄、乾燥したカバーガラスとのアガロース溶液を補充して下さい。 </li><li>アガロース溶液を30分間室温で放置する。 </li><li>一方、（ステップ2）細胞試料を準備します。 </li><li>慎重にゲルパッドの上部からカバーガラスを剥がす。ゲルパッドの上部に1.0μlの洗浄された細胞培養を追加します。培養のための〜2分をゲルパッドに吸収されるのを待ちます。それはとても長いゲルパッドoverdriesその待たないようにすることが重要であるが、十分な長さのセルが正しくゲルパッドに付着していること。理想の待機時間は、温度や湿度によって異なります。 </li><li>待っている間に、別の予備洗浄​​したカバーガラスを乾燥させてください。 </li><li>カバーガラスとMicroaqueductスライドがよく合っていることを保証する新たなカバーガラスでサンプルをカバーしています。 </li><li>メーカーの指示に従って、温度制御された成長チャンバーを組み立てます。現在では、顕微鏡でイメージング（ステップ5）に使用することができます。 </li></ol> 5。イメージングとタイムラプスムービーの取得<ol><li>メーカーの指示（ 例えばレーザーは実験前〜0.5時間ウォームアップする必要があるかもしれません）以下のレーザーと顕微鏡をオンにします。 </li><li>顕微鏡ステージ上のインサートに組み付け室をロックします。成長室の温度は、37℃又は他の所望の成長温度での客観的なヒーターを設定します。 </li><li>イメージング室温以上の場合、フォーカスまたはジェルパッドは、通常約15分初期温度シフト後のために大幅にドリフトします。実際には、関心のある領域を見つけ、実験のために必要に応じてイメージングスクリプトを変更するには、この時間を使用します。 </li><li>顕微鏡で細胞を見つけると、事前に定義され、整列撮像領域内のそれを中心にした後に各セルの位置を格納します。複数のセルは、各画像取得時間ウィンドウに結像させることができる。 m以上のタイムラプスイメージング用任意の世代は、画像化された細胞は最初に他のコロニーが成長中に撮像領域を入力しないように、少なくとも百数μmで、他の細胞から分離されていることを確認してください。 </li><li>ので、6エクスポージャー（ 図5）内のほぼ全ての蛍光分子が光退色、レーザー励起強度を調整します。 </li><li>自動イメージングスクリプト/ジャーナルを使用して、 図3の実験的なワークフローで記述されたアルゴリズムを使ってタイムラプスデータを取得する。 </li></ol>

<ol>
	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., 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+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

私たちには開示しません。

CoTrAC方法は、従来のN末端またはC末端蛍光タンパク質融合体がタンパク質活性を妨害する可能性のある他のタンパク質の産生を測定することが一般化することができる。 CoTrAC戦略は、現在の方法に比べて3特有の利点があります。まず、共同翻訳融合は蛍光レポーターの1分子がリアルタイムでタンパク質生産の正確なカウントを可能にする、目的のタンパク質の各分子のために生成されるこ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>アガロース（低融解温度） </td><td>ロンザ</td><td> 50100 </td><td></td></tr><tr><td>のMilli-Q H 2 O </td><td></td><td></td><td></td></tr><tr><td> 5×M9塩</td><td></td><td></td><td>以下のレシピは9に記載</td></tr><tr><td> 20％グルコース</td><td></td><td></td><td></td></tr><tr><td> MgSO 4を </td><td></td><td></td><td></td></tr><tr><td> CaCl 2を </td><td></td><td></td><td></td></tr><tr><td> 50×MEMアミノ酸溶液</td><td>インビトロジェン</td><td> 11130-051 </td><td></td></tr><tr><td>温度制御成長室 ステージアダプタ</td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td>客観ヒーター</td><td> Bioptechs </td><td>モデルは、顕微鏡の対物レンズに依存</td><td></td></tr><tr><td>スライドをMicroaqueduct </td><td> Bioptechs </td><td> 130119から5 </td><td></td></tr><tr><td>マイクロカバーガラス</td><td> VWR </td><td> 40CIR-1 </td><td>ソースが困難な場合があります。Bioptechsからも利用可能</td></tr><tr><td>ガラス/スライドガスケットをカバー</td><td> Bioptechs </td><td> FCS2 0.75ミリメートル</td><td></td></tr><tr><td>蛍光顕微鏡</td><td>さまざまな</td><td>セットアップ例：コヒーレントイノーバ308Cアルゴンイオンレーザー、オリンパスIX-81顕微鏡、オリンパスPlanApo 100X NA 1.45客観、Metamorphソフトウェア</td><td>レーザー励起、自動XYZステージ、スクリプト化されたイメージングとオートフォーカスが可能な自動化ソフトウェア、resolvinことのできる光学系を持っている必要がありますgシングル蛍光タンパク質</td></tr><tr><td> EM-CCDカメラ</td><td>さまざまな</td><td>セットアップ例：アンドールIXON DU-898 </td><td>バックグラウンドを超える単一の蛍光タンパク質を検出するために十分に低いノイズを持っている必要があります</td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

我々は、画像への開裂により蛍光顕微鏡法、共同翻訳活性化（CoTrAC）タンパク質の機能を乱すことなく、単一分子の精度で生細胞中のタンパク質分子の製造を記述している。この方法によれば、シーケンシャル、5分の時間枠で、1セル内に産生されるタンパク質分子の数を数えるようになります。それは、生きた細胞内の単一の蛍光タンパク質分子を検出するために十分に敏感である〜0.5から1キロワット/ cm 2のレーザー励起パワー密度を持つ蛍光顕微鏡を必要とします。膜標的化配列、高速熟成、黄色蛍光蛋白質とプロテアーゼ認識配列：このメソッドで使用される蛍光レポーターが3つの部分から成ります。記者が翻訳目的のタンパク質のN末端に融合されています。細胞は、温度制御された顕微鏡ステージ上に成長させられる。 5分おきに、細胞内の蛍光分子（およびそれ以降の共同撮像される蛍光画像を分析することによってunted）、続いて、新しく翻訳されたタンパク質は、次の測定にカウントされるようにphotobleached。 この方法から得られた蛍光画像は、各画像の蛍光スポットを検出し、個々のセルに割り当てること、その後の細胞系譜への細胞を割り当てることによって分析することができます。与えられたセルにタイムウィンドウ内で産生される蛋白質の数は、単一蛍光分子の平均強度によってスポットの統合された蛍光強度を割ることによって計算されます。我々は、単一の5分の時間ウィンドウで0から45分子の範囲内で発現レベルを測定するために、このメソッドを使用していました。この方法は、私たちがλリプレッサーCIの式で騒音を測定するために有効になっており、システム生物学の他の多くの潜在的なアプリケーションを持っています。

λリプレッサーCIの生産を追跡CoTrAC実験から得られた典型的な結果を図4および図5に示されている。この実験では、12個のコロニーは、5分間隔で撮像した。各時点で、コロニーは最初autofocusedたと撮像領域内の中央。次に、フォーカス/中心位置が格納されていたと明視野画像を取得した。ステージはその後細胞（位相差または微分干渉コントラスト（DIC）光学系なし2?...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

我々は、画像への開裂により蛍光顕微鏡法、共同翻訳活性化（CoTrAC）、タンパク質の機能を乱すことなく、単一分子の精度で生細胞中のタンパク質分子の製造を記述している。このメソッドは、転写因子、λリプレッサーCIの確率の発現動態を追跡するために使用されています<sup&gt; 1</sup&gt;。

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

single-molecule-imaging-gene-regulation-vivo-using-cotranslational

Research

JoVE Journal

Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

9.8K ビュー.  Johns Hopkins University School of Medicine. 我々は、画像への開裂により蛍光顕微鏡法、共同翻訳活性化（CoTrAC）、タンパク質の機能を乱すことなく、単一分子の精度で生細胞中のタンパク質分子の製造を記述している。このメソッドは、転写因子、λリプレッサーCIの確率の発現動態を追跡するために使用されています 1。

ビデオ: Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

核輸送の1分子イメージング

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

切断断片の検出と電流測定を組み合わせることにより、上皮性ナトリウムチャネル（ENaCの）のタンパク質分解活性の実証

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

1分子FRETによるDNAループを研究

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

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) ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

部位特異的DNA切断のための並列高スループット単一分子運動アッセイ

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...