この作品は、ヒューマン·フロンティア·サイエンス·プログラム助成RGP0024によって資金を供給された。

1。 DNAテンプレートの調製とin vitro転写 <ol><li>目的の遺伝子は、任意のT7および/または、例えばプラスミドに基づくSP6プロモーターに複製されます。興味のあるRNCを得るためには、標的ポリペプチドのC末端は、SECM FXXXXWIXXXXGIRAGP 7の逮捕誘導シーケンスを追加することによって拡張されています。目的のポリペプチドフラグメントは、リボソームトンネルの外に押し出すことを保証するためには、標的タンパク質のC末端部分は、少なくとも30のアミノ酸12から14によって拡張する必要があります。柔軟性のグリシン - セリン豊富なリンカーは、すべての可能なコンフォメーションの制約を回避するために、タンパク質とSECM逮捕·シーケンスの間に導入することができます。 </li><li> in vitro転写では、テンプレートDNAは、ORFの下流を切断する制限酵素で直線化されるべきである。一つは、アガロースゲル電気泳動で制限酵素消化製品を実行することにより、プラスミドDNAの完全な線形化を確認する必要があります。 </li><li>線形化されたプラズマidは、さらにin vitro転写反応のために使用されます。テンプレートDNAの異なる濃度は 、in vitro転写のために必要な最適なDNA濃度を識別するためにテストすることができます。一般的にAmbionのMEGAscript高利回り転写キット（Ambion社/ライフテクノロジーズ、Grand Island、NY）を、1μgの直鎖状DNAの収率40から60μgのmRNAと。in vitro転写メーカーの指示（Ambion社/ライフテクノロジーズ、ニューヨーク州グランドアイランド）に続いて行われます。 </li><li> in vitro転写に続いて 、mRNAは、製造元の指示（AmbionのMEGAscript高利回り転写キット、Ambion社/ライフテクノロジーズ、グランドアイランド、NY）によると塩化リチウム沈殿により精製されています。 </li><li> mRNAの整合性は、さらに、アクリルアミドまたはアガロースゲルを用いて電気泳動によって確認されています。 </li></ol> （2）in vitro翻訳 RTS 100 E.大腸菌HYキット（5首相、ゲイザーを使用してin vitro翻訳のためにsburg、MD）は次の手順に従います。 <ol><li>メーカーの指示に続くin vitro翻訳反応で 100μlの準備をします。簡単に言えば、ヌクレアーゼフリーの水で24μlのアミノ酸混合物を引いたメチオニン（1mM）を、リボヌクレアーゼインヒビターの10単位（Invitrogen社/ライフテクノロジーズ、グランドアイランド、NY）、放射性の20μCiの[35 S]（-メチオニンのMPバイオメディカルを追加ソロン、オハイオ州）、20μlの反応ミックスは、24μlの再構成バッファとE.の24μlを大腸菌ライセート。 </li><li>暖かい翻訳反応を事前に5分間30℃で反応をインキュベートします。 10〜15分間30℃での翻訳反応とインキュベートするmRNAの1〜2μgのを追加します。 </li><li>氷の上で翻訳反応を置くことによって反応を停止します。 </li></ol> 3。 in vitro翻訳反応でから新生ポリペプチドの単離<ol><li> RNCを分離するには、翻訳反応は、20mM HEPES-KOH pHが7の5〜30％のショ糖勾配の4.5ミリリットルの上に積層されている0.5、15 mMのMgCl 2、100mMの酢酸カリウム、1mMのDTT及び41000 rpmでベックマン·コールター社のSW55-Tiローター、4℃で2時間Cを使用して遠心分離した。 </li><li>遠心分離後、ショ糖密度勾配は異なるリボソーム集団とそれに関連する新生鎖を分離する分画する。分離は、ISCO UA-6吸光度検出器を用いて254nmで連続記録とISCOプログラマブル密度勾配システムを使用して監視されます。 </li><li> 70Sリボソームを含む画分（s）を収集し、さらに実験の目的に応じて分析されます。 </li></ol> 4。トリス - トリシンSDS PAGEでのリボソームに結合したタンパク質の観察 SECM拡張タンパク質はリボソーム70Sに接続されたままであるかどうかを確認するには、勾配分画を回収し、次のように扱われました。 <ol><li>各画分中のタンパク質は10％の最終濃度になるようにトリクロロ酢酸（TCA）を添加することにより沈殿させ、4℃overniでインキュベートしたGHT。 </li><li>一晩インキュベートした後、試料をペレットに15分タンパク質を14,000×gで遠心分離した。以下の遠心上清を除去し、ペレットをアセトンを含有する溶液で2回洗浄した。1 mMのトリス-HCl pHは7.6（4:1）。ペレットは、さらに空気が乾燥した後、SDS-PAGEローディング緩衝液に溶解した。 </li><li>処理された試料が解決され、トリス - トリシンSDS-PAGEにより分析した。 </li><li>電気泳動後、ゲルを固定し、真空ゲル染色を使用して、オートラジオグラフィーにかけ乾燥させた。新生ポリペプチドの分布はphosphoimagingを用いて観察した。 </li></ol> 5。代表的な結果ここでは、安定した70Sリボソームに結合したフルレングスウシγ-Bクリスタリンの分離を説明する実験を提示します。 図1は、ウシγ-BクリスタリンのRNCの分離に必要な手順を示しています。 γ-BクリスタリンのC末端が32アミノ酸tで全体的な拡張されましたoは、リボソームトンネルからその全長タンパク質の押し出​​しを確保する、これはまた、融合ポリペプチドのC末端は非常に配置されたSECM失速シーケンスが含まれています。 in vitro翻訳に続いて 、70Sリボソームは、ショ糖密度勾配遠心法（図2.1）により単離した。蛋白質が安定してリボソームに結合したままであることを確認するためには、分数を含む70Sをプールした、脱塩、バッファーが交換され、ショ糖密度勾配遠心の追加ラウンド（図2.2）に供した。 図2に示す結果が明確にSECMを効率的にγ-BクリスタリンのRNCの翻訳停止を誘導することを示唆している。 <img alt="図1" src="/files/ftp_upload/4027/4027fig1.jpg" /> 図1。 RNCの分離に必要な手順の概略説明。ウシγ-BクリスタリンのC末端が32アミノ酸配列によって拡張されました。この拡張C-末端領域にもSECM逮捕配列を含む。 SECM配列を有するγ-BクリスタリンはpIVEX 2.3（T7ベース）プラスミドにクローニングした。 in vitro転写用テンプレートDNAをXbaIで直線化されました。線形化されたテンプレートは、さらにin vitro転写のために使用されていました。 DNAテンプレートのT7は、T7プロモーターの下流に位置する、γ-Bクリスタリン遺伝子を転写するT7 RNAポリメラーゼによって認識されている。 mRNAは、塩化リチウム沈澱法を用いて精製した。精製したmRNAはその後in vitro翻訳のために使用されていました。以下のインキュベーションは、翻訳反応は、5〜30％のショ糖勾配の上に重層し、2時間41000回転、4℃でベックマン·コールター社のSW55-Tiローターで遠心分離した。 <spクラス= &quot;pdflinebreak&quot;&gt; <img alt="図2" src="/files/ftp_upload/4027/4027fig2.jpg" /> 図2。ウシγ-Bクリスタリンの分離が安定して70Sリボソームに結合したRNCは1μgのmRNAを100μlの反応E.で混合した大腸菌 S30抽出システム20μCiの[35 S]でプロトコルの項で述べたように-メチオニン、15分間30℃でインキュベートした。以下のインキュベーションは、翻訳反応は、20mMのHEPES-KOH pHは7.5、15 mMのMgCl 2、100mMの酢酸カリウム、1mMのDTTに5〜30％のショ糖勾配の上に重層し、41000で、ベックマン·コールター社のSW55-Tiローターで遠心分離した。 rpmで2時間、4℃。速度沈降に続いて、勾配がアンロードされ、リボソームプロルが得られた。データはPeakTrakプログラム（ISCO勾配密度勾配分画システム）によって記録された。画分を回収し、タンパク質は、CのTris-T 6％、16.5％のTにTCA沈殿させ、解決されましたricine PAGEゲル15。ゲルを乾燥し、オートラジオグラフィー用に公開されました。次の暴露は、ゲルは台風9410イメージングスキャナを使用してスキャンした。 図2.1は、フルレングス、γ-Bクリスタリンは、70Sリボソームの分画に存在することを示している。したがって、SECM失速シーケンスは、安定したウシγ-BクリスタリンのRNCの分離を可能にします。 図2.2のデータは、明確に分離されたRNCのは確かに安定していることを示しています。現在の実験では、ショ糖密度勾配遠心分離の最初のラウンド後の70S画分をプールし、スクロース、20 mMのHEPES-KOH pH7.5で、15mMのを含む溶液中に緩衝液交換に続いてアミコンウルトラ-4遠心式フィルターユニット（ミリポア）を使用して、削除されましたのMgCl 2、100mMの酢酸カリウム、1mMのDTT。このサンプルは、さらに5〜30％のショ糖密度勾配遠心分離によって分画し、追加のラウンドを行った。各画分は処理され、 図2.1のように分析した。

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利害の衝突が宣言されません。

再現可能な結果を得るためには、in vitro転写と翻訳のために使用されるコンポーネントの品質と濃度が非常に重要です。慎重に処理した場合、我々は、in vitro転写と翻訳の抽出物で市販されている使用していると、彼らは効率的かつ再現可能な結果を与える。 mRNAの品質は翻訳に影響を与えることがないので、in vitro翻訳のためにそれを使用する前に、mRNAの整?...

<table border="1"><tbody><tr><td> 試薬/キットの名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> MEGAscript T7高収率転写キット</td><td> Ambionの</td><td> AM1333 </td></tr><tr><td> RTS 100大腸菌HYキット</td><td> 5内閣総理</td><td> 2401100 </td></tr><tr><td>リボヌクレアーゼ阻害剤</td><td>インビトロジェン</td><td> 15518012 </td></tr><tr><td>トランス[35 S] -ラベル</td><td> MPのバイオメディカル</td><td> 0151006 </td></tr><tr><td>アミコンウルトラ-4遠心式フィルターユニット</td><td>ミリポア</td><td> UFC801008 </td></tr><tr><td>貯蔵燐光体オートラジオグラフィー</td><td> GEヘルスケア</td><td>台風9410可変モードイメージャ</td></tr><tr><td>密度勾配分画システム</td><td>ダインイスコ株式会社</td><td> ISCOプログラマブル密度勾配系</td></tr><tr><td>スクロース勾配CENtrifugation </td><td>ベックマン·コールター</td><td>オプティマL-90 K取超遠心</td></tr></tbody></table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

広範な研究は、細胞内のタンパク質のフォールディングの共同翻訳プロセス1から5であることを示唆している十分な証拠を提供しています。しかし、その機能的なフォームを実現するための共同翻訳折りたたみ時のポリペプチド鎖が続くことを正確な経路はまだ謎です。このプロセスを理解し、共同翻訳の折り畳み中間体の正確な立体構造を決定するために、それは彼らのさらなる構造解析を可能にするために所定のサイズの新生鎖を保有するRNCの分離を可能にする技術を開発することが不可欠です。 SECM（分泌モニター）は、170アミノ酸E.です。 SECM-セカオペロン6の下流セカ（分泌駆動）ATPaseの発現を調節する大腸菌の蛋白質。 Nakatogawaと伊藤は当初、SECMタンパク質のC末端領域17アミノ酸長いシーケンス（150-FSTPVWISQA QGIRA G P-166）への十分かつ必要であることがわかったこれにより、安定したリボソームのP部位7-9結合したペプチジル-グリシル-tRNAを生成する、Gly165でSECM伸びの停滞が発生します。さらに重要なのは、それはこの17アミノ酸長いシーケンスは、このように所定の大きさ7の新生鎖を保有するRNCの生産を可能にする事実上すべてのフルレングスおよび/ ​​または切り捨てられたタンパク質のC末端に融合させることができることがわかった。したがって、標的タンパク質に融合または挿入したときに、SECMストールシーケンスは、ポリペプチド鎖の伸長の逮捕を生成し、E.の in vivo の両方で安定したRNCを生成する大腸菌細胞と無細胞系の in vitro インチショ糖密度勾配遠心法は、さらにRNCを分離するために利用されています。 孤立したRNCは、共同翻訳の折り畳み中間体の構造的および機能的特徴を分析するために使用することができます。最近、この手法は成功した10,11、いくつかのリボソーム結合した新生鎖の構造への洞察を得るために使用されている。ここでは、ウシγ-BクリスタリンのRNCの分離SECMに融合し 、in vitro 翻訳系で生成について説明します。

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

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

ここでは今定期的に安定的に結合したリボソーム新生鎖複合体（のRNC）を分離するために使用される手法について説明します。この手法は、17アミノ酸長いSECM &quot;逮捕シーケンスは&quot;原核生物の翻訳伸長を停止することができるという発見を利用しています（<em&gt; E.大腸菌</em&gt;実質的に任意のタンパク質（またはC末端に融合させた）に挿入された）システムでは、。

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 ...

using-secm-arrest-sequence-as-tool-to-isolate-ribosome-bound

Research

JoVE Journal

Biology

12.3K ビュー.  Cleveland State University. ここでは今定期的に安定的に結合したリボソーム新生鎖複合体（のRNC）を分離するために使用される手法について説明します。この手法は、17アミノ酸長いSECM "逮捕シーケンスは"原核生物の翻訳伸長を停止することができるという発見を利用しています（ E.大腸菌実質的に任意のタンパク質（またはC末端に融合させた）に挿入された）...

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

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

微細加工後のアレイ検出器（mPADs）：機械的な力を単離するためのアプローチ

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

生物学

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

マイクロインジェクション分子およびそれらの真核生物の細胞内ターゲットを視覚化するツールに相関光学および電子顕微鏡（クレム）


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

宿主植物の揮発性物質のスクリーニングツールとしてElectroantennographicバイオアッセイ

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

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 Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

アフリカツメガエルは、翻訳減損を識別するためのモデルとして、 アフリカツメガエル

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

BrdUでのChIP-スロット西洋技術を用いて哺乳動物細胞中で新生DNAに結合したタンパク質の検討

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

個々の細胞タイプの転写プロファイリングのためのツールと​​してのレーザ支援顕微解剖（LAM）

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

磁気活性化細胞を分離し、滑膜液由来間葉系幹細胞が家兎モデルからの浄化戦略を並べ替え

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

マウスから脳ペリサイトを分離する高出力法

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

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、

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、