著者らは、これらの方法が開発されており、チューリッヒ連邦工科大学の生化学研究所のマティアスピーターと彼のグループに感謝します。この作品は、スイス国立科学財団によってサポートされています。

1。顕微鏡測定<ol><li>酵母で合成培地5mlに植菌。 </li><li> 30℃で一晩細胞を成長させる。 </li><li>翌朝一晩培養物のOD 600を測定します。 </li><li> 600 0.05にOD 5ミリリットル合成培地中で一晩培養を希釈します。 </li><li>少なくとも4時間、30℃で希釈した培養を育てる。 </li><li>合成培地に集中して3倍（0.6 M）のNaCl溶液を調製する。 </li><li> PBSにコンカナバリンA（ConAを）の1 mg / mLの溶液を調製する。 </li><li>ろ液〜ウェルスライドにConAを溶液200μl。 </li><li> 30分間放置してみましょう。 </li><li>のConA溶液を除去。 </li><li>ウェルに150μlのH 2 Oを追加します。 </li><li> H 2 Oを削除</li><li>細胞培養のOD 600を測定します。 </li><li> 1.5ミリリットルマイクロチューブに= 0.02 OD 600で細胞培養液300μlを準備します。 </li><li> 45秒間、水浴中で細胞を超音波処理する。 </li><li>渦簡単にマイルcrotube。 </li><li> 45秒間、水浴中で細胞を超音波処理する。 </li><li>簡単に渦マイクロチューブ。 </li><li>ウェルに、細胞培養液200μlを加える。 </li><li>細胞がウェルの底に沈殿させるために30分待ちます。 </li><li>顕微鏡上でうまくスライドを配置します。 </li><li>記録される蛍光チャンネル用照明設定を選択します。 </li><li> （：細胞の適切なセグメンテーションを可能にするために、焦点面下の3程度やや焦点のうち、1）2明視野画像の照明設定を選択します。 </li><li>タイムラプス測定のための時間間隔を選択します。 </li><li>画像への視野を選択します。 </li><li>数フレームの取得を開始します。 </li><li> 0.6MのNaClを培地100μlを追加し、取得を一時停止します。 </li><li>画像を再開する。 </li></ol> 2。フローサイトメトリー測定<ol><li>酵母で合成培地5mlに植菌。 </li><li>一晩30℃で成長する。 </li><li>対策OD一晩培養の600翌朝。 </li><li> OD 600 0.1に5ミリリットル合成培地中で一晩培養を希釈します。 </li><li> 0.2〜0.4のOD 600に到達するために、少なくとも4時間、30℃で成長する。 </li><li> H 2 Oにシクロヘキシミド溶液1 mg / mlのを準備します</li><li>合成培地に集中して3倍（0.6 M）のNaCl溶液を調製する。 </li><li>測定対象の各時点での0.6 MのNaCl培地100μlで1 1.5ミリリットルマイクロチューブを準備します。 </li><li>時間0で、各マイクロチューブに細胞培養液200μlを加える。 </li><li> 30℃で振とうしながらインキュベート</li><li>時刻tでの、マイクロチューブに30μlのシクロヘキシミドを追加します。 </li><li>すべての希望する時点について、手順2.11。 </li><li>少なくとも45分間、すべてのマイクロチューブをインキュベートし、振とうしながら30℃まで3時間。 </li><li> 400μlのPBSでFACSチューブを準備します。 </li><li> 1分間水浴中で細胞を超音波処理する。 </li><li>簡単に渦マイクロチューブ。 </li><li>息子1分間水浴中で細胞をicate。 </li><li>簡単に渦マイクロチューブ。 </li><li>各マイクロチューブからそれに対応する、FACSチューブに100μlの細胞培養を追加します。 </li><li>検出のために488nmの励起レーザーと30分の530 nmのバンドパスフィルタを選択します。 </li><li>テスト非発現し、完全に彼らは検出器の感度の範囲に入るかどうかを確認するためにサンプルを表現する。 </li><li>取得した各サンプルについて、10,000個の細胞を測定します。 </li></ol>

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	<li>Gasch, A. P., Spellman, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+expression+programs+in+the+response+of+yeast+cells+to+environmental+changes.">Genomic expression programs in the response of yeast cells to environmental changes.</a> Molecular biology of the cell. 11 (12), 4241-4257 (2000).</li><li>de Nadal, E., Zapater, M., Alepuz, P. M., Sumoy, L., Mas, G., Posas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAPK+Hog1+recruits+Rpd3+histone+deacetylase+to+activate+osmoresponsive+genes.">The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes.</a> Nature. 427 (6972), 370-374 (2004).</li><li>Ghaemmaghami, S., Huh, W. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Wu, J. -. Q., Pollard, T. 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B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li><li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells.">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li><li>Carpenter, A. E., Jones, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome biology. 7 (10), R100 (2006).</li><li>Chernomoretz, A., Bush, A., Yu, R., Gordon, A., Colman-Lerner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Cell-ID+1.4+with+R+for+microscope-based+cytometry.">Using Cell-ID 1.4 with R for microscope-based cytometry.</a> Current protocols in molecular biology. Chapter 14, Unit 14.18 (2008).</li><li>Dimopoulos, S., Mayer, C., Rudolf, F., Stelling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+Single+Cell+Segmentation+in+a+Variety+of+Microscopy+Images.">Automatic Single Cell Segmentation in a Variety of Microscopy Images.</a> Current protocols in molecular biology. , (2012).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Current Opinion in Chemical Biology. 7 (5), 557-562 (2003).</li><li>Hersen, P., McClean, M. 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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

顕微鏡検査 ConAを持つウェルの治療は、細胞の適切な画像化を確保するための重要なステップです。のConAをPBS（5 mg / ml）を、低溶解性を有するため、濾過工程は、フィルタリングされていない溶液中に存在し、撮像を妨害する大きな凝集物を除去することができる。細胞が処理された表面に比較的強く付着し、誘導溶液の添加は、刺激の前と後の継続的な追跡を可能にし、...

伝達カスケードを介したシグナル伝達は、多くの場合、タンパク質の発現で絶頂に達する。この発現プロファイルの特徴付けは、生物学的経路の機能を理解する上で重要な要素です。アップレギュレートされたタンパク質およびその活性化の動態のスペクトルの同定は、例えば、マイクロアレイ、ノーザンブロットまたはウェスタンブロット1-3のような様々な技術によって達成することができる。しかしながら、これらの技術は、細胞の集団全体の応答を平均化する。タンパク質の発現の微調節を理解するためには、単一細胞レベルでの測定値を収集することが望ましい。理想的には、これらの測定は、根本的な経路の数学モデルを開発するために適し定量的データを提供すべきである。 顕微鏡およびフローサイトメトリー、理想的な定量的な単一細胞の測定値を提供するのに適している二つの技術、である。蛍光タンパク質とタンパク質の内因性のタグ付けはできるBeは、それらの発現レベル4を定量するために使用。タンパク質の末端における大きな蛍光部分の付加は、それを非機能的にすることができるので、それは、蛍光構築物の発現を駆動するプロモーターに基づいて、特定の発現レポーターを生成するために、多くの場合望ましい。このレポータータンパク質は、セルラーシステムに対して外因性であるため、セルに行われているシグナル伝達事象に影響を与えることはない。 顕微鏡法およびフローサイトメトリーの両方が広く酵母シグナリング分野で使用されてきた。例として、コールマン·ラーナーらは相関単一細胞では、酵母接合シグナル伝達カスケード5のノイズを定量化するために顕微鏡によって具体的かつ構成的に発現記者を交配の発現、Acarのと制御ネットワークを研究するフローサイトメトリーを使用し、同僚GAL遺伝子6の発現を制御する。以前の研究7において、我々はcombinatiを使用している出芽酵母では高浸透圧グリセロール（HOG）経路からの発現の出力を研究するため、これら2つの技術の上。このマイトジェン活性化プロテインキナーゼ（MAPK）経路は、超浸透圧ストレスによって誘発される。それは、およそ300の遺伝子の発現を生じる転写プログラムを誘導するために、細胞の核に移行しHOG1 MAPKの活性化をもたらす。このプロセスを研究するために、我々は、四重ビーナス蛍光タンパク質（STL1のp-QV）の発現を駆動STL1プロモーター（HOG1活性8に応答して特異的に誘導される遺伝子）に基づく発現レポーターを設計した。測定はフローサイトメトリー、蛍光レポーターを表現する人口のほんの一部に中間ストレスレベル（0.1MのNaCl）で細胞の2集団が存在することを発見した。今後も、この動作を調査するために顕微鏡を使用し、タンパク質発現におけるこのノイズは本質的な要因9に支配されたことを発見した。我カップLDさらにHOG1活動の同様のレベルを有する細胞が著しく異なる発現の結果を表示できることを確認します。これら2つの技術の組み合わせは、私たちはストレス応答遺伝子の遅いリモデリングは、単一細胞レベル7での発現の結果に影響を与えたかを証明することができました。 本論文では、顕微鏡によるタンパク質発現の定量化の一例として、超浸透圧ショックにより誘導されたP STL1-QVレポーターの式を使用し、フローサイトメトリー。 0.2 MのNaClストレスを受ける同じ株を、両方の技術を用いて研究した。これは、私たちは、これらの2つの高度に補完的な技術のいくつかの重要な違いを強調することができます。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Yeast Nitrogen Base</td><td>ForMedium</td><td>CYN6202</td><td></td></tr><tr><td>CSM amino-acid mix</td><td>ForMedium</td><td>DCS0011</td><td></td></tr><tr><td>Concanavalin A</td><td>GE Healthcare</td><td>17-0450-01</td><td></td></tr><tr><td>Cycloheximide</td><td>Fluka Aldrich Sigma</td><td>C7698</td><td>Toxic</td></tr><tr><td>ySP9</td><td></td><td></td><td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td></tr><tr><td>pSP34</td><td></td><td></td><td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td></tr><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Ti-Eclipse</td><td></td></tr><tr><td>Microscope control software</td><td>Micro-manager</td><td>Ver 1.4.11</td><td></td></tr><tr><td>Incubation chamber</td><td>LIS</td><td>The Box</td><td></td></tr><tr><td>Fluorescence light source</td><td>Lumencor</td><td>SpectraX</td><td></td></tr><tr><td>Camera</td><td>Hamamatsu</td><td>Flash 4.0</td><td></td></tr><tr><td>Flow cytometer</td><td>BD</td><td>FACS calibur</td><td></td></tr><tr><td>Sonicator bath</td><td>Telesonic</td><td>TUC-150</td><td></td></tr><tr><td>8-well-slide </td><td>Thermo Lab-tek</td><td>155409</td><td></td></tr><tr><td>96-well-plate</td><td>Matrical bioscience</td><td>MGB096-1-2-LG</td><td></td></tr><tr><td>FACS tube</td><td>BD Falcon</td><td>352054</td><td></td></tr></tbody></table>

temporal quantification of mapk induced expression in single yeast cells

単一細胞レベルでの遺伝子発現の定量化は、集団レベルで実行される測定に不明瞭新規調節機構をが明らかになりました。二つの方法に基づいて、顕微鏡およびフローサイトメトリーは、このようなデータを取得することができる方法を示すために提示される。酵母における高浸透圧グリセロールMAPK経路の活性化の際に誘導される蛍光レポーターの発現は、例として使用される。各方法の具体的な利点が強調表示されます。フローサイトメトリーは、細胞（10,000）を多数測定し、蛍光タンパク質の成熟遅い速度の独立したタンパク質発現の動力学を直接測定を提供する。顕微鏡による生細胞のイメージングは​​、少数の細胞におけるレポーターの成熟した形での測定に限定された対照的である。しかし、この技術によって生成されたデータ·セットは、複数のレポーターの組合せ及び空間的及び時間的情報のおかげで非常に豊富であることができる個々の細胞から得られた。これら二つの測定法の組み合わせはシグナル伝達経路によってタンパク質発現の調節に関する新たな洞察を提供することができます。

顕微鏡検査発現レポーターSTL1のp-QV（ySP9 7）を有する酵母細胞は、ウェルスライドの底部に取り付けられ、顕微鏡下に置いた。細胞は、撮像セッションの過程の間に直接ウェルに応力媒体を添加することにより刺激した。これは、私たちは、経路誘導前の細胞のいくつかの画像を取得し、刺激後、彼らの運命をたどることができます。この場合、細胞を10分間...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

フローサイトメトリーおよび顕微鏡検査に基づいて、二つの相補的な方法は、酵母におけるMAPK経路の活性化によって誘導される遺伝子発現の動態を、単一細胞レベルで、定量化を可能にする提示されている。

シングル酵母細胞中のMAPK誘導発現の時間的な定量

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

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Research

JoVE Journal

Biology

8.5K ビュー.  University of Lausanne. フローサイトメトリーおよび顕微鏡検査に基づいて、二つの相補的な方法は、酵母におけるMAPK経路の活性化によって誘導される遺伝子発現の動態を、単一細胞レベルで、定量化を可能にする提示されている。 

ビデオ: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

生物学

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

S の酵母酵母の低酸素応答中に時間をかけての mRNA のレベルを測定

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

遺伝学

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

酵母4 チオウラシルの定量化とラベリング代謝新しく合成された mRNA RNA ポリメラーゼ II の活性のためのプロキシとして

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

高齢者の細胞の伸長によって酵母年表寿命の定量化

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

ベーカー酵母サッカロミセス・セレビシエにおけるミトコンドリアゲノム発現産物の表示と可視化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

生化学

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

出芽酵母の蛍光タイムラプス作品を取得し、移植片を使用したシングルセルダイナミクスの解析

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

タバコ一過性発現系を使用したMAPKシグナル伝達経路の空間再編のイメージング

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

分裂酵母における細胞質分裂のイベントの時空間解析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...