この作品は、バイオテクノロジーおよび生物科学研究会議（助成金31/C15982）、保健研究、イノベーションのためのカナダ財団、ブリティッシュコロンビア州の知識開発基金、健康研究のためのマイケルスミス財団（助成金CJRへのカナダの協会によってサポートされていましたローウェン）、およびサイトのファイト。

材料および方法： <ol><li>酵母菌株： <ul><li> Acp1 - GFP（GFP：：彼）：Acp1のC末端GFP -タグは、プラスミドpKT128を（GFPとHIS5を含む）を用いてPCRを介した相同組換えにより生成されました。陽性形質転換体は、共焦点顕微鏡とコロニーPCRにより確認した。ブーンラボ（トングとブーン、2006）から：歪みの背景には、BY7043（STE2pr - lue2lyp1Δhis3Δ1leu2Δ0ura3Δ0met15Δ0：MATアルファcan1Δ）であった。 </li><li>欠失変異体の配列（DMA）（XXX：：KanR）：それは、非必須遺伝子の約5,000の欠失変異体の配列です。これらの非必須遺伝子のすべては、G418にノックアウトされました。配列の歪みの背景には、BY4741（MATA his3_1 leu2_0 met15_0 ura3_0）です。私たちのDMAは、もともとブーンラボからだ。 </li></ul></li><li> DMAでAcp1 - GFPを組み込む： GFPの配列を作るための以下のプロトコルは、修正を加えた合成遺伝子解析（SGA）技術（トンとブーン、2006）を改作したものです。レプリカピニングのすべての手順は、シンガーローター高密度アレイ化ロボットシステムを使用することによって行われます。また、マニュアルのピン配置または液体ベースの高密度アレイ化ロボットを使用することができます。 略語：YPD、酵母エキス - ペプトン - デキストロース、SD、合成ドロップアウト、LRK、ルー/アルギニン/リジンドロップアウトメディア、LHRK、ルー/彼/アルギニン/リジンドロップアウトメディア 準備 <ol><li> YPDプレートに一晩Acp1 - GFPの培養5 mlを注いでAcp1 - GFPの細胞の芝生を育てる。板は炎によって、または生物学的なドラフト内で十分に乾燥することができます。 </li><li> 30˚Cで一晩インキュベートする。 </li><li>プレート当たり1536コロニーにロボット、配列Acp1 - GFP株を使用した。同時に、レプリカはYPD + G418プレートに固定することによって、DMAの新しいコピーを準備する。 </li><li> 30˚Cで一晩培養する。 交配と二倍体の選択 </li><li>レプリカのピン配置やYPDプレート上のコロニーを上に敷設によるDMAとAcp1 - GFP株をかん。 </li><li> 30˚Cで一晩培養する。 </li><li>二倍体細胞を（MATA / MATalpha）を選択するために彼の+ G418プレート - SD上にレプリカプレートコロニー。 </li><li> 30˚Cで一晩培養する。 Sporulationgと発芽 </li><li>減数分裂胞子の形成を誘導する炭素源および窒素源の削減レベルを含む強化された胞子形成のメディア上にレプリカプレート二倍体コロニー、。 </li><li> 25˚C 5日間の最小のプレートをインキュベートする。 </li><li> LRKメディアにレプリカプレーティングしたコロニーでMATA減数分裂の子孫を発芽し、二日間、30˚Cで培養する。 </li><li>このメディアは、胞子からの半数体細胞の発芽を誘発する。 LUE2オープニングリーディングフレーム（ORF）はMATA -特異的STE2プロモーターに統合されているため、発芽すべての半数はMATAになります。 両方の対立遺伝子の選択 </li><li>それは今でGFPの対立遺伝子と、単一の変異型対立遺伝子の両方を選択する必要があります。これを達成するために、マタ細胞は、遺伝子の欠失（：：kanR XXX）を運ぶ半数体細胞のために選択LRK + G418メディア 、上にレプリカメッキされています。 </li><li> 30˚Cで一晩培養する。 </li><li>またAcp1 - GFPを（：：彼のGFP）保有：最後に、MATA減数分裂子孫は、単一の変異株の増殖を選択するためにLHRK + G418メディア （：kanR XXX）上にレプリカメッキです。これらのプレートは˚Cまでの3ヶ月間4℃保存することができます。 </li></ol> GFPの配列を作るための以下のプロトコルは、修正を加えた合成遺伝子解析（SGA）技術（トンとブーン、2006）を改作したものです。レプリカピニングのすべての手順は、シンガーローター高密度アレイ化ロボットシステムを使用することによって行われます。また、マニュアルのピン配置または液体ベースの高密度アレイ化ロボットを使用することができます。 略語：YPD、酵母エキス - ペプトン - デキストロース、SD、合成ドロップアウト、LRK、ルー/アルギニン/リジンドロップアウトメディア、LHRK、ルー/彼/アルギニン/リジンドロップアウトメディア 準備 </li><li>顕微鏡： <ol><li>プレートから直接Acp1 - GFPを発現し、目的の変異体（s）を選ぶ。 </li><li> 30℃で細胞を成長° YPDまたはSDのC - 初期対数増殖期に彼のメディア。 </li><li> GFPのfiltersetを使用して共焦点顕微鏡により視覚化する。期待される結果の例は、図1に示します。 </li></ol></li></ol> SGAメディア： このプロトコルで使用されている増殖培地（YPD）と選択培地（SD）は、日常的に酵母分子生物学で使用されています。詳しい説明については、 酵母のメソッド （アンベルクら、2005）を参照してください。 LRK、LHRKメディア（400mlの合計） <table border="0"><tbody><tr><td>順番に追加</td><td>量は</td></tr><tr><td>硫酸アンモニウムと酵母窒素塩基</td><td> 2.7グラム</td></tr><tr><td>アミノ酸ミックス</td><td> 0.8グラム</td></tr><tr><td>寒天</td><td> 8グラム</td></tr><tr><td>のdH 2 O </td><td> 360 mLの</td></tr><tr><td colspan="2">オートクレーブ</td></tr><tr><td> 20％ブドウ糖</td><td> 40ミリリットル</td></tr><tr><td colspan="2"> 65˚Cに冷却</td></tr><tr><td> 100 mg / mLのカナバニン</td><td> 0.2ミリリットル</td></tr><tr><td> 100 mg / mLのthialysine </td><td> 0.2ミリリットル</td></tr><tr><td colspan="2">プレート当たり50mlを注ぎ、混ぜる</td></tr></tbody></table> LRK、LHRK + G418メディア​​（400mlの合計） <table border="0"><tbody><tr><td>順番に追加</td><td>量は</td></tr><tr><td>硫酸アンモニウムなし酵母窒素塩基</td><td> 0.7グラム</td></tr><tr><td>アミノ酸ミックス</td><td> 0.8グラム</td></tr><tr><td>グルタミン酸1ナトリウム</td><td> 0.4 </td></tr><tr><td>寒天</td><td> 8グラム</td></tr><tr><td>のdH 2 O </td><td> 360 mLの</td></tr><tr><td colspan="2">オートクレーブ</td></tr><tr><td> 20％ブドウ糖</td><td> 40ミリリットル</td></tr><tr><td colspan="2"> 65˚Cに冷却</td></tr><tr><td> 100 mg / mLのカナバニン</td><td> 0.2ミリリットル</td></tr><tr><td> 100 mg / mLのthialysine </td><td> 0.2ミリリットル</td></tr><tr><td> 200 mg / mlのG418 </td><td> 0.4ミリリットル</td></tr><tr><td colspan="2">プレート当たり50mlを注ぎ、混ぜる</td></tr></tbody></table>濃縮された胞子形成培地（400mlの合計） <table border="0"><tbody><tr><td>順番に追加</td><td>量は</td></tr><tr><td>カリウム酢酸</td><td> 4グラム</td></tr><tr><td>酵母の抽出</td><td> 0.4グラム</td></tr><tr><td>デキストロース</td><td> 0.2グラム</td></tr><tr><td>胞子形成アミノ酸ミックス</td><td> 0.04グラム</td></tr><tr><td>寒天</td><td> 8グラム</td></tr><tr><td>のdH 2 O </td><td> 360 mLの</td></tr><tr><td colspan="2">オートクレーブ</td></tr><tr><td colspan="2"> 65˚Cに冷却</td></tr><tr><td> 200 mg / mlのG418 </td><td> 0.4ミリリットル</td></tr><tr><td colspan="2">プレート当たり50mlを注ぎ、混ぜる</td></tr></tbody></table>

<ol>
	<li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual. , (2005).</li><li>Tong, A. H., Boone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+analysis+in+Saccharomyces+cerevisiae.">Synthetic genetic array analysis in Saccharomyces cerevisiae.</a> Methods Mol Biol. 313, 171-192 (2006).</li></ol>

このメソッドは、様々な変異体背景にミトコンドリアマーカー、Acp1 - GFPを組み込む効率的に助けることができる。それは、ロボットシステムの使用に依存しており、簡単にロボットシステムで使用するために採用することができます。この手順は、マーカーの他のタイプを組み込むために使用することができます。例えば、ERを視覚化するために、我々は日常的にマーカーErg11 - GFPを使う。?...

<table><tbody><tr><td>SGA media</td><td></td><td></td><td>The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.</td></tr></tbody></table>

a high-throughput method to globally study the organelle morphology in s. cerevisiae

タンパク質の局在や細胞小器官の形態を調べるためにハイスループット法は、タンパク質相互作用を研究するための効果的なツールであり、分子経路の包括的な理解を達成するの​​に役立ちます。出芽酵母では、非必須遺伝子の欠失配列の発展とともに、我々は世界的に小胞体（ER）およびミトコンドリアを使用してGFP（または変異型）マーカー異なる遺伝子背景のようなさまざまな細胞小器官の形態を学ぶことができます。しかし、それぞれの単一変異に組み込んだGFPマーカーは、個別に労働集約的なプロセスです。ここで、我々は日常的に我々の研究室で使用されている手順を説明します。高密度の酵母アレイと薬剤選択のテクニックを処理するためのロボットシステムを使用することによって、我々は、大幅に必要な時間を短縮し、再現性を向上させることができます。簡単に言えば、我々はロボットレプリカ確保することにより、4672非必須遺伝子の欠失変異体の高密度配列にGFP -タグ付きミトコンドリアマーカー（Apc1 - GFP）を渡ります。二倍体の選択、胞子形成、発芽およびデュアルマーカーの選択を通じ、我々は両方の対立遺伝子を回復する。その結果、それぞれの半数体の単一変異体は、そのゲノム遺伝子座に組み込まApc1 - GFPが含まれています。今、我々はすべての非本質的な変異体バックグラウンドにおけるミトコンドリアの形態を学ぶことができます。このハイスループットアプローチを使用して、私たちは便利な研究とゲノム全体の設定の継承と細胞小器官の形成に関与する経路と遺伝子を描くことができる。

Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

A high-throughput method to globally study the organelle morphology in S. cerevisiae

GFP融合タンパク質は、広く共焦点顕微鏡による細胞小器官を可視化するために使用されます。しかし、細胞小器官の形態に影響を与える変異のスクリーニングは通常、個々の変異誘発を必要とし、時間のかかる作業です。ここで、我々は同時に、酵母のほぼ5000の非必須遺伝子のオルガネラ- GFPマーカーを組み込む方法を示しています。

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

a-high-throughput-method-to-globally-study-organelle-morphology-s

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.3K ビュー.  University of British Columbia - UBC. GFP融合タンパク質は、広く共焦点顕微鏡による細胞小器官を可視化するために使用されます。しかし、細胞小器官の形態に影響を与える変異のスクリーニングは通常、個々の変異誘発を必要とし、時間のかかる作業です。ここで、我々は同時に、酵母のほぼ5000の非必須遺伝子のオルガネラ- GFPマーカーを組み込む方法を示しています。

ビデオ: A high-throughput method to globally study the organelle morphology in S. cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

出芽酵母サッカロミセスセレビシエにおけるサブソキアドタンパク質の局在化の評価

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

生化学

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

生物学

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 ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

液滴ベースのRNAシーケンシングによるハイスループット酵母株フェノタイピング

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

生物工学

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母の4D顕微鏡検査

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

成長と遺伝子発現の不均一性を測定するマイクロコロニーのハイスループットライブイメージング

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

S. cerevisiaeの減数分裂を研究するためのタイムラプス顕微鏡とタンパク質のステージ特異的核枯渇の使用

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...