The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

蛍光ホスト細胞株の1世代<ol><li> 2×10 の 6細胞/ウェル6ウェルプレートに1日目プレート293T細胞（または他のレトロウイルスパッケージングライン）、〜75％コンフルエント翌日にします。必要に応じて、各レトロウイルス用プレートの重複ウェルを使用します。 </li><li> 2日目を吸引細胞と2 mlを加え、新鮮な増殖培地（DMEM + 10％FBS + 2mM L-グルタミン）。リポフェクタミン2000または同様の試薬 ​​を用いて、製造業者の指示に従って5-8μgのレトロウイルスベクター（GFPを含む）、パッケージングベクターのそれぞれ（ 例えば 、pVSVg）で細胞をトランスフェクトします。 </li><li> 37℃のCO 2インキュベーターで4〜6時間のためのDNAで細胞をインキュベートし、続いて1ミリリットルの成長培地で培地を交換してください。 </li><li> 37℃のCO 2インキュベーター中で 48時間、細胞をインキュベートします。 </li><li>ウェル10 5細胞 /のおおよその密度で6ウェルプレート上で3日目プレートの標的細胞（ 例えば 、HeLa細胞）、〜50％コンフルエントであるために次の日。倒立顕微鏡でGFP蛍光をモニターすることにより、293T細胞で陽性トランスフェクションを確認してください。 </li><li> 4日目は、ピペットを用いて293T細胞からレトロウイルス含有上清を収集し、0.45μmの酢酸セルロースシリンジフィルターを通して上清をフィルタリングします。 </li><li> HeLa細胞から培地を除去し、16 / mlのポリブレンを含む0.5 mlの増殖培地を追加します。賢明ドロップ、24時間、37℃のCO 2インキュベーターで細胞を培養する細胞にレトロウイルスのフィルタリング〜0.5ミリリットルを追加します。 4℃で（重複ウェルからすなわち 、）残りのレトロウイルスを保管してください。 </li><li>二連のウェルは、余分なレトロウイルスを生成するために行われた場合は5日目には、シリアル残りのレトロウイルス粒子を感染させるためにステップ1.7を繰り返します。 </li><li>選択抗生物質（例えば、500 / mlのネオマイシン）を含む10cmの皿に1：6日目通過は、HeLa細胞1をトランスフェクトしました。 </li><li>形質導入した細胞は、選択antibioの存在に戻って成長するのに十分な時間を待って細胞は、選択抗生物質（ 例えば 、200 / mlのネオマイシン）の低い濃度で、標準的な技術によって増殖させることができ、その後、チック。 注：必要に応じて、細胞はまた、クローン、この時点で理想的な明るさのために選択することができます。 </li></ol> GFP発現クラミジアトラコマティスの2世代<ol><li> 2倍のCaCl 2バッファー（20 mMトリスpHは7.4、100 mMのCaCl 2を）と4SPバッファー（0.4 Mショ糖、16mMのの Na 2 HPO 4）を準備します。 </li><li> 1日目解凍冷凍C.トラコーマ株式氷上で、すぐに50μlの2倍のCaCl 2バッファーに10μlの細菌を希釈。よく混ぜ、3μgのプラスミドDNA（ 例えば 、PASK-GFP-mKate2-L2）を追加し、25℃で30分間インキュベートします。 </li><li>このステップの間に、遠心管にMcCoy細胞のT-75フラスコをトリプシン処理することにより、宿主細胞を準備します。 5分間50×gで細胞懸濁液を遠心し、上清を捨てます。 Resuspen1XのCaCl 2緩衝液の適切な容積中のd細胞ペレットを4×10 7細胞/ mlの最終濃度を得ました。 </li><li> （ステップ2.2）から30分間インキュベートした後、形質転換混合物管（総体積200μlの）にMcCoy細胞を用意100μlを添加し、25℃でさらに20分間インキュベートします。 2ミリリットルの成長培地で6ウェルプレートとオーバーレイから単一のウェルに、この形質転換細胞の混合物の100μlのを追加します。 2日間、CO 2インキュベーター中、37℃でインキュベートします。 </li><li> 3日目を溶解McCoy細胞は 、Cに感染しました1ミリリットルのdH 2 Oで培地を交換し、曲がっ千μlのピペットチップを用いて細胞を掻き取ってトラコーマ 。 Cの効率的な細胞溶解および放出のための5回1ミリリットル4SPバッファを追加して、〜27.5 G針を通してサスペンションを渡しますトラコーマ 。 </li><li> C.を含む細胞溶解物を2mlの転送たてメッキMCCのコンフルエントな単層を含むT-75フラスコにトラコマチスオイ細胞; （FBSなし）8 mlのDMEMを添加し、Cを可能にするために25℃で2時間インキュベート細胞に付着するトラコーマ 。 </li><li>吸引し細胞および10 U / mlのペニシリンGと1 / mlのシクロヘキシミドを補充した新鮮な増殖培地を追加します。 37℃のCO 2インキュベーター中で 48時間、フラスコをインキュベートします。 </li><li> 4日目は、細胞が感染していると介在物が異常クラミジアが含まれている光学顕微鏡で確認してください。標準の光学顕微鏡に明るい空胞、および直径2μm以上である介在物内にリング状の物体などの異常クラミジア体として介在物を特定します。 </li><li> 2ミリリットルのdH 2 Oで培地を交換し、懸濁液中に細胞をこすり取ることにより、5日目を溶解文化。 2ミリリットル4SPバッファを追加し、約5倍の27.5 G針を通してサスペンションを渡します。 </li><li> 6ウェルプレート中で単一ウェルでMcCoy細胞の新鮮​​な単層を感染させるために、細胞溶解物の2ミリリットルを使用してください。 25℃で2時間溶解液で細胞をインキュベートし、吸引し、新鮮な成長を追加ペニシリンGおよびシクロヘキシミドを含む培地。 </li><li>細胞の一般的な健康状態に応じて、3〜5日間、37℃のCO 2インキュベーター中で細胞をインキュベートします。 </li><li>繰り返し（異常な体を欠いている）通常の探している介在物が出現するまで、6ウェルプレート中の新鮮なウェルに2.9から2.10 1回以上、必要に応じて、継代培養を繰り返します。この時点でことを確認するために倒立蛍光顕微鏡を使用するC.トラコーマは mKate2（赤）を発現します。 </li><li> T-150フラスコ中で増殖させ、感染したMcCoy細胞またはHeLa細胞からのクラミジアの形質転換体を採取することにより、たとえば、高力価のクラミジア株を生成するため、感染した培養物をスケールアップ。 </li></ol> 3. クラミジアで細胞を感染させます<ol><li> IFUの決意とマルチウェルスクリーニングのための、例えばガラスボトムディッシュまたはチャンバースライドの高解像度画像化のために、または24ウェルプレートのための所望の培養容器にプレートGFP-HeLa細胞、。 </li><li>あちこち解凍Cを含む禅チューブトラコマティス、C。 muridarum、またはC.氷上ニューモニエ例えばMOI = 1、感染の所望の多重度（MOI）にHBSS中に希釈します。 </li><li>使用される特定のクラミジア株に基づいて静的または遠心支援の感染症のいずれかを実行します。 <ol><li> C.での感染についてトラコマチス LGV血清型L2またはC. muridarumは 、HBSSでGFP-HeLa細胞を洗浄し、25℃で2時間希釈した細菌とインキュベートします。吸引し、HBSSで細胞を洗浄し、37℃のCO 2インキュベーター中で増殖培地中で細胞をインキュベートします。 </li><li> C.トラコマチス血清型DまたはCと感染症肺炎連鎖球菌は 、HBSSでGFP-HeLa細胞を洗浄し、細胞に希釈した細菌を追加します。卓上遠心機で、スイングバケットロータ取付のマルチウェルプレートホルダーにマルチウェルプレートまたは（マルチウェルプレートの蓋にテープで固定）ガラスボトムディッシュを置きます。 </li><li> 1時間25℃で900×gで遠心分離した細胞。 </lI&gt; <li> 1時間、37℃のCO 2インキュベーター中で遠心分離し、場所から培養容器を外します。 </li><li>安全キャビネット内を吸引細胞は、HBSSで細胞を2回洗浄し、新鮮な増殖培地を追加します。 37℃のCO 2インキュベーター内で場所細胞。 </li></ol></li></ol> クラミジア介在物の4生細胞の可視化<ol><li> CO 2インキュベーターからクラミジア感染GFP-HeLa細胞を削除し、フェノール5 +赤％のFBSを含まないRPMIで培地を交換してください。 </li><li> 20倍以上のオイル対物レンズを用いて倒立蛍光顕微鏡（ニコンエクリプスのTi-Eまたは類似の）上のマウント細胞。 </li><li>大きなブラックホール（ クラミジア介在物）を含む緑色の細胞：イメージングソフトウェア（Volocity 6または類似）を使用して、に焦点を当て、特定クラミジアは、GFP-HeLa細胞を感染させました。 </li><li>マークイメージングソフトウェアを使用して、関心のクラミジアに感染した細胞を含む領域のXYZ場所（巻ocity 6または類似）。手動XYステージ表示モードでしばらく「ポイントの追加」をすることによって、これを実行します。このように、XYZ座標は、各マークされた場所のために保存されています。 </li><li>取得設定]ダイアログボックスで、緑（GFP、ヒーラ細胞質ゾル）と赤（mKate2、 トラコーマクラミジア ）チャネル、およびチャネルごとに新しいフレームごとに5分の割合でを取得するソフトウェアを設定します。 </li><li>取得プロトコルを使用して画像シーケンスを取得」キャプチャ/録音」ボタンをクリックすることで、上記で入力しました。 </li></ol>生細胞内封入成長パラメータの5定量<ol><li>所望の時間感染後（ 例えば 、24、48 HPI）でインキュベーターからクラミジア感染GFP-HeLa細胞を除去し、倒立蛍光顕微鏡に取り付けます。注：高NAの20Xや40X空気目的は、24ウェルプレートのために最も適しており、40X油目的は、チャンバースライドをお勧めします。このお尻のためのいずれかの基板上に細胞を増殖させますAY。 </li><li>介在物は、その最大径であり、鮮明なエッジをもたらす細胞のミッドプレーン、に焦点を当てています。 </li><li>各ウェル（ウェル当たり少なくとも10）に多くの分野のための画像を取得します。ウェルは、通常、複製または実験変数を表します。 </li><li>目で（介在物は、GFP-HeLa細胞における黒区画されている）、手動で介在物の個数を列挙したり、自動的に介在物を特定し、カウントするイメージングソフトウェア・アルゴリズムを使用しています。 <ol><li>蛍光強度によって、GFP-HeLa細胞を探す（コマンド「オブジェクトの検索」）を、細胞の相対強度に基づいて、蛍光閾値強度を調整します。 </li><li> 52μm以下だけ大きいものを維持するサイズのガイドラインを使用して、選択したオブジェクトをフィルタリングします。これは、「人口1」です。 </li><li>入力として「人口1 &#39;を使用して、コマンド「オブジェクトの穴フィル」を適用します。このステップは、「人口2 &#39;を生成します。 </li><li> P &#39;から「人口1」を減算し「人口3 &#39;と指定積極マークさクラミジア介在物を、生成する「opulation 2。 </li><li>例えば、24時間以上のために感染した細胞内封入体のためには25μm2よりも大きなオブジェクトを保持するために、「人口3 &#39;（コマンド「人口フィルター」を）にサイズのフィルタを適用します。これらの介在物がはるかに小さいなどの前に18時間早ければ感染の時間については、設定サイズのフィルタは、4μm2でより大きなオブジェクトを保持します。 「介在物」として指定されたオブジェクトの集団でのサイズフィルタリング結果。 </li><li>イメージングソフトウェアを使用して、例えば、画像から封入番号、封入サイズ、及び包含外周の定量的データを計算します。 </li></ol></li></ol>

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著者は、彼らが競合する金融利害関係を有していないことを宣言します。

ここでは、リアルタイムの可視化とクラミジア介在物の分析のための蛍光宿主細胞を生成するための実験的な戦略について説明します。この液胞の可視化手法は、点灯トラックと定量的に細胞の集団間または経時単一細胞におけるクラミジア介在物の動的特性を測定するための強力な能力を付与する。蛍光タンパク質標識細胞におけるクラミジア介在物が著しく十分に定義され?...

クラミジアは性感染症、骨盤内炎症性疾患、失明、肺炎、おそらくアテローム性動脈硬化症1-4を含む、世界の健康に大きな負担を惹起細胞内細菌の種によって引き起こされる感染症。液胞の中から、宿主細胞と相互作用するクラミジアの能力は、（封入と呼ばれる）、それらの成功した細胞の感染および宿主のための重要な決定要因です。包含は、クラミジアの成長を可能にし、動的にクラミジア 5の全体の2〜3日の発達サイクルを通じて変更された新たな病原性の区画です。クラミジアの偏性細胞内性質は直接含めることのユニークな生物学を研究するため、特に研究コミュニティに多くの課題を提示。主要なハンディキャップを効率的に蛍光アプローチによって、細胞内のクラミジアやその含有物のいずれかを可視化することができないことでした生きた細胞内のES。最近の発見は、Cを GFP発現を生成するための手段を最終的に明らかにしましたトラコーマ 6;しかし、この発見は、まだ包含の特異的な標識には至っていません。いくつかの技術は、細菌や介在物7,8の標識に記載されているが、彼 ​​らは、このような光退色に対して非特異性、仮初や感受性などの欠点があります。私たちのグループによる重要な発見は、宿主細胞9 GFP発現を使用して含めることを照明するための新たな戦略を確立しました。この戦略は、合理的にダ10 520よりも大きい分子に包接膜の固有の不透過性を利用します。細胞を安定的に特定の細胞質の蛍光タンパク質（例えば、GFPまたはmCherryを）を発現するように操作された場合、 クラミジア介在物は、蛍光の彼らの完全な排除による著しい明快で見ることができます。この逆イメージング戦略は、すべてのクロロフィルのための介在物の即時の可視化を可能にしますamydia種と容易に関心のほとんど宿主細胞に適合させることができます。その有用性の実証として、この方法は、 クラミジア属 9のための細胞の出口経路を明らかにし、定義するために以前に使用されました。 ここでは、さらにこの方法が行われ、包接成長のダイナミクスについての重要な定量的データを得るために活用することができる方法を示しています。また、効果的にコストのかかる抗体ベースの列挙法の代わりにすることができ、そのようなクラミジア 11 mKate2発現などの他の蛍光標識と並行して使用することができます。ツールのこの強力な組み合わせは、宿主細胞を生きた内部クラミジア封入体膜の物性の探査を可能にします。

<table><tbody><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668</td><td></td></tr><tr><td>Opti-Mem</td><td>Invitrogen</td><td>31985</td><td></td></tr><tr><td>Polybrene</td><td>Sigma</td><td>H9268</td><td></td></tr><tr><td>0.45 &amp;micro;m filters</td><td>Fisher</td><td>09-719D</td><td></td></tr><tr><td>G418</td><td>Invitrogen</td><td>10131</td><td></td></tr><tr><td>HBSS</td><td>Invitrogen</td><td>14025</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>11995</td><td></td></tr><tr><td>RPMI 1640</td><td>Invitrogen</td><td>11875</td><td></td></tr><tr><td>RPMI 1640 w/o phenol red</td><td>Cellgro</td><td>17-105-CV</td><td></td></tr><tr><td>Penicillin G</td><td>Sigma</td><td>13752</td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma</td><td>C7698</td><td></td></tr><tr><td>Glass bottom dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Chamber slides, Lab-Tek II</td><td>Nunc</td><td>154526, 154534</td><td></td></tr></tbody></table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

サイトゾル蛍光タンパク質 （例えば、GFP）を発現する哺乳動物細胞を生、感染細胞培養物においてクラミジア介在物の照明を可能にするように設計することができます。 クラミジアの感染の際に、介在物は、宿主細胞（ 図1）における黒点として容易に見ることができます。蛍光欠けている介在物の透明度は、ビューおよび/ ​​または治療（ 図1）</st...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

視覚化し、定量化するために蛍光タンパク質を使用しました<em&gt;クラミジア</em生細胞内&gt;液胞の成長ダイナミクス

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

7.5K ビュー.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

ビデオ: Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

生化学

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

蛍光寿命イメージングによる細胞内のスネアを可視化

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

免疫・感染学

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

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

蛍光共焦点顕微鏡を用いた単純ヘルペス ウイルス 1 蛋白質の核-細胞質転流の時空間解析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

蛍光顕微鏡法による、複数世代にわたってライブ哺乳類細胞におけるプラスミドの複製を監視

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication.
Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

This protocol utilizes fluorescent promoter-reporters, live-cell microscopy, and individual inclusion extraction in a directed forward genetic approach to identify and isolate developmental mutants of Chlamydia trachomatis.

クラミジア・トラコマティスにおける発生変異体の同定と分離に対する生細胞前方遺伝的アプローチ

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

培養細胞における小胞体サブドメインの可視化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

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

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

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

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

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

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

視覚化し、定量化するために蛍光タンパク質を使用しました

要約

さらに動画を探す

この動画の章

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

蛍光寿命イメージングによる細胞内のスネアを可視化

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

蛍光共焦点顕微鏡を用いた単純ヘルペスウイルス 1 蛋白質の核-細胞質転流の時空間解析

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

蛍光顕微鏡法による、複数世代にわたってライブ哺乳類細胞におけるプラスミドの複製を監視

クラミジア・トラコマティスにおける発生変異体の同定と分離に対する生細胞前方遺伝的アプローチ

培養細胞における小胞体サブドメインの可視化

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

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

視覚化し、定量化するために蛍光タンパク質を使用しました

要約

さらに動画を探す

この動画の章

蛍光住セル完全の栄養細胞周期の遅い成長社会細菌Myxococcus ザンサス

蛍光寿命イメージングによる細胞内のスネアを可視化

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

蛍光共焦点顕微鏡を用いた単純ヘルペス ウイルス 1 蛋白質の核-細胞質転流の時空間解析

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蛍光共焦点顕微鏡を用いた単純ヘルペスウイルス 1 蛋白質の核-細胞質転流の時空間解析