We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

The authors declare no competing interests.

酵母細胞におけるエンドサイトーシスイベントの定量化のためのpHluorinのアプリケーションは、蛍光タグは、タンパク質（ 図1A）の細胞質尾部に融合された貫通貨物を利用します。ここに記載のアッセイのために、貨物はpHluorinタグがニュートラルな環境にさらされたときに明るく蛍光であるが、pHluorinタグは酸性条件に遭遇したときに急冷になります。したがって、pHluorinタグ付...

小胞輸送は、真核細胞内小器官の同一性および機能を維持する上で重要な役割を果たし、個々の細胞区画のタンパク質および膜組成を調節するための重要なメカニズムです。エンドサイトーシスを介して生成された小胞は、その後のリサイクルまたはリソソームに標的化するための表面から膜およびタンパク質を除去に対し、原形質膜で、エキソサイトーシス小胞の融合は、細胞の表面に新たなタンパク質や膜を実現します。このように、エンドサイトーシスは、栄養摂取のためおよび細胞外環境への応答のために重要です。エキソサイトーシスおよびエンドサイトーシスは、形質膜の表面積を調節するため、および損傷を受けたタンパク質の代謝回転を可能にするようにバランスされています。 酵母および哺乳動物細胞での研究には、専用の様々なことに応答して、エンドサイトーシスに関与するタンパク質の大規模な数、ならびに特定の貨物の内在化を促進する複数のエンドサイトーシス経路またはその行為を同定していますvironmental条件。最もよく研​​究経路は、クラスリンおよびサイトゾルアクセサリータンパク質は、発生期のエンドサイトーシス小胞を安定化させるためにコート構造に集合したクラスリン依存性エンドサイトーシス（CME）、です。出芽酵母サッカロマイセス・セレビシエにおける実験は、力学と多くのエンドサイトーシスタンパク質1-3のための募集のために重要な洞察が得られています。特に、CMEの機械は非常に進化の過程で保存されているような酵母におけるCME関連タンパク質の大部分は人間のオルソログを持っていること。このように、出芽酵母は、高等真核生物に保存されているエンドサイトーシスのメカニズムを理解するための重要なツールとなっています。例えば、出芽酵母を用いた研究では、追加のエンドサイトーシスのアクセサリーの動員に続いて、CME構造（酵母における皮質アクチンパッチと呼ばれる）の形成と成熟がクラスリンと貨物結合アダプタータンパク質で始まる、多くのタンパク質の順次募集を伴う決定しましたタンパク質、ARP2 / 3媒介アクチン重合の活性化、および小胞の切断の1,2に関与するタンパク質の動員。皮質アクチンパッチにCME機械タンパク質の募集は、高度に秩序とステレオタイプのプロセスであり、多くのタンパク質のための募集の正確な順序は、CMEの機械の他の構成要素に関して確立されています。重要なことは、最近の研究では、哺乳動物細胞4でクラスリン被覆ピットでのCMEのタンパク質のための募集の同様の順序を確認しています。 CMEに加えて、多くの細胞型は、原形質膜5-7の小胞形成および内在化を促進するための代替メカニズムに頼る一つ以上のクラスリン非依存性エンドサイトーシス（CIE）の経路を有します。酵母では、我々は最近、低分子量GTPase Rho1とその活性化グアニンヌクレオチド交換因子（GEF）、ROM1 8,9を利用CIE経路を同定しました。 Rho1は、アクチン重合1を促進するためにformin Bni1を活性化させます酵母12でクラスリン非依存性エンドサイトーシスは、このフォームに必要とされる0,11、。また、タンパク質のα-アレスチンファミリーは、CME 13-17を通じて貨物のユビキチン化およびその後の内在化を促進し、またおそらくCIE 18に関与するタンパク質との直接的な相互作用を介して、CIE経路を介して貨物の内在化を促進するために、ユビキチンリガーゼRSP5を募集します。 CIE経路の様々なもRho1オルソログ、RhoAの9,19に依存しているクラスリン依存せず、食作用経路を含めた哺乳動物細胞、中に存在します。哺乳類CIEにおけるRhoAの役割は十分に理解されていません。従って、出芽酵母での研究は、哺乳類のCIEに適用可能な追加の機械的な洞察を提供してもよいです。 エンドサイトーシス事象の正確な定量は、エンドサイトーシスを調節する特定のタンパク質の役割に関する重要な情報を提供することができ、および貨物の内在またはprogressio上の変異の影響を明らかにすることができますエンドサイトーシス経路を介して、n個。この目的のために、生化学的方法は、リガンドの取り込みを監視するために、またはエンドサイトーシス積荷タンパク質の分解の速度を測定するために利用することができます。生細胞において、関心対象の貨物に緑色蛍光タンパク質（GFP）及びその変異体の融合は、貨物輸送の直接可視化を可能にします。 GFPは、（酵母または液胞）リソソームでの分解に耐性があるのでしかし、GFPタグ貨物は、エンドサイトーシスの定量化のための使用が制限されています。また、GFP蛍光は、pHの変化に部分的にのみ敏感であり、液胞ルーメン20,21内で検出可能なままです。その結果、GFPタグの蛍光は、貨物の残りの部分が劣化したずっと後に液胞に固執し、貨物の強度の全細胞ベースの定量化が原因液胞の長期的なGFP蛍光に不正確になることがあります。 液胞GFP蛍光の欠点を克服するために、我々は、以前にsuperecliptic pHを使用しましたluorin、中性pHで明るく蛍光を発するが、そのようなリソソーム液胞/ 20,22,23の内腔などの酸性環境下で蛍光を失うGFPのpH感受性変異体です。エンドサイトーシス貨物の細胞質尾部にpHluorinタグを配置することは、原形質膜でとpHluorinタグが細胞質（ 図1A）に露出したまま初期エンドソーム、上の荷物の可視化を可能にします。初期エンドソームが成熟するにつれ、エンドソームは、多胞体（MVBs）24を生成し 、エンドソームの内腔に出芽小胞への輸送のために必要な複雑な（ESCRT）機械パッケージ表面局在貨物を仕分け。内部MVB小胞に組み込まれている貨物については、pHluorinタグは小胞内腔に向いています。内部MVB小胞が酸性化されています。彼らはMVBs 20に成熟としてこのように、pHluorinタグ付きカーゴは、初期エンドソームの蛍光を失います。液胞とMVBのその後の融合は、MVB腔の内容を提供します分解のために、とpHluorinタグは液胞内腔の酸性環境中で急冷したまま。 本稿では、酵母における細胞質pHluorinタグで貨物を用いた定量的エンドサイトーシスアッセイの詳細な説明を提供します。 pHluorinタグ貨物を発現する株は、特定の貨物の性質および輸送の挙動に応じて、運動及び/又はエンドポイントアッセイに使用することができます。さらに、いくつかのpHluorinタグ貨物は、フローサイトメトリーなどのハイスループット分析法に適しています。重要なことは、pHluorinタグは、野生型と変異株におけるエンドサイトーシス機能のエンドサイトーシスとの比較の定量化を可能にする、生きた細胞内のエンドサイトーシス事象を研究するための汎用性の高いツールです。

<table><tbody><tr><td>Adenine</td><td>Sigma</td><td>A8626-25G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Bacto-agar</td><td>Fisher</td><td>BP1423-2</td><td>Use for preparation of plate media</td></tr><tr><td>BD Difco Yeast Nitrogen Base (without amino acids)</td><td>BD</td><td>291920</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>Concanavalin A</td><td>Sigma</td><td>C5275-5MG</td><td>Use for coating of chamber slides</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>BP350-1</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>L-Histidine</td><td>Fisher</td><td>BP382-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Leucine</td><td>Acros</td><td>125121000</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Lysine</td><td>Fisher</td><td>BP386-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tryptophan</td><td>Fisher</td><td>BP395-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tyrosine</td><td>Acros</td><td>140641000</td><td>Use for preparation of amino acid mixture</td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass (8-well)</td><td>Thermo Scientific</td><td>155411</td><td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Axiovert 200 inverted microscope</td><td>Carl Zeiss</td><td>Custom Build</td><td></td></tr><tr><td>100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td><td>Carl Zeiss</td><td></td><td>Objective should be 100X, 1.4NA or higher</td></tr><tr><td>Sensicam</td><td>Cooke Corporation</td><td></td><td>Camera should have 12-bit or higher dynamic range</td></tr><tr><td>X-Cite 120PC Q Illumination Source</td><td>Excelitas Technologies</td><td></td><td></td></tr><tr><td>Slidebook 5 software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></td><td>http://imagej.nih.gov/ij/</td></tr></tbody></table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

緑色蛍光タンパク質（GFP）およびその変異体は、広くタンパク質の局在や、細胞骨格の再構築と生きた細胞内小胞輸送などのイベントのダイナミクスを研究するためのツールを使用しています。キメラGFP融合物を用いた定量方法は、多くの用途のために開発されています。しかし、GFPは、タンパク質分解に対して多少耐性があるので、その蛍光は、エンドサイトーシス経路に貨物輸送の定量化を妨げる可能性がリソソーム/液胞に持続します。エンドサイトーシスとエンドサイトーシス後の人身売買のイベントを定量化するための別の方法はsuperecliptic pHluorin、酸性環境中で急冷されるGFPのpH感受性の変異体を使用しています。多胞体（MVBs）およびリソソーム/液胞の内腔への配信への貨物の取り込み時に蛍光の減衰における膜貫通貨物タンパク質結果の細胞質尾部にpHluorinのキメラ融合。このように、液胞蛍光の消光はquantifiを容易にエンドサイトーシスとエンドサイトーシス経路における初期の事象のカチオン。本稿では、フローサイトメトリーを用いて、蛍光顕微鏡を介したエンドサイトーシスの定量化のためのpHluorinタグ付き貨物だけでなく、集団ベースのアッセイを使用する方法について説明します。

構成的に内在化エンドサイトーシス貨物タンパク質STE3の定常状態の局在化および定量化 生きている細胞、STE3、酵母におけるA因子フェロモン受容体の細胞質C末端テイルを有するキメラGFPとpHluorin融合の局在化pHluorinタグ貨物はエンドサイトーシスの定量化のために利用できることを実証するために、比較しま...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

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

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 ...

applications-phluorin-for-quantitative-kinetic-high-throughput

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.8K ビュー.  The Johns Hopkins University. 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.

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

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

細胞培養およびひよこ胚における軸索誘導受容体のダイナミクス評価におけるpHluorinの使用

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

神経科学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

生物学

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

細胞イメージングおよびデータ分析：SH-SY5Y神経芽腫細胞における神経伝達物質小胞のダイナミクスを評価するためにTIRFMとpH感受性GFP-プローブ

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

液胞と細胞質のpHの測定<emインビボで&gt;</em&gt;酵母細胞懸濁液に

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols 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 ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

細胞内局在や人身売買を監視するpHluorinタグ付き受容体を利用します

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

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 ...