我々は、様々なFP-TUB1プラスミドを共有するためのケリー・ブルーム（ノースカロライナ大学）、カイアング・リー（NCI）、スティーブン・マーカス（コロラド州立大学）、およびエルマー・シーベル（理学部ハイデルベルク）を感謝します。私たちは、スライドチャンバーの調製方法で私たちを訓練するためのメリッサ・ガードナー（ミネソタ大学）に感謝しています。この作品は、国立衛生研究所（NIH）（JKM）にR01GM112893-01A1と（CE）はT32GM008730を付与することにより、サポートされていました。

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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

出芽酵母モデルは、経時画像単一微小管に能力および酵母遺伝学のツールを使用してチューブリン及び微小管調節を操作する能力を含む、 インビボ設定において微小管ダイナミクスの高分解能測定値を収集するための主要な利点を提供します。 コンカナバリンAでコーティングされたチャンバーは、溶融寒天パッドを含む前述の装置、より多くの利点を提供します...

微小管は、αβチューブリンタンパク質サブユニットからなる細胞骨格ポリマーであり、細胞内輸送、細胞分裂、形態形成、および運動性を含む細胞状況、多種多様に使用されています。これらの多様な役割のための微小管ネットワークを構築するために、細胞は、慎重に、いつどこで微小管の形を調整する必要があります。この規則は、遊離のサブユニットに微小管ポリマーまたは逆アセンブル微小管にαβチューブリンサブユニットを組み立てるのいずれかことが反応を制御することによって達成されます。これは、微小管ダイナミクスとして知られています。 微小管分野の主要な目標は、αβチューブリンサブユニットおよび/または微小管にチューブリンと結合する外因性制御因子の研究を含む微小管ダイナミクスを調節する分子メカニズムを解明することです。十分に確立された実験的なアプローチは、多くの場合に、COM、精製αβチューブリンタンパク質を用いてインビトロでこのシステムを再構築することです精製した外因性の規制当局とbination。これは有用なアプローチではあるが、再構成されたシステムでの微小管ダイナミクス生きた細胞で観察されたものから強く異なることは明らかです。例えば、微小管は、より速く成長し、 インビトロよりもインビボにおいても遅い縮小します。これらの違いは、細胞内の既知の外因性調節因子1の可用性、ならびにまだ未定義の要因に起因し得ます。したがって、微小管の規制当局およびネイティブ細胞の状況でのダイナミクスを混乱させる変異体の活性を測定することが重要です。 後生動物モデルは、微小管機能と高次組織を調査するための有力なシステムであることが証明されているが、いくつかの実用的な懸念が厳しく、微小管ダイナミクスの正確な測定のために、これらのモデルの有用性を制限します。まず、数十からセル当たり数千人に及ぶ微小管の高い数は、それが困難な自信を持ってすることができます時間をかけて個々の微小管を追跡します。多くの研究は、エンド結合（EB）家族の中でタンパク質のような選択的に微小管の両端に局在するタンパク質を、画像化することによってこの課題に対処します。しかし、これらのタンパク質は後生動物のみ2で成長している微小管の両端に局在することが知られています。したがって、これらのタンパク質の有用性は間接的にしかそのような破局の周波数と動力学の他の側面を、測定しながら直接、増殖速度を測定するように制限されます。第二に、ゲノム編集技術の進歩にもかかわらず、安定的に蛍光標識したチューブリンを発現するかまたは選択的にチューブリンまたは微小管のレギュレータを操作するために変異を導入した細胞を作成することは重要な課題です。また、後生動物における多くのチューブリンイソ型の存在は、変異は、個々のチューブリン遺伝子に影響を与える方法の研究を混乱させる。 出芽酵母系は、in vivo microtubu に測定するために、いくつかの重要な利点を提供しますルダイナミクス。酵母は、個々の微小管の可視化を可能に簡略化微小管ネットワークを持っています。酵母では、微小管は、核膜3中に埋め込まれている紡錘体極体（のSPB）、として知られているセンターを組織から発します。 SPBは、微小管4,5の核γチューブリン小複合体のための足場として機能します。 SPBは、微小管、スピンドル微小管とアストラル微小管の2つのクラスを核形成します。核質にスピンドル微小管プロジェクトと動原体微小管を経由し、極間微小管6を重ね経由してスピンドルを安定化させるための染色体に取り付けるために重要です。対照的に外側にサイトゾルに、アストラル微小管プロジェクト及び紡錘体微小管の緻密なネットワークに比べて比較的まれです。有糸分裂の際、事前に後期の細胞は、SPBのいずれかから発せられるだけで1-2アストラル微小管を持っています。これらは私のように存在してndividual微小管ではなく、バンドル7のように。有糸分裂時のアストラル微小管の役割は、つぼみネックとして知られている母親とつぼみのコンパートメントとの間の接合部の中に核とスピンドルを移動させることです。この動きは、向かって、最終的に芽ネック8に核とスピンドルを引く、アストラル微小管に力を生成する明確に定義された経路を含みます。 酵母系の別の利点は、比類のない精度で微小管調節剤及びチューブリンサブユニットを調査するために使用することができ、その遺伝学の有用性です。単一βチューブリン遺伝子（TUB2）および2つのαチューブリン遺伝子（TUB1とTUB3）：酵母はまた、チューブリンアイソタイプの簡略化されたレパートリーを有します。変異は、容易にこれらの遺伝子に導入し、それによって均質なチューブリン集団9、10で研究することができます。広くは数多くあります利用できるラベリング微小管のための構造、およびこれらは、安定な発現のための染色体座での統合のために標的とすることができる（説明を参照）。 この方法の全体的な目標は、微小管ダイナミクスの高分解能測定のための4つの次元（X、Y、Z、およびT）で酵母細胞を生きた画像単一微小管です。酵母細胞における蛍光標識チューブリンの恒常、低レベルの発現のための構築物を統合するための方法が記載されています。撮像前に、生きている細胞は、長期的なイメージングのための細胞を安定化するためにレクチンコンカナバリンAで被覆したスライドチャンバーに取り付けられています。画像取得のための最適なパラメータ、ならびにデータ分析のためのワークフローは、また、記載されています。

<table><tbody><tr><td>DOB (dropout bases)</td><td>Sunrise science&amp;nbsp;</td><td>1650</td><td></td></tr><tr><td>CSM-Leu</td><td>Sunrise science&amp;nbsp;</td><td>1005</td><td></td></tr><tr><td>Agar</td><td>Ameresco</td><td>N833</td><td></td></tr><tr><td>100 mm polystyrene plates</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>ssDNA (Samon Sperm)&amp;nbsp; in sterile DiH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;D7656</td><td>resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Synthetic Complete Media&amp;nbsp;</td><td>Sunrise science&amp;nbsp;</td><td>1459-100</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L7647</td><td>resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>Microscope Coverslips</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td>paraffin film</td></tr><tr><td>VALAP (Equal parts of Vaseline, lanolin and paraffin)</td><td></td><td></td><td>melt at 75 &amp;ordm;C before use</td></tr><tr><td>forceps&amp;nbsp;</td><td>Fisher Scientific</td><td>16-100-106</td><td></td></tr><tr><td>Poyethylene glycol (PEG) 3350</td><td>Sigma-Aldrich&amp;nbsp;</td><td>202444</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ti E inverted Perfect Focus microscope</td><td>Nikon Instruments</td><td>MEA53100</td><td></td></tr><tr><td>1.45 NA 100X CFI Plan Apo objective</td><td>Nikon Instruments</td><td>MRD01905</td><td></td></tr><tr><td>Piezo electric stage&amp;nbsp;</td><td>Physik Instrumente</td><td>P-736</td><td></td></tr><tr><td>Spinning disk scanner</td><td>Yokogawa</td><td>CSU10</td><td></td></tr><tr><td>Laser combiner module</td><td>Agilent Technologies</td><td>MCL400B</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor Technology</td><td>iXon Ultra 897&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NIS Elements software</td><td>Nikon Instruments</td><td>MQS31100</td><td></td></tr><tr><td>Microsoft Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks, Inc</td><td></td><td></td></tr><tr><td>ImageJ64</td><td>NIH</td><td></td><td>Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.</td></tr><tr><td>Bio-Formats Importer plug-in</td><td>Open Microscopy Environment</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pUC19-LEU2::GFP-TUB1</td><td></td><td>pSK1050</td><td>reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)</td></tr></tbody></table>

high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast

ダイナミック微小管は、多くの細胞プロセスの基本である、と微小管ダイナミクスの正確な測定は、細胞が、これらのプロセスを調節し、どのように遺伝的変異インパクトレギュレーション方法への洞察を提供することができます。後生動物モデルにおける微小管ダイナミクスの定量化は、遺伝子操作上の高い微小管の密度および制限などの関連する課題の番号を持っています。これとは対照的に、出芽酵母のモデルは、これらの課題を克服する利点を提供しています。このプロトコルは、酵母生細胞における単一微小管のダイナミクスを測定する方法を説明しています。蛍光タグ付けされたチューブリンを発現する細胞は、安定したタイムラプス画像の取得を可能にする、組み立てられたスライドチャンバーに接着されます。高速の詳細なガイドは、四次元画像の取得はまた、共焦点画像スタック内の動的微小管の特性を定量化するためのプロトコルと同様に、提供されます。この方法では、従来の酵母の遺伝学と組み合わせて、PROV定量チューブリンサブユニットの活性を変化させる微小管調節剤または変異の効果を評価するための一意に適しているアプローチのIDE。

生きた酵母細胞において微小管ダイナミクスを測定する遺伝子の変異は、微小管調節剤またはチューブリンサブユニット衝撃重合及び解重合速度、ならびにこれらの状態間の遷移の周波数をエンコードする方法を評価するための魅力的なツールを提供します。 図1ディスプレイ野生型細胞におけるアストラル微小管ダイナミクスの時系列と（430Δtub2-）?...

Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

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

出芽酵母は、その強力な遺伝学とその微小管細胞骨格のシンプルさにin vivoでの微小管ダイナミクスを研究するための有利なモデルです。以下のプロトコルを変換し、培養酵母細胞、共焦点顕微鏡画像を取得し、かつ定量的に酵母生細胞における微小管ダイナミクスを分析する方法について説明します。

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 ...

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Universidad de Cartagena UDEC

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JoVE Journal

Biology

9.2K ビュー.  University of Colorado School of Medicine. 出芽酵母は、その強力な遺伝学とその微小管細胞骨格のシンプルさにin vivoでの微小管ダイナミクスを研究するための有利なモデルです。以下のプロトコルを変換し、培養酵母細胞、共焦点顕微鏡画像を取得し、かつ定量的に酵母生細胞における微小管ダイナミクスを分析する方法について説明します。 

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

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

TIRF顕微鏡による インビトロでの 架橋小管と単一微小管のダイナミクスの同時可視化

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

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 ...

生物学

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

分裂酵母セクシャルライフサイクルの顕微鏡観察

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

球状​​エマルジョン液滴での基本的な紡錘体の再構成

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

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 ...

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 ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

単極有糸状スピンドルにおけるスピンディスク顕微鏡による微小管ダイナミクスの測定

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

細胞下構造の超分解能ライブ細胞イメージング

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

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

要約

さらに動画を探す

この動画の章

TIRF顕微鏡によるインビトロでの架橋小管と単一微小管のダイナミクスの同時可視化

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

分裂酵母セクシャルライフサイクルの顕微鏡観察

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

球状エマルジョン液滴での基本的な紡錘体の再構成

分裂酵母における細胞質分裂のイベントの時空間解析

酵母の4D顕微鏡検査

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

単極有糸状スピンドルにおけるスピンディスク顕微鏡による微小管ダイナミクスの測定

細胞下構造の超分解能ライブ細胞イメージング

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

要約

さらに動画を探す

この動画の章

TIRF顕微鏡による インビトロでの 架橋小管と単一微小管のダイナミクスの同時可視化

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

分裂酵母セクシャルライフサイクルの顕微鏡観察

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

球状​​エマルジョン液滴での基本的な紡錘体の再構成

分裂酵母における細胞質分裂のイベントの時空間解析

酵母の4D顕微鏡検査

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

単極有糸状スピンドルにおけるスピンディスク顕微鏡による微小管ダイナミクスの測定

細胞下構造の超分解能ライブ細胞イメージング

TIRF顕微鏡によるインビトロでの架橋小管と単一微小管のダイナミクスの同時可視化

球状エマルジョン液滴での基本的な紡錘体の再構成