我々は、様々な発達の時点で動物の姿を提供するためJames Clark氏に感謝します。C.本研究ではエレガンス株は研究資源のためのNIHナショナルセンター（NCRR）によってサポートされ線虫遺伝学センターから入手した。この作品は、ニューヨーク市立大学からJLとCSDにCIRG 1817でサポートされていた、とNIH 1R15GM073678-01およびCSDへ1R15GM097692-01で我々は、原稿上のコメントのための博士ウィリアム·J·ライス博士ナタリアホルツマン、とメリッサSilvestriniに感謝します。この作品は、ニューヨーク市立大学（S.熊）の大学院センターから博士号のための要件の部分的な達成で行った。

1。標的遺伝子配列を運ぶのRNAi給餌プレートの準備<ol><li>市販のRNAiライブラリー（ 例えば、ソースバイオサイエンスライフサイエンスから）使用している場合は、1.4に進みます。代わりに、標準的なクローニングプロトコルによって、ベクトルL4440、8 RNAiのプラスミド、一般的に使用されるワームに標的遺伝子配列のクローンを作成します。 </li><li>細菌株HT115（DE3）を、25μg/ mlのカルベニシリンおよび12.5μg/ mlのテトラサイクリンを含むLB寒天プレート上で培養して組換えプラスミドに変換し、将来の使用のために単一クローンをピックします。 </li><li>一方、実験の対照として使用する細菌株HT115に空のベクターL4440を変換します。 </li><li> 37℃で一晩100μg/ mlのアンピシリンを含む1ミリリットルLBブロス℃の文化換えと空の両方L4440-運ぶ細菌テトラサイクリンは、それがRNAiの効率を低下させるという事実のために追加されません。 </li><li>一晩cultuに100μg/ mlのアンピシリンを持つ別の5mlのLBブロスを追加37で別の4から6時間インキュベート再、℃ </li><li> RNAiのワームプレートにシード0.5ミリリットル換えまたは空L4440-運ぶ細菌培養。クローン名を持つすべてのプレートにラベルを付けます。 </li><li>プレートをマークし、37℃で一晩インキュベート℃の細菌の芝生を育てるために、これらは標的遺伝子配列の有無を問わずRNAiの板である。少なくとも2つのプレートはそれぞれの条件のために準備されるべきです。 </li></ol> 2。 RNAiの給餌プレート上の培養ワーム<ol><li>炎が白金線ワームピックの先端を消毒。 6月10日4令幼虫（L4）は、組換えまたは空L4440を含む各RNAiのプレートに雌雄同体迎えに選ぶワームを使用します。動物は食物源として細菌を使用します。 </li><li>雌雄同体が20℃（C. elegansのための標準的な培養条件）で一晩成長させ、彼らは翌日、若年成人になるでしょう。 </li><li>新たに対応して標識し、準備したRNAiプレートに6月10日、若年成人を転送します。 </li><li>てみましょう大人は4-6時間、卵を産み、その後板からすべての大人を削除し、このステップでは、子孫を同期させることです。母親が削除されたら、時間をカウントし始める。これは時間がゼロになります。 </li><li> 20でプレートをインキュベート℃の動物は特定の発達段階に成長するようにするためには、このプロトコルでは、我々は表現型分析のための若年成人を選択します。通常の速度で開発ノックダウンのために、72時間のインキュベーションは、若年成人になるための動物のために十分である。しかし、様々な遺伝子が異なる動物の発達に影響を与える。 RNAiは、異なる速度で成長すると動物が発生した場合は、若年成人が完了外陰部の開発と子宮の2-6胚（肥沃な場合）とL4は過去24時間以外のものは、それ以下に識別することができます。他の発達段階を識別するために、治験責任医師は、ガイドとして性腺と外陰部の開発を使用する必要があります。 </li></ol> 3。 RNAiに処理されたワームのスコアボディサイズ表現型<ol><li>スライドガラス上に色のついたラベルテープの2つのレイヤーを配置します。のメイク2seのガラススライド。次に、テープを持つ2つのスライドの間に新しいスライドガラスを配置します。水中2％アガロースを溶かし、新しいスライドガラスの中央に一滴を適用し、次にアガロースは前述の9記載されている2％の薄い層を作るために上に第2のスライドガラスを押してください。 </li><li>アガロースが固化した後、上部スライドガラスを取り外すこと。アガロースパッド付きのスライドは、ワームをロードする準備ができています。クローン名とラベルのスライド。 </li><li>アガロースパッドに10μlの25mMのNaN 3を追加して、それを固定するために、NaN 3溶液中に30から40の動物を選択してから、上にカバースリップを入れて、組換えと空の両方がRNAi処理した動物をL4440-運ぶための繰り返し。 </li><li>画像2.5倍の対物レンズを用いて解剖顕微鏡下ワーム、ここで我々は、ソフトウェアQcaptureをサポートするとライカのデジタルカメラを使用しています。 </li><li>キャリブレーションでは、最初の標準マイクロメートル定規のイメージをキャプチャします。 [イメージ-Proソフトウェアで画像を開いて、選択して &quot;測定&quot;をクリックし、 &quot;キャリブレーション&quot;を選択してください&quot;空間キャリブレーションウィザード&quot;。 &quot;アクティブな画像をキャリブレーション&quot;を選択した状態で、 &quot;次へ&quot;をクリックします。入力キャリブレーションの名前と空間参照の単位がメートルに設定されていることを確認してください。そして、 &quot;作成基準校正&quot;ボタンをチェックし、 &quot;次へ&quot;をクリックします。をクリックして &quot;描画基準線。&quot;基準スケールバーが表示されます。マイクロメータの両端に一致するスケールバーの位置を変更し、参照が千単位を示すことを示している。 &quot;OK&quot;ボタン、 &quot;次へ&quot;をクリックし、 &quot;完了&quot;。 </li><li>ソフトウェアが校正されると、ワームの画像を開きます。次に、 &quot;測定&quot;メニューの下に選択して、 &quot;空間を選択&quot;をクリックし、 &quot;キャリブレーション&quot;を選択します。プルダウンメニューでは、マイクロメータ校正用に作成したファイル名を選択します。次に、 &quot;測定&quot;メニューの下に、 &quot;測定&quot;を選択してください。開いたウィンドウで、自由に描画ツールを選択し、コンピュータのマウス（ 図1）と尾部に頭から動物の体の中心を通る線をたどる。長さがウィンドウに報告されます。今標的遺伝子のRNAiと空L4440ベクトルで処置した対照動物と投与動物の体の長さ：あなたはデータの2つのグループがあります。 </li><li> Microsoft Excelファイル、または他の適切な統計ソフトに長さの測定値をエクスポートします。各サンプルの平均値と標準偏差を計算することにより、データを分析します。その後、スチューデントのt検定を用いてサンプルを比較します。 </li></ol>

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特別な利害関係は宣言されません。

このプロトコルでは、Cのボディサイズ欠損変異株の同定のための私達の方法を記述RNAiの給餌によってエレガンス 。この方法では、TGF-βシグナル伝達コンポーネントの識別にも適用可能である。このような成分が高度に進化15を介して保存されているので、このような画面は、すべての後生動物ではTGF-βシグナル伝達の分子機構の解明に関連しています。このような画面...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> RNAiのワームプレート 17.7グラムのWORMメディアミックス 60グラム寒天 3リットルにH 2 Oを追加します。オートクレーブ。 3ミリリットル1M IPTG（滅菌）および3ml 25 mg / mlのカルベニシリンを（滅菌）を追加してから、60ミリメートルペトリ皿に注ぐ。 ワームプレート 17.7グラムのWORMメディアミックス 0.6グラムの硫酸ストレプトマイシン 60グラム寒天 3リットルにH 2 Oを追加します。オートクレーブ。 60ミリメートルペトリ皿に注ぐ。 WORMメディアミックス 55グラムのトリス-Cl 24グラムのトリス-OH 310グラムバクトペプトン 800mgのコレステロール 200グラムのNaCl 徹底的に混ぜるとチャンクを避けるようにしてください、この粉末の混合物を使用する前に何ヶ月も保存することができます。 2％アガロース 0.5グラムアガロース 広告dは25ミリリットルのdH 2 O電子レンジでの加熱溶解するまで。 1Mイソプロピルβ-D-1-チオガラクトピラノシド（IPTG） 2グラムのIPTG IN10ミリリットルdH 2 Oを溶かす 1MのNaN 3 6.5グラムのNaN 3 100mlにdH 2 Oを追加 25mMのNaN 3を 2.5ミリリットル1MのNaN 3 100mlにdH 2 Oを追加 線虫の遺伝学センターから線虫（Caenorhabditis elegans） プラスミドL4440（アンピシリン耐性遺伝子を運ぶ） 菌株HT115（DE3）（IPTG誘導性T7ポリメラーゼを含んでいます。またテトラサイクリン耐性遺伝子を運ぶRNアーゼIII遺伝子（dsRNAse）を欠損する）16 60ミリメートルペトリ皿 100ミリメートルペトリ皿 14ミリリットルの丸底ファルコン培養管 スライドガラス カバーガラス 1から20μlのピペッター 20から200μlのピペッター 200-1000μLピペッター 1から200μlのピペットチップ 200-1000μlのピペットチップ 1.5mlのエッペンドルフチュー​​ブ 白金線ワームピック</td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

二本鎖RNA媒介干渉（RNAi）は1月3日 、標的遺伝子の発現をノックダウンする効果的な戦略である。それは植物、無脊椎動物と脊椎動物を含む多くのモデルシステムに適用されています。 生体 4,5 でRNAiを達成するためのさまざまな方法があります。一過性かもしれない代わりに特定の標的遺伝子（RNAiのオリゴ）から二本鎖オリゴヌクレオチドを​​合成し、例えば、標的遺伝子がRNAiのベクトルに変換することができ、その後永久に、または一過性に遺伝子ノックダウンの効果を達成するために、細胞株や初代培​​養細胞に形質転換標的遺伝子を沈黙させる細胞株や初代培​​養細胞に形質転換し、または合成さ​​れた二本鎖RNA分子が生体に微量注入することができる。線虫C.以来虫、細菌が標的遺伝子に対する二本鎖RNAを発現する動物の餌、食料源として細菌を使用して実行可能な戦略6を提供しています。ここではRNAiの給餌を提示体の大きさの表現型を獲得するための方法。 Cのボディサイズ虫は主に 、TGF-βによって調節されている-のような配位子DBL-1ので、このアッセイは、TGF-βシグナル伝達成分7の識別に適しています。私たちは、RNAi給餌実験を再現する2 RNAiの過敏株を含む様々な菌株を使用していました。我々の結果は、RRF-3株は私たちに最高の期待されるのRNAi表現型を与えることを示した。方法は、実施が容易で再現性があり、容易に定量化される。さらに、我々のプロトコルでは、特殊な装置の使用を最小限に抑えているので、小さな実験室や主に学部機関の人に適しています。

私たちの研究では、DBL-1/TGF-β経路による体の大きさの規制に重点を置いています。野生型動物7,10,11に比べ短いボディの長さも含めて、小さなボディサイズでDBL-1経路の結果、の機能の喪失。したがって、Cの画面虫の体の大きさの変異体は、TGF-βシグナル伝達成分と修飾子を識別することが可能である。 DBL-1シグナルが細胞表面の受容体と細胞内のSmadシグナルトランスデ...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

我々は、標的遺伝子とスコアボディサイズの表現型をノックダウンするのRNAi送り技法を使用する方法を示し<em&gt; Cエレガンス</em&gt;。このメソッドは、DBL-1/TGF-βシグナリングによるボディサイズ調節に関与するものなど興味のある潜在的な遺伝的要素を識別するために大規模なスクリーンを使用することができる。

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた<em&gt; Cエレガンス</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

using-rna-mediated-interference-feeding-strategy-to-screen-for-genes

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JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

12.2K ビュー.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). 我々は、標的遺伝子とスコアボディサイズの表現型をノックダウンするのRNAi送り技法を使用する方法を示し Cエレガンス。このメソッドは、DBL-1/TGF-βシグナリングによるボディサイズ調節に関与するものなど興味のある潜在的な遺伝的要素を識別するために大規模なスクリーンを使用することができる。

ビデオ: Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

潜在的なパーキンソン病の遺伝子を検証するための線虫のドーパミンニューロンの変性のアッセイのアプリケーション

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

生物学

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体染色中<em&gt; C.虫</em&gt;「フリーズクラッキング」使用

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

大規模な遺伝子ノックダウン中<em&gt; C.虫</em&gt;堅牢な機能喪失表現型を生成するためのdsRNA摂食ライブラリの使用

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

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 ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

筋肉の細胞内コンパートメントを評価するための方法<em&gt; C。エレガンス</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

の発達過程を追跡し、定量化<em&gt; C。エレガンス</em&gt;オープンソースのツールを使用しました

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C.エレガンスにおけるRANペプチド毒性の測定

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

カエノハブディティス・エレガンスを使用して組織特異的シャペロン相互作用をスクリーニングする

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...