著者らは、図の作成の支援のためにルネScriwanekに感謝します。 FRがエコー（700.59.003）、ALWオープンプログラム（821.02.017や822.02.014）、DFG-NWO協力（DN82-303）とZonMW VICI（016.130.606）の補助金によってサポートされています。

1。スフェロプラストの準備<ol><li>実験の設計によって決定された適切な培地10ml中で30℃で一晩酵母をインキュベートする。 </li><li>翌日、設計しない限り、光度計を用いて600nmで（OD 600）での培養物の光学密度を測定する0.2〜0.4のOD 600に同じ培養培地中で細胞を希釈し、指数増殖期にそれらを成長させる実験は、異なる条件を必要とします。培養がOD 1-2の600を持っている時に細胞が指数増殖期にある。 </li><li> 50mlチューブ中で5分間、3,500×gで遠心分離することによって細胞の10 OD 600当量を収集する。遠心分離の後、上清を捨てる。 </li><li> 100mMのPIPES（pH9.6）中の5mlの10mMジチオスレイトール、10分間30℃でインキュベートして細胞ペレットを再懸濁。 </li><li> 5分間3500×gで再び遠心分離して細胞を回収します。上清を捨てる。 </li><li>細胞を再懸濁Inは1 Mソルビトールおよび溶菌酵素5mgを含有する（実験の設計によって決定される）、そして穏やかに30分間振とうしながら30℃で混合物をインキュベートする培地5ml。 </li><li>スフェロプラストに対応するペレット分画を回収し、5分間300×gで細胞懸濁液を遠心分離する。上清を捨てる。 </li><li>氷冷たいメディアの960μlのフェロプラストを再懸濁し、1Mのソルビトールを含む（メディア実験の設計によって決定される）、アイス冷2 mlマイクロチューブに混合物を移す。 </li></ol> 2。ナノ金取り込み<ol><li>ピペットを使用して、静かに水40μlに再懸濁させ、正に帯電しナノゴールド粒子の4ナノモルでフェロプラストをミックス。混合物の最終容量を1mlとする必要がある。 </li><li> 15分間氷上で得られた細胞懸濁液を置きます。 </li><li>室温でサスペンションを転送し、Eによりナノゴールド内在化を可能にするために必要な時間のためにそれをインキュベートndocytosis。 注：15分の取り込みが全体エンドソームシステム（初期および後期エンドソーム）をマークすることを許可する一方、5分間の取り込みは、主に、細胞内小胞と早期エンドソーム区画のラベル付けにつながる。長いインキュベーション時間（30分以上）も、空胞を標識することを許可します。 NOTE：パルスチェイス標識実験は、この方法を行うことができない。 LEの分析のためにラベル付けする際にそのため、ナノゴールドもPMおよびinEEにあります。 </li></ol> 3。固定と分割<ol><li> 2倍濃度の固定液を1ml添加​​することにより、ナノゴールド取り込みを停止する[4％パラホルムアルデヒド（PFA）、0.1 M PHEM緩衝液中の0.4％グルタルアルデヒド（GA）（20mMのPIPES、50mMのHEPES、pH6.9の、20mMのEGTA、4のMgCl 2）]を細胞懸濁液に1Mソルビトールを含有する。室温でチューブを保った。 </li><li>優しく（これがフェロプラストを維持するために可能にする30分の間にmicocentrifugeチューブを手動で数回転倒懸濁液）してから25秒間1700×gで二度遠心する。 </li><li> 1Mソルビトールを含有し、ゆっくりと回転するホイール​​上で室温で2時間インキュベート上澄みを捨てたと（0.1M PHEM緩衝液中の2％のPFA、0.2％GA）の新鮮な標準強度固定液1ミリリットルを追加することにより、固定液を交換してください。 </li><li>以前に説明したように6クライオ切片化のための細胞を処理する。 NOTE：ナノゴールド取り込みおよび銀増強する手順については、指定されたプロトコルに記載の周期的な酸処理を行うことがないようにしている。過ヨウ素酸処理は、より良いセル壁26を透過することであるが、この構造は、スフェロプラストの生成中に除去されている。 </li><li>以前8に記載されているようUCTウルトラを用いたドライダイアモンドナイフで-120℃で50 nmの薄凍結切片を切り取り、50はニッケルグリッドメッシュコーティングされたホルムバール炭素上に置きます。 </li></ol>ナノ金粒子映像設備のための4。銀強化lization 注：反応混合物を製造するための手順は、基本的には、製造業者が示す1です。このプロトコルを使用して実用的な適応は、凍結切片上で効果的かつ信頼性の高い銀増強手順を行う。 <ol><li>冷凍庫から銀増強キットを削除し、37℃インキュベーターまたは水浴中で解凍。解凍した場合には、使用するまで、暗い部屋の中に置かれ24℃のインキュベーター（4.7参照）でキットを配置。 </li><li>暗い部屋で熱板を置き、24℃の最終温度まで温める</li><li>表面保護、光沢のある面を上にして加熱プレートの上部を覆い、テープで固定します。 </li><li> Benchkoteにパラフィルムを配置し、暗所にパラフィルムの端を見ることができるようにするために黒のマーカーでエッジをマーク。 </li><li>熱板の温度を監視するBenchkoteの上に温度計をテープで固定します。徐々に温度を調整しない場合は、24℃ </li><li>ナンプラー蒸留水や内部の銀増強キットで50ミリリットルチューブをCE。 </li><li>再蒸留水を用いて小型のペトリ皿を埋め、37℃で予め温め試料は30分間、上下左右、水にグリッドを配置します。 </li><li>この4.7ステップ1より多くの時間を繰り返します。 </li><li>収納ボックスにグリッドを転送し、暗い部屋にそれらをもたらす。 </li><li>加熱プレート上に固定されているパラフィルム上に置かれ24℃の蒸留水を数滴に（下に試料側に）、それらを渡すことによって、再びグリッドを洗浄します。 </li><li> 9追加の蒸留水の水滴が銀増強反応後の洗浄に、パラフィルム上で準備ができていることを確認してください。 </li><li>光をオフにして、すべてのライトが消えていることを確認してください。赤色光をオンにします。 </li><li> 24℃のインキュベーターから銀増強キットのAとBのソリューションを取り出します。 </li><li>最初のA液6滴と1.5メートルのB液を6滴入れLマイクロチューブ、気泡を作るために避けてガラスパスツールピペットでよく混ぜる。溶液は非常に厚く、それが最終的に手動で簡単にそれをボルテックスする前にピペットで混合しなければならない。 </li><li> 24℃のインキュベーター中でAとBのソリューションを戻し、C溶液を取り出します。 </li><li> A / BミックスにC溶液6滴を追加します。その後、パスツールピペットで最初再び混合し、次いでボルテックスにより、気泡を作ることは避けてください。 </li><li>パラフィルム上（〜20μL/ドロップ）滴を配置することで、すぐに最終的な混合物を使用してください。 </li><li>クリーン、耐磁ピンセットで、得られることを望んだ機能拡張（金粒子のつまり 。サイズ）によっては6〜15分間混合物（ 例えば 、銀強化反応）にグリッドを配置します。 </li><li>混合物からグリッドを取り外し、洗浄液を24℃で低下し、二重蒸留水の上に渡します。まず、急速に連続して6滴での使用、ドロップあたりの7分のインキュベーション時間で3滴、ライトをオンにする前に。 </li><li>次のステップは、EM調査のために、結果を可視化するために免疫金標識または膜染色27のいずれかに進みます。 </li></ol>

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The authors have nothing to disclose.

免疫電子顕微鏡は、これらのタンパク質が存在するキャリアや細胞小器官の超微細構造の分解能でタンパク質の局在を組み合わせることが可能な技術である。酵母エンドソームシステムを研究する際に、その区画は蛍光顕微鏡のように断続構造が表示されますので、これは特に重大である。これは、それらを区別することは困難である。このような理由から、EMによって検出可能と時間依存?...

エンドソーム系は、1,2受容体リソソーム酵素ソーティング受容体のトラフィッキング及び原形質膜（PM）のリサイクルを含む複数の重要な細胞の役割を果たしている主要な膜選別装置である。エンドソームは、3つの異なるコンパートメント、 すなわち分割されている。初期エンドソーム（EE）、後期エンドソーム（LE）と、リサイクルエンドソーム。この分類は、特定のマーカータンパク質に及び、それらの形態で、それらに到達するためにエンドサイトーシスされた物質のにかかる時間に基づいている。膜は、午後から内部化すなわちタンパク質と脂質二重層は、分解のためにエンドソームを経由してリソソームに送達するか、または再循環する。膜はまた、ゴルジ体からエンドソームに輸送し、同様に、いずれのリソソームに継続するか、戻ってゴルジに検索することがされています。さらに、タンパク質は、エンドソーム境界膜の形成に至る過程から内方出芽管腔小胞に分類することができLEのサブカテゴリ、多胞体。 酵母エンドソーム系は、高い真核細胞のものよりも比較的複雑である。酵母エンドソームは、EEとLEに分かれています。哺乳動物細胞とは対照的に、それらはリサイクルエンドソームでなく、組織特異的なリソソーム関連オルガネラを含まない。そのため彼らは、エンドソーム人身売買ルート3,4の少ない複雑なネットワークを持っている。そのため酵母が表現され、今でもエンドソーム系に膜輸送の基礎となる原則のいくつかを研究するための有利な実験系を表しています。この利点は、エンドソーム経路に関与する多数の遺伝子が最初に酵母5での遺伝子スクリーニングで単離されているという事実によって強調される。 野生型および変異細胞における酵母エンドソーム系は広く生化学的および蛍光顕微鏡法アプローチを用いて研究してきたが、超微細構造レベルでの調査のみであった最小限。エンドソーム小器官のほとんどは彼らの明確な同定6困難に蛍光顕微鏡で断続構造として検出されるため、形態学的分析は、酵母において特に関連する。残念ながら、酵母エンドソームタンパク質マーカーを認識し、抗血清の限られた数は、（IEM）準備7月10日免疫電子顕微鏡で働いている。いくつかのタンパク質については、この問題は、目的の遺伝子の内因性のタグ付けとそれ7,11,12を検出するためのタグを認識する抗体を使用することによって回避されている。多くの場合、しかし、タンパク質が低いため発現レベルのIEMによって検出されない。この方法は細胞小器官の形態/機能にミス局在および/または変更を誘導することができるので、その過剰発現は、解決策ではありません。このように、電子顕微鏡EMによって検出可能なプローブを用いたエンドサイトーシス区画のラベルは、効果的なオプションです。これは、特にPR場合に最適なソリューションです。OBEは、特定の細胞小器官6を迎えます時に知ることができ、時間依存的にエンドサイトーシス経路を、入力している。 酵母スフェロプラスト（細胞壁が酵素的に除去されている、すなわち酵母）によって正に帯電したナノ金の取り込みに成功した酵母エンドソーム区画10は識別するのに使用されている。これらの粒子は強く、生体膜を構成する負に帯電した脂質に結合する。このようにPMと正に帯電したナノゴールドアソシエイツは、エンドサイトーシスによって細胞に浸透し、空胞に到達する前に、EEとLEを通過する。これらの小さな金粒子は、しかしながら、EMによって見られるように適切なサイズを有していない。彼らは目に見えるレンダリングするには、その大きさは、金型プローブ13〜15の周囲に銀や金の析出につながる化学反応によって拡大することができる。我々が開発され、首尾よく実行するために細胞内徳安法に基づくIEMアプローチを適用しているローカリゼーションは、8,16を研究。この方法では、形態学8,17-24の優れた分解能で酵母の準備に免疫金標識化を行うことができます。我々はまた、酵母エンドソーム系コンパートメント6のナノゴールドラベルとこのIEMプロトコルを組み合わせた手順を確立しています。我々は、形態学的に欠陥6,25エンドソーム輸送で変異体をエンドソームの異なるサブクラスを特徴とする超微細構造を調べてきたこのアプローチを使用する。また、我々は、このナノゴールド標識は異なるエンドソーム小集団に、目的のタンパク質の分布を探索する可能性を提供免疫金ラベル付けと組み合わせることができることを実証した。ここでは、正に帯電したナノゴールドと酵母エンドソームシステムの標識は、実質的にどのように実行されるか提示します。

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nanogold labeling of the yeast endosomal system for ultrastructural analyses

エンドソームは、真核細胞の主要な膜ソートのチェックポイントの一つであり、彼らは主に、細胞膜やゴルジ体からのタンパク質のリサイクルや破壊を調節する。その結果、エンドソーム系は、細胞の恒常性を維持する上で中心的な役割を果たしており、小胞輸送によって相互接続オルガネラのこのネットワークに属する遺伝子の変異は、がんや神経生物学的障害を含む深刻な病状を引き起こす。これは、エンドソーム系の生物発生と組織のメカニズムを理解することは、プライム関連性のある。酵母Saccharomyces cerevisiaeが 、この作業において極めて重要となっている。特にこのモデル生物のエンドソームシステムにラベルを付け、超微細構造レベルで解析するために、我々はここで銀増強反応によりこれらの粒子の可視化に続いてスフェロによって正に帯電したナノ金の取り込みのための詳細なプロトコルを提示する。この方法はあまりにも貴重なものですエンドソーム輸送における欠陥を有する突然変異体の形態学的検査のためのL。また、それのみならず、超微細構造検査のために適用可能であるが、それはまた、タンパク質の局在の調査のための免疫金ラベル付けと組み合わせることができる。

提示されたプロトコールに従って、酵母エンドソーム系の形態は、送信EMによってアクセスすることができる。 図1に達し、したがって、ナノゴールドで標識されているエンドソーム区画の異なるタイプを示している。銀増強ナノ金は明らかに電子密度の高い粒子として見ることができます。銀増強反応のために確立された最適な設定は、酵母細胞の超微細構造を妨害しない5〜15 ...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

酵母、 サッカロマイセス·セレビシエは 、エンドソーム系の生合成と機能を調節する遺伝子を同定し、研究するための重要なモデル生物となっている。ここでは、超微細構造の研究のためのエンドソーム区画の特異的標識のための詳細なプロトコルを提示する。

超微細構造解析のための酵母エンドソーム系のナノゴールドラベル付け

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Research

JoVE Journal

Biology

10.2K ビュー.  University Medical Center Utrecht. 酵母、 サッカロマイセス·セレビシエは 、エンドソーム系の生合成と機能を調節する遺伝子を同定し、研究するための重要なモデル生物となっている。ここでは、超微細構造の研究のためのエンドソーム区画の特異的標識のための詳細なプロトコルを提示する。 

ビデオ: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

ライブ固定化酵母細胞の可視化のための高速手法

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

生物学

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

切断者のためのOsseointegratedインテリジェントインプラント設計システムの生体電気分析

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

の蛍光標識ショウジョウバエハートの構造

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人間のDNA修復タンパク質の遺伝的研究は、モデルシステムとして酵母を使用

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

複数の遺伝子欠失を持つ酵母菌株の世代のためのグリーンモンスタープロセス

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

相互作用するリガンドおよび/またはアンタゴニストと、哺乳動物核内受容体残基を同定するために逆酵母ツーハイブリッドシステム

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...