We thank Stephan Uphoff for discussions. 
R.C. was supported by Linacre College, Oxford University. A.P. was supported by the German Academic Exchange Service (DAAD), the German National Academic Foundation and EPSRC. M.S. was supported by the Wellcome Trust. A.N.K. was supported by a UK BBSRC grant (BB/H01795X/1), and a European Research Council Starter grant (261227).

<ol>
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The authors have nothing to disclose.

多くのパラメータは、細胞のエレクトロポレーションおよび対象の生物学的システムと実験（細胞レベルまたは単一分子分析）の正確な性質に応じて、データ取得中に変化させることができる。細菌にDNAをエレクトロポレーションする際に、例えば、標識されたdsDNAフラグメントの0.25〜5ピコモル（事前に光退色を必要とすることなく、 すなわち 、）直接の単一分子の検出を可能にする、低い内在化効率をもたらす。 5 pmolのdsDNAの上方には、細胞は、単一細胞分析のための体制が適し、高負荷になる傾向がある。すべての標識されたDNAはまた、DNAのストック溶液から遊離染料（未反応のフルオロフォア）の痕跡を除去するために、先にゲル精製しなければならない。また、特にsmFRET実験のためのDNAの分解、潜在的な問題は、非天然の核酸、またはそのようなヘアピンループとしてエキソヌクレアーゼアクセス可能な末端を保護するモチーフがDNAを使用することによって対処することができる。 もうadjustablエレクトロポレーションの電子パラメータは、エレクトロポレーションの際に印加される電界強度である。低い電界強度（〜1kVの/ cm）は、単分子研究のための適切な低充填効率につながる。 （1.8 kVの/センチまで）より高い電界強度が積載効率を増加させる。しかしながら、エレクトロポレーション後の電界強度と細胞生存率との間に逆相関がある（ 図4参照）。参考のために、細菌および酵母エレクトロポレーションのために使用される通常の電界強度は〜1.5 kVの/ cmである。時定数は、すぐに任意のアーキング現象がキュベットで起こるように低下​​するので、この減衰の長さを表す時定数は、追従するための便利なパラメータである。通常の設定の下では、時定数は4ミリ秒以上でなければなりません。低い値は、低い積載効率あるいは非ロードされた損傷を受けた細胞につながる。ほとんどのエレ​​クトロポレーターは、両方の曲に変更することができる（例えば、「パルス切り捨て」または「パルス状」のような）他の自​​由度を提供セル·ローディングおよび生存。私たちは、その膜は（単一脂質二重層）は実際それほど複雑ではないので同じような手順では、また、適切なエレクトロの設定を用いて哺乳動物細胞への標識生体分子の内在化を可能にするはずである、細菌や酵母の両方にこの方法を適用し、エレクトロポレーションは、すでにこのような細胞で使用されているため21。 標識されたタンパク質を内在化すると、すべての遊離の色素は、標識されたタンパク質のストック溶液エレクトロポレーションの前に除去する必要がある。遊離色素分子は、それらの小さいサイズに、目的のタンパク質よりも優先的に内在化し、（それらの予想より速い拡散にもかかわらず）は、データ分析の間を区別するのが困難であることができる。有機的に標識されたタンパク質のサンプルのためのガイドは、エレクトロポレーションのために適切であるとして、遊離色素の残存量が2％未満であるべきである22（SDS-PAGEの蛍光スキャニングを用いて検出）。このプロセスは、特に重要であるいくつかの分子は、エレクトロポレーション、細菌や酵母の外膜に付着することがありますように。この点において、陰性対照試料は、（任意の蛍光標識された生体分子と共にインキュベートもエレクトロポレーションされていない細胞、 図2）は、空のセルの自己蛍光のレベルとして、理想的な低エレクトロポレーションした細胞よりも明らかに低い細胞当たりの蛍光強度を表示すべきである。 二本鎖DNAの場合と同様に、標識されたタンパク質の内在化効率は、エレクトロポレーションの前に細胞に加え、生体分子の量にリンクされる。しかしながら、そのような大きさおよび電荷などの他のパラメータは、内在化において役割を果たす。小さ ​​なタンパク質は、（kDaの98まで）より大きなタンパク質に対して、高い内部移行効率を示す成功し、より低い効率（ 図5）26と内部移行することができる。タンパク質の等電点、細胞膜との相互作用の可能性、また、他の物理化学的パラメータエレクトロポレーション時の影響セル·ローディング。その結果、ユーザーは標識タンパク質（&gt;50μM）の高い初期濃度が成功ロードするための最善の機会を与えることを知って、自分のシステムのための実験を​​最適化する必要があります。エレクトロポレーションも細胞（ラベルまたは非標識のいずれか）に、タンパク質および他の生体分子を導入することにより、細胞機能を撹乱し、分析するための新しいツールを提供しています。存在するT7 RNAポリメラーゼの実験（ 図5C）、我々は電気穿孔法を用いてインビボで遺伝子発現を変更することができる生体分子を導入することができ、実験のような例。 単一分子蛍光実験を行う際には、カバースリップの表面（〜100nm）を上記の薄切片内の励磁のみフルオロフォアで最高の信号対雑音比を提供するように、TIR照明は、通常、他の照明モードよりも好まれる。しかし生きた微生物の中に拡散イメージングラベル生体分子が再かもしれない（ 大腸菌用0.8ミクロンまで）帖より深い照明。高い信号対雑音比を維持しながら、より深い照明は、HILOモードで達成される。一方、広視野画像は、ユーザが高いレーザパワー全体ロードされたセルを光退色し、生産ユニタリ強度によって初期細胞の蛍光強度を割ることによって内在化される分子の数を推定された段階的な光退色の分析のために特に重要である単一分子（単一光漂白工程と、 図3）による。それらの軌道がセル全体の体積をカバーする場合でも、広視野イメージングは​​また、関心のある拡散分子を局在化するために、長期的な分子の追跡に必要とされる。 このプロトコルでは、エレクトロポレーション、細胞内で核酸を送達するための生物学者や生化学者のための標準的な技術は、種々の細胞型蛍光生体分子を送達するための簡単​​な方法を構成する方法を提示する。目小説、ハイスループット技術は、それらのネイティブ環境で標識された分子を観察するユニークなツールを提供しています。広い波長範囲をカバーするフルオロフォアで標識された生体分子の付加では、エレクトロポレーションは、非天然のヌクレオチドおよびアミノ酸、金属キレート剤、架橋剤、及びケージング基のような多くの化学基で修飾された分子を送達することができる。対象の生物学的システムは、細胞の発達に必須ではない場合には内在化後に観察されたタンパク質は、細胞内タンパク質プールのすべて（またはほとんど）を表すことを保証する、標的タンパク質をコードする遺伝子はまた、削除することができる（またはノックダウン） 。本質的には、エレクトロポレーションは、「移植」生きている細胞へのin vitroでのバイオコンジュゲートの柔軟性、したがって、合成生物学、システム生物学の取り組み、および生体内の単一分子検出に利益をもたらすことができます。

生きた細胞内部の大部分の蛍光の研究では、GFP 1などの蛍光タンパク質（FPS）、とのタンパク質融合に依存します。これらの蛍光タグは、遺伝子発現または膜輸送2-7のようなプロセスに関与するタンパク質のコピー数、拡散パターンまたは局在化の研究を可能にする。 FPSが高いラベリング特異性、容易な実装を提供し、様々な光物理的および化学的性質1を有する変異体の大規模な在庫でご利用いただけます。しかし、有機蛍光色素分子は、in vitroで彼らの大きな光安定性のための実験（最大のFPよりも安定100倍）8,9、小さいサイズ（最大のFPよりも小さなボリュームを100倍）と分子内ラベリングを容易にするために主要な選択肢のまま（主にシステイン残基の使用による）。これらの要因はすべて、単一分子蛍光のために特に重要であり、FRETが10を検討する 。 いくつかの内部移行方法のCOMBIN有機標識およびin vivo検出に有利で ​​るが過去10年間に導入されている。しかしながら、このような方法は、非天然アミノ酸15の使用を必要とするか、 例えば、（大きな単一膜の真核細胞に限定され、比較的大きなポリペプチドタグ（ 例えば 、TMP、HALO、または20kDaのSNAPタグ）11-14を使用するいずれか。 、ロード、注射器のロード、マイクロインジェクション）16-19をこすり。 このプロトコルは、新規な、 インビボでの観察と結合する有機蛍光体の長所を簡単かつ高スループットの内在化方法を記載している。この技術を開発するために、我々は、Eのように、一般に微生物をロードするために、プラスミドDNA 20,21を用いて細胞を形質転換するために使用されるエレクトロポレーションの手順を適合さcoliまたはS.有機的にラベル生体分子と出芽酵母 。プロトコルは4簡単な手順で構成されています。ラベルの生体分子と細胞のインキュベーション、エレクトロポレーション、細胞の回収、および細胞洗浄は、非内在化生体分子を除去する。ここで、我々は、細胞ベースの単一分子蛍光を研究するために、このエレクトロポレーションプロトコル、ならびに細胞イメージングおよびデータ分析プロセスを提示し、信号をFRET。 エレクトロポレーションは、生体分子（ 図1）20,21細胞に進入できるような一過性の膜孔を形成するために低イオン強度細胞懸濁液を横切って高電圧電場を放電に依存している。ただ、プラスミドDNAと細菌または酵母の形質転換と同様に、細胞は、それらのelectrocompetencyを確実にするために電気穿孔する前に用意しなければならない。この手順は、水で数回の洗浄工程からなる、膜透過性を増加させ、エレクトロポレーションキュベット中でのアーク放電を回避するために、細胞溶液のイオン強度を低下させる。このプロトコルでは、細胞は、（PROTOCOL参照：1.1）を下記のように調製することができるか、商業プロバイダから購入S。 <img alt="図1" src="/files/ftp_upload/52208/52208fig1.jpg" /> 図1：内在化プロトコルの概略図左から右へ：エレクトロコンピテント細胞（二重に標識されたDNA断片と、この例では、細菌）のアリコートに標識された生体分子の数マイクロリットルを加える。氷上で1から10分間インキュベートし、予め冷却したエレクトロポレーションキュベットに移す。エレクトロポレーションし、すぐ後に、細胞に0.5〜1ミリリットル豊富な培地を追加します。細胞が回復させるために37℃（または生物によって必要な温度、 例えば、酵母のための29℃）でインキュベート。余分な非内部化標識分子を除去するための5回の洗浄工程を行う。 PBS緩衝液およびピペットアガロースパッド上に10μlの100〜200μlの最終ペレットを再懸濁。 （広視野モードまたはHILOモード）蛍光顕微鏡での清掃カバーガラスと画像とのパッドをカバーしています。 図1）を洗浄する前に、リカバリを可能にする富栄養培地中でインキュベートする。非内在化標識生体分子の過剰な第1の塩のかなり高い濃度といくつかの界面活性剤を含有する緩衝液中で洗浄することにより除去される（3.3 PROTOCOL参照 ）。塩の存在は、そうでなければ、外膜に付着も非内部化ラベル生体分子によって形成され、非特異的な静電相互作用を破壊。同様に、洗浄緩衝液中の界面活性剤の存在は、非特異的疎水性相互作用を破壊する。 DNAの内在化は、（ 図2）簡単ですがエレクトロポレーションを用いて標識されたタンパク質を内部移行する際、予防措置が取られる必要がある。まず、有機的に標識されたタンパク質のストック·サンプルは、まだ遊離色素の小さな割合が含まれる場合があります。遊離色素分子は、タンパク質よりもはるかに小さいので、優先的に内在化されるかもしれない。観察に内在蛍光分子の大部分は、目的のタンパク質に対応するように、初期のタンパク質試料は、〜2％遊離色素（ 図5）22未満含まれている必要があります。非内在化さ標識タンパク質の過剰もエレクトロポレーション後、外側細胞膜に固執することができます。この現象は、タンパク質特異的であり、それぞれの新しいタンパク質をチェックする必要がある。我々は（：3.3.3プロトコルを参照してください）ロードされた細胞試料からの非内部化タンパク質の除去を可能にするいくつかのオプションを提案する。 最後に、細胞をリン酸緩衝液を少量に再懸濁し、蛍光顕微鏡でその画像化を可能にする、アガロースのパッド上にピペット。アガロースパッド上固定化は、SIMPLですEとそれらの完全性を損​​なうことなく、カバースリップ上の細胞を画像化する効率的な方法。パッドは、低蛍光培地が含まれている必要があります。 細胞イメージングを行うことができるいずれかの広視野、全内部反射蛍光（TIRF）またはHILO（高傾斜及び積層光学シート）を使用して顕微鏡検査。 HILO構成では、レーザビームは、TIRFよりも試料に深く浸透し、まだ大きな信号対雑音比23を可能にする、広視野用として試料全体を点灯しない。使用されるレーザパワーと時間分解能に応じて、内在化生体分子は、（段階的に、光退色分析を用いて、 図3）をカウント局在化、または24-28を追跡することができる。フルオロフォアのFRET対で二重に標識された構築物の内在化は、単一細胞または単一分子レベル（ 図6）でのFRETの両方の定量化を可能にする。 異なるパラメータを変化させることができる所望の出力に応じて、及び生物学的システムを研究。まず、細胞当たりの内在化材料の量は、標識された生体分子の濃度を変えることによって調整することができるエレクトロポレーションの前（ 図2）を細胞に添加した。エレクトロポレーションの電界強度も荷重効率および細胞生存率の両方に影響する。予想されるように、増加する電界強度と積載効率が増加しながら、エレクトロポレーションした細胞の生存率は、（ 図4A）を減少させる。両方のパラメータは、エレクトロポレーションの後にロードされ、分裂細胞​​の割合を記録することによって定量することができる。蛍光イメージングと相まって、この生存率アッセイはまた、 生きた細胞内に内在化生体分子の観察を検証し、数世代（ 図4B）にわたって連続観測を可能にします。 要約すると、このプロトコルは、に蛍光標識DNAおよびタンパク質分子の内在化を可能にする大腸菌またはS. cerevisiaeの 26。有機フルオロフォアで標識された個々の分子は、一桁長い時間スケールのためのFPよりも高い時空間解像度で追跡することができる。最後に、この方法は、広視野、TIRFおよび共焦点検出、ならびにALEX（レーザー励起28,29を交互に）としてパルス励起スキームと互換性がある。

<table><tbody><tr><td>ElectroMax DH5-alpha Comptent cells</td><td>Invitrogen</td><td>11319-019</td><td>or any other commercial or lab-mage electrocompetant bacteria or yeast.</td></tr><tr><td>EZ Rich Defined Madia</td><td>Teknova</td><td>M2105</td><td>low fluorescence rich media</td></tr><tr><td>MicroPulser Electroporation Apparatus&amp;nbsp;</td><td>Biorad</td><td>165-2100</td><td>or any classical electroporator for microorganism transformation</td></tr><tr><td>Certified Molecular Biology agarose</td><td>Biorad</td><td>161-3100</td><td>low fluorescence agarose for agarose pad</td></tr><tr><td>Microscope coverslips No 1.5 thickness</td><td>Menzel</td><td>BB024060SC</td><td>remove background particles by heating slides in furnace at 500 &amp;deg;C for 1h</td></tr><tr><td>Single-molecule fluorescence microscope</td><td>Home-built</td><td></td><td>described in REFs</td></tr><tr><td>Localization software</td><td>Custom-written, available online</td><td></td><td>MATLAB and C++ software package that can be adapted for localization analysis.&amp;nbsp;</td></tr><tr><td>Tracking software</td><td>Available online</td><td></td><td>MATLAB implementation by Blair and Dufresne.&amp;nbsp;</td></tr></tbody></table>

internalization and observation of fluorescent biomolecules in living microorganisms via electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Watch this Scientific Journal Video about Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation at JoVE.com

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

エレクトロポ生きた微生物で内在化および蛍光生体分子の観測

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

internalization-observation-fluorescent-biomolecules-living

Nanyang Technological University

Research

JoVE Journal

Biology

16.9K ビュー.  University of Oxford. Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

ビデオ: Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

単一分子複合体の可視化インビボでの高度な蛍光顕微鏡を用いて

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

生物学

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

哺乳動物細胞への機能性細菌エフェクターのエレクトロポレーション

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

免疫・感染学

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

日和見菌病原体への蛍光タグ付き遺伝子のバイオリスティック変容<em&gt;クリプトコックス·ネオフォルマンス</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

細胞の電気融合を蛍光顕微鏡で可視化

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

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 ...

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and mediates the delivery of protein and DNA substrates to target cells, a process generally requiring direct cell-to-cell contact. We have recently solved the structure of the Dot/Icm apparatus by cryo-electron tomography (cryo-ET) and showed that it forms a cell envelope-spanning channel that connects to a cytoplasmic complex. Applying two complementary approaches that preserve the native structure of the specimen, fluorescent microscopy in living cells and cryo-ET, allows in situ visualization of proteins and assimilation of the stoichiometry and timing of production of each machine component relative to other Dot/Icm subunits. To investigate the requirements for polar positioning and to characterize dynamic features associated with T4SS machine biogenesis, we have fused a gene encoding superfolder green fluorescent protein to Dot/Icm ATPase genes at their native positions on the chromosome. The following method integrates quantitative fluorescence microscopy of living cells and cryo-ET to quantify polar localization, dynamics, and structure of these proteins in intact bacterial cells. Applying these approaches for studying the Legionella pneumophila T4SS is useful for characterizing the function of the Dot/Icm system and can be adapted to study a wide variety of bacterial pathogens that utilize the T4SS or other types of bacterial secretion complexes.

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

レジオネラ肺球菌ドット/Icm分泌系の空間的特徴を解決するためのライブ細胞イメージングとクライオ電子断層撮影の適用

The Dot/Icm secretion system of Legionella pneumophila is a complex type IV secretion system (T4SS) nanomachine that localizes at the bacterial pole and ...

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 ...

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 ...

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 ...

Introduction of Bacterial Effector Proteins into Mammalian Cells by Electroporation

Source: Sontag, R. L. et al., <a href="https://app.jove.com/v/52296">Electroporation of Functional Bacterial Effectors into Mammalian Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the introduction of bacterial effector proteins into mammalian cells by electroporation. The intracellular localization of the bacterial effector proteins is detected by confocal microscopy following staining with specific fluorophore-conjugated antibodies.

エレクトロポレーションによる細菌エフェクタータンパク質の哺乳動物細胞への導入

Source: Sontag, R. L. et al., Electroporation of Functional Bacterial Effectors into Mammalian Cells. J. Vis. Exp. (2015)
In this video, we demonstrate ...