Name: applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing
Uploaded: 2016-07-06T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

The authors have nothing to disclose.

分泌系、および細菌エフェクタータンパク質の宿主細胞の細胞質へのこれらのシステムの輸送は、多くの病原性細菌は、一意複製ニッチ内感染を確立するために利用する重要な病原性要素です。多くの研究グループの焦点は、細菌のエフェクターおよび宿主タンパク質およびこれらのエフェクターは、宿主細胞経路上に持っている影響力の間の相互作用を調査することでした。非常に限られ?...

Q熱リケッチアは人獣共通ヒト感染のQ熱の原因となるユニークな細胞内病原体です。この疾患は、多くの場合、暴露1の後に心内膜炎の年として現れる生命を脅かす慢性感染症に無症候性抗体陽転から延びる臨床症状の広いスペクトルに関連しています。ヒトへの感染は、反芻動物感染症、特に、乳牛、羊やヤギのための主要なリザーバと、主に汚染されたエアロゾルの吸入を介して行われます。これらの動物におけるコクシエラ感染は通常、無症候性であるが、感染は流産を引き起こす可能性と出産流体と胎盤内のかなりの細菌負荷は、ローカル環境1を汚染することができます。このような汚染が公衆衛生と農業産業の両方に持つことができる巨大な負担の例は、最近オランダ2で発生したQ熱の流行で観察されました。 2007との間2010年には、Q熱の4,000以上のヒト症例が診断され、この流行はヤギ農場3の重大な汚染に連結されていました。さらに、 コクシエラは厳しい罹患率と死亡率4を引き起こす細菌や必要な低感染量の環境安定性に米国疾病対策予防センター、によって分類され、潜在的な生物兵器です。 コクシエラは、2つの段階に存在する：天然源から単離されたフェーズIの生物は、非常に病原性であり、フェーズIIの生物は非常に生体内で減衰されます。例えば、 コクシエラバーネッティナインマイルフェーズIの生物のいくつかのin vitroで継代した後、フェーズII細菌は切り捨てリポポリサッカライド（LPS）5で得られた不可逆的な染色体欠失を含有する製造されました。この株、C.バーネッティ NMIIは、組織培養モデルにおけるI相に表現型が類似しており、安全なモードを提供します研究室5にコクシエラの病因を研究する研究者のためのリットル。近年では、いくつかのブレークスルーは急速にコクシエラの遺伝学の分野を進めてきました。最も顕著なのは、純粋メディア（酸性クエン酸、システイン媒体- ACCM-2）の開発は、液体と6,7固形培地上の両方でコクシエラの無細胞成長を可能にしました。これは、誘導性遺伝子発現システム、シャトルベクターとランダムトランスポゾンシステムを8月11日を含むコクシエラために利用可能な遺伝子ツールの直接的な改善をもたらしました。最近では、標的遺伝子の不活性化のための2つの方法は、特定の病原性遺伝子の候補12を検査するための道を切り開いて、開発されてきました。 肺胞マクロファージによる内在続いて、 コクシエラは膜結合区画内に高い数字に複製は液胞（CCV）を含むCoxiella-と呼ばれます。 CCVは、エンドサイトーシス人身売買番目のホストが必要ですラフ初期および後期エンドソームはリソソーム由来の細胞小器官13内に成熟するまで。このプロセスを通じて、CCVは、どちらかが一過現れるかを含め、液胞に関連付けられているが、Rab5の、Rab7、CD63およびLAMP-1 13-15、これらに限定されないままと宿主因子を取得します。宿主細胞内でコクシエラのレプリケーションが完全に機能するドット/ ICMタイプIVB分泌系（T4SS）8,16,17に完全に依存しています。この分泌系はancestrally共役システムに関する多タンパク質構造であり、細菌タンパク質を送達するために、両方の細菌および液胞の膜にまたがる、宿主細胞質18に、エフェクターと呼びます。 コクシエラ T4SSは、機能的には十分に特徴付けタイプレジオネラニューモ 19,20のIVBドット/ ICM分泌系に非常に類似しています。 コクシエラが酸性リソソーム由来に達するまで興味深いことに、T4SSとその後のエフェクター転座の活性化は発生しませんオルガネラ、約8時間の感染後17,21。現在までに、130以上のドット/ ICMのエフェクターは9,17,22-24を同定されています。これらのエフェクターの多くは、おそらく宿主細胞内コクシエラの複製中に重要な役割を果たしています。しかし、わずか数エフェクターは機能的に25-29を特徴づけられています。 本研究では、宿主細胞の細胞質（ 図1）内のβラクタマーゼ活性を介して（以下BLAM基板と呼ぶ）CCF2-AM FRET基質の切断に依存する蛍光に基づく転位アッセイを利用します。目的の遺伝子は、構成的発現を提供するレポータープラスミドのTEM-1βラクタマーゼ（BLAM）に融合されます。 BLAM基質はFRET対を形成する2つのフルオロフォア（クマリンおよびフルオレセイン）で構成されています。フルオレセインおよびエフェクター転座の不在下で緑色の蛍光発光のFRETのクマリン結果の励起;しかし、Blaの場合M-エフェクター融合タンパク質が宿主細胞質に転位され、得られたβラクタマーゼ活性切断励起以下の青色蛍光発光を生成するFRETペアを分離BLAM基板のβラクタム環、。この転座アッセイはよくC.含むさまざまな細胞内および細胞外の細菌の範囲からエフェクタータンパク質を同定するためのアプローチとして実証されていますバーネッティ 、L.ニューモ 、L. longbeachae、 トラコーマクラミジア 、腸管病原性E.大腸菌 、 サルモネラ菌およびブルセラ 17,30-35。 コクシエラエフェクター転上の特定の宿主因子の役割を決定するために、我々は、特定の低分子干渉RNA（siRNA）で、RNA干渉として知られている遺伝子サイレンシングのための十分に確立された方法を利用します。もともと線虫（Caenorhabditis elegans）で同定され、RNA干渉は、先天性defeに使用保存された内因性細胞プロセスであり、ウイルスだけでなく、遺伝子調節36,37に対してNSE。配列特異的なsiRNAの結合後、mRNAの分解は、特異的な遺伝子サイレンシングまたはノックダウン38で得られた（RNA誘導サイレンシング複合体）RISCを介して行われます。この研究では、siRNAは、エンドサイトーシス経路の重要な調節因子である2つのホストタンパク質、Rab5AとRab7Aを標的化するために使用しました。エフェクター転上Rab5AとRab7Aサイレンシングの影響は、Cを使用して確認しましたバーネッティ pBlaM-CBU0077。それは以前にコクシエラ 17のドット/ ICMの分泌系によって転位することが示されたようCBU0077を選択しました。 siRNAの遺伝子サイレンシング、ここでは説明fluorescencebased転座アッセイの両方を利用して、我々はコクシエラによってエフェクタータンパク質の転位に宿主因子の役割を確立するために始めています。このアプローチは、類似secretioを有し、両方の細胞内および細胞外の細菌の広い範囲に適用することができますエフェクタータンパク質のトランスロケーションを担当するN個のシステム。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>C0759</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S4641</td><td>ACCM-2 medium component</td></tr><tr><td>Potassium phosphate</td><td>Sigma</td><td>60218</td><td>ACCM-2 medium component</td></tr><tr><td>Magnesium chloride</td><td>Calbiochem</td><td>442611</td><td>ACCM-2 medium component</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080</td><td>ACCM-2 medium component</td></tr><tr><td>Iron sulfate</td><td>Fisher</td><td>S93248</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td>ACCM-2 medium component</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C6852</td><td>ACCM-2 medium component</td></tr><tr><td>Bacto-neopeptone</td><td>BD</td><td>211681</td><td>ACCM-2 medium component</td></tr><tr><td>Casamino acids</td><td>Fisher</td><td>BP1424</td><td>ACCM-2 medium component</td></tr><tr><td>Methyl beta cyclodextrin</td><td>Sigma</td><td>C4555</td><td>ACCM-2 medium component</td></tr><tr><td>RPMI + Glutamax</td><td>ThermoFisher Scientific</td><td>61870-036</td><td>ACCM-2 medium component</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td>For bacterial culture</td></tr><tr><td>ON-TARGETplus Non-targeting Control Pool (OTP)</td><td>Dharmacon</td><td>D-001810-10-05</td><td>Non-targeting control</td></tr><tr><td>siGENOME Human PLK1 (5347) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-003290-01-005</td><td>Causes cell death; measure of transfection efficiency</td></tr><tr><td>siGENOME Human RAB5A (5968) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-004009-00-0005</td><td></td></tr><tr><td>siGENOME Human RAB7A (7879) siRNA - SMARTpool</td><td>Dharmacon</td><td>M-010388-00-0005</td><td></td></tr><tr><td>5x siRNA buffer</td><td>Dharmacon</td><td>B-002000-UB-100</td><td>Use sterile RNase-free water to dilute 5x siRNA buffer to 1x siRNA buffer</td></tr><tr><td>DharmaFECT-1 Transfection Reagent</td><td>Dharmacon</td><td>T-2001-01</td><td>For transfection</td></tr><tr><td>Opti-MEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>51985-034</td><td>Reduced-serum medium used for transfection</td></tr><tr><td>DMEM + GlutaMAX</td><td>ThermoFisher Scientific</td><td>10567-014</td><td>For cell culture and infection</td></tr><tr><td>Heat-inactivated fetal calf serum (FCS)</td><td>Thermo Scientific</td><td>SH30071.03</td><td>Can use alternate equivalent product</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td>For storage of Coxiella strain</td></tr><tr><td>PBS</td><td></td><td></td><td>For cell culture</td></tr><tr><td>0.05% Trypsin + EDTA</td><td>ThermoFisher Scientific</td><td>25300-054</td><td>For cell culture</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>For dilution of samples and standards for qPCR</td></tr><tr><td>Quick-gDNA Mini Prep</td><td>ZYMO Research</td><td>D3007</td><td>Can use alternate equivalent product to extract gDNA</td></tr><tr><td>SensiFAST SYBR No-ROX Kit</td><td>Bioline</td><td>BIO-98020</td><td>Can use alternate equivalent qPCR master mix product for qPCR reaction</td></tr><tr><td>LiveBLAzer FRET-B/G Loading kit with CCF2-AM</td><td>Invitrogen</td><td>K1032</td><td>For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)</td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106469</td><td>Probenicid solution component</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma</td><td>71505</td><td>Probenicid solution component</td></tr><tr><td>di-Sodium hydrogen orthophosphate</td><td>Merck</td><td>106586</td><td>Probenicid solution component</td></tr><tr><td>Probenicid</td><td>Sigma</td><td>P8761</td><td>Probenicid solution component</td></tr><tr><td>DRAQ5 Fluorescent Probe Solution (5 mM)</td><td>ThermoFisher Scientific</td><td>62251</td><td>Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS.&amp;nbsp;</td></tr><tr><td>4% PFA (paraformaldehyde) solution in PBS</td><td>Sigma</td><td>P6148</td><td>Fixing solution for HeLa 229 cells</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430639</td><td>For growth of bacterial strain</td></tr><tr><td>96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile</td><td>Corning</td><td>3603</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>430641</td><td>For growth of HeLa 229 cells</td></tr><tr><td>175 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask with vented cap</td><td>Corning</td><td>431080</td><td>For growth of HeLa 229 cells</td></tr><tr><td>Haemocytometer</td><td></td><td></td><td>For quantification of HeLa 229 cells</td></tr><tr><td>Tear-A-Way 96 Well PCR plates</td><td>4titude</td><td>4ti-0750/TA</td><td>For qPCR reaction</td></tr><tr><td>8-Lid chain, flat</td><td>Sarstedt</td><td>65.989.002</td><td>For qPCR reaction</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;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bench top microfuge</td><td></td><td></td><td></td></tr><tr><td>Bench top vortex</td><td></td><td></td><td></td></tr><tr><td>Orbital mixer</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td>For pelleting bacterial culture</td></tr><tr><td>Nanodrop</td><td></td><td></td><td>For gDNA quantification</td></tr><tr><td>Mx3005P QPCR machine</td><td>Aligent Technologies</td><td>401456</td><td>For quantification of Coxiella genomes in 7 day culture</td></tr><tr><td>ClarioSTAR microplate reader</td><td>BMG LabTech</td><td></td><td>For measurement of fluorescence</td></tr></tbody></table>

applying fluorescence resonance energy transfer (fret) to examine effector translocation efficiency by coxiella burnetii during sirna silencing

Q熱リケッチア 、Q熱の原因物質は、複製ニッチを確立するために、タイプIVドット/ ICM分泌系に依存して細胞内病原体です。エフェクターのコホートは、ホストプロセスを操作し、複製のためのユニークなリソソーム由来の液胞の確立を可能にするために宿主細胞にこのシステム通って移動しています。特定の遺伝子のsiRNAを用いたサイレンシングし、βラクタマーゼ活性に依存するFRETベースの基板を用いて、エフェクター転位の測定：ここで提示された方法は、2つのよく確立された技術の組合せを含みます。これら二つのアプローチを適用すると、我々は、細菌の分泌システム機能およびエフェクター転における宿主因子の役割を理解し始めることができます。本研究では、Rab5AとRab7A、両方のエンドサイトーシス人身売買経路の重要な調節因子の役割を調べました。我々は、エフェクターの減少タンパク質をもたらすいずれかの発現をサイレンシングすることを実証translocation個の効率。これらの方法は、容易にまた分泌系を利用する他の細胞内および細胞外の病原体を検査するように変更することができます。このようにして、細菌のエフェクター転座に関与する宿主因子の全体像を明らかにすることができます。

この研究のために、C. CBU0077が以前コクシエラドット/ ICMの分泌系17の転エフェクターであることが示されているようにバーネッティ pBlaM-CBU0077株を選択しました。感染7日C.におけるゲノム/ mLの総数に先立ちバーネッティ pBlaM-CBU0077文化は、定量PCRを使用して列挙した。 図4は、定量PCR、次の両方の標準およびサンプ...

Watch this Scientific Journal Video about Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing at JoVE.com

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

細菌性病原体とそれらのホスト間の相互作用を調査することは、生物学的研究の重要な領域です。ここでは、BLAM基板を用いたsiRNAの遺伝子サイレンシング中にQ熱リケッチアによってエフェクター転座を測定するために必要な技術を記載しています。

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用<em&gt; Q熱リケッチア</em&gt;のsiRNAサイレンシング中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

applying-fluorescence-resonance-energy-transfer-fret-to-examine

Research

JoVE Journal

Immunology and Infection

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

10.6K ビュー.  University of Melbourne. 細菌性病原体とそれらのホスト間の相互作用を調査することは、生物学的研究の重要な領域です。ここでは、BLAM基板を用いたsiRNAの遺伝子サイレンシング中にQ熱リケッチアによってエフェクター転座を測定するために必要な技術を記載しています。 

ビデオ: Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

ミリ秒時間スケールでのタンパク質複合体のダイナミクスを調べるため体外蛍光共鳴エネルギー移動の使用

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

生化学

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins with the ability to inhibit the host plant RNA silencing pathway. Unlike virus effector proteins, only several secreted effector proteins have showed the ability to suppress RNA silencing in bacterial, oomycete, and fungal pathogens, and the molecular functions of most effectors remain largely unknown. Here, we describe in detail a slightly modified version of the co-infiltration assay that could serve as a general method for observing RNA silencing and for characterizing effector proteins secreted by plant pathogens. The key steps of the approach are choosing the healthy and fully developed leaves, adjusting the bacteria culture to the appropriate optical density (OD) at 600 nm, and observing green fluorescent protein (GFP) fluorescence at the optimum time on the infiltrated leaves in order to avoid omitting effectors with weak suppression activity. This improved protocol will contribute to rapid, accurate, and extensive screening of RNA silencing suppressors and serve as an excellent starting point for investigating the molecular functions of these proteins.

Here, we present a modified screening method that can be extensively used to quickly screen RNA silencing suppressors in plant pathogens.

植物病原体の分泌されたエフェクターからのRNAサイレンシング抑制剤のスクリーニングと同定

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...

遺伝学

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

免疫・感染学

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

解析<em&gt;腸炎エルシニア</em&gt;エフェクター転β-ラクタマーゼエフェクター融合を用いて宿主細胞に

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

近接ライゲーションアッセイを用いたC.エレガンス生殖細胞系列におけるリボヌクレオプロテイン複合体集合体のSITU検出

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

発生生物学

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

遺伝子調節の1分子蛍光イメージング<emインビボ&gt;</em&gt;開裂により同時翻訳アクティベーションの使用（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

生物学

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

高コンテンツ、オープンソースソフトウェアでサポートされているユニバーサル顕微鏡データからの蛍光マーカー、個々の細胞定量のワークフロー

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within &#60;10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) is an imaging technique for detecting protein interactions in living cells. Here, a FRET protocol is presented to study the association of histone-modifying enzymes with transcription factors that recruit them to the target promoters for epigenetic regulation of gene expression in plant tissues.

蛍光共鳴エネルギー移動による 生体内 におけるヒストン修飾酵素と転写因子の相互作用の解明

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides.
This video describes the basics behind chemical mediated transfections and introduces some of the most commonly-used reagents, including charged lipids, polymers, and calcium phosphate. Each step is described from the preparation of cells for transfection through analysis of transfection efficiency. Additionally, the applications section of this video-article describes the use of electroporation and a biolistic transfection as alternative methods for introducing nucleic acid into mammalian cells. It also describes an advanced use of transfection where co-transfection of interfering RNA and DNA are introduced as a way to down-regulate a naturally occurring protein while at the same time producing a mutant variant of it within the same cell.

Transfection: Inserting Genetic Materials into Mammalian Cells

トランスフェクション入門

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用

要約

さらに動画を探す

この動画の章

ミリ秒時間スケールでのタンパク質複合体のダイナミクスを調べるため体外蛍光共鳴エネルギー移動の使用

植物病原体の分泌されたエフェクターからのRNAサイレンシング抑制剤のスクリーニングと同定

発生とのマルチ表現型ハイコンテンツスクリーニング

解析

近接ライゲーションアッセイを用いたC.エレガンス生殖細胞系列におけるリボヌクレオプロテイン複合体集合体のSITU検出

遺伝子調節の1分子蛍光イメージング

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

高コンテンツ、オープンソースソフトウェアでサポートされているユニバーサル顕微鏡データからの蛍光マーカー、個々の細胞定量のワークフロー

蛍光共鳴エネルギー移動による生体内におけるヒストン修飾酵素と転写因子の相互作用の解明

トランスフェクション入門

によってエフェクター転効率を検査するための蛍光共鳴エネルギー移動（FRET）を適用

要約

さらに動画を探す

この動画の章

ミリ秒時間スケールでのタンパク質複合体のダイナミクスを調べるため体外蛍光共鳴エネルギー移動の使用

植物病原体の分泌されたエフェクターからのRNAサイレンシング抑制剤のスクリーニングと同定

発生とのマルチ表現型ハイコンテンツスクリーニング

解析

近接ライゲーションアッセイを用いたC.エレガンス生殖細胞系列におけるリボヌクレオプロテイン複合体集合体のSITU検出

遺伝子調節の1分子蛍光イメージング

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

高コンテンツ、オープンソースソフトウェアでサポートされているユニバーサル顕微鏡データからの蛍光マーカー、個々の細胞定量のワークフロー

蛍光共鳴エネルギー移動による 生体内 におけるヒストン修飾酵素と転写因子の相互作用の解明

トランスフェクション入門

蛍光共鳴エネルギー移動による生体内におけるヒストン修飾酵素と転写因子の相互作用の解明