私たちは、貴重なアドバイスのためにビクターGJロジャースに大変感謝しております。我々は非常に密接な共同作業のために、この研究のヘルプは遼グループのすべてのメンバーに感謝します。この研究は、米国国立衛生研究所（JLにグラントAI076504）によってサポートされていました。

1。プラスミド構築<ol><li> PCRにより遺伝子のオープンリーディングフレームを増幅し、PCRII-TOPOベクターにPCR産物をクローニングします。 </li><li>配列決定することによって製品を確認して、クローンをコードするcDNA CyPet-（プリSUMO1）YPet、CyPet-SUMO1、YPetおよびN-末端ヒスチジンタグ付きpET28（b）のベクトルにSENP1の触媒ドメイン。 </li></ol> 2。タンパク質の発現と精製<ol><li> pET28とBL21（DE3）株の大腸菌細胞を形質転換（b）にCyPet-（プリSUMO1）YPet、CyPet-SUMO1、YPetとSENP1の触媒ドメインをコードするベクター。 </li><li> 600nmで0.5の光学濃度に到達するために37°C（250 rpmで振とう）で3時間2×YT培地で形質転換された細菌を増殖させた。 </li><li>タンパク質の発現を誘導し、25℃で16時間振盪100μMのイソプロピル-β-D-ガラクトシド（IPTG）（最終濃度）を追加°200rpmで、C。 </li><li> centrifugによって細菌を収穫4℃、10,000 rpmでation℃で5分間及び20mMのトリス-HCl（pH7.4）、50mMのNaCl、5 mMイミダゾールのバッファーで再懸濁します。 </li><li> MiSonics 4000ソニケーターを用いて25Wの電力設定で5秒間隔で10分間、細胞を超音波洗浄し、4℃で35000×gで遠心分離することにより、それらを収集℃で30分間。 </li><li> 1Lの培養のための500ミリリットルNi-NTAビーズを使用して列を設定し、カラムに上清を移す。 </li><li>倍の20mMトリス-HCl（pH7.4）、500mMのNaCl、10mMのイミダゾール緩衝液を用いて樹脂を洗浄します。 </li><li> 20mMのトリス-HCl（pH7.4）、500mMのNaCl、500 mMイミダゾール、20 mMのトリス-HCl（pH7.4）、50mMのNaClおよび1mMジチオスレイトール（DTT）でのバッファに透析のバッファーで溶出タンパク質4℃で一晩。 </li><li> Bradford法により精製したタンパク質の濃度を決定する。タンパク質濃度測定のための代替方法は、イメージングと続か次にクーマシーブルーで染色したSDS-PAGEゲル電気泳動することとなります定量。 </li></ol> 3。定量的なFRETスペクトラム解析<ol><li>アッセイのための一般的な戦略は、（ 図1）シグナリングFRETに基づくものであった。 FRET対を、CyPetとYPetは、事前SUMO1、それぞれ、N末端とC末端にタグ付けされていた。 SENP1 SUMO1のC末端のGly-Glyのサイトで切断融合タンパク質CyPet-（プリSUMO1）YPetとは、このように、YPetとSUMOの尾を解放します。信号がCyPetからの発光の増加とCyPet励起波長でYPetの排出の劇的な減少、その結果、破壊されたFRET。 </li><li>絶対FRET誘起YPetの排出、CyPet直接排出とYPet直接排出（ 図 ：波長414 nmの光で励起すると、530nmでCyPet-（プリSUMO1）YPetの総蛍光発光は、次の3つのソースから導出することができる2）。 </li></ol><img alt="式（1）" src="/files/ftp_upload/4430/4430eq1.jpg" /> どこフロリダ414分の530 &lt;/ sub&gt;の414 nmで励起し530nmの総蛍光発光は、FLはFRETが絶対にFRETシグナルであり、 フロリダ州CyPet（続き）は 414 nmで励起CyPet直接排出であり、 フロリダ州YPet（続き）は YPetです414 nmで励起直接排出。の添字（続き）が拠出を意味します。 <ol start="3"><li> αの一定比率と414 nmで励起した場合に530 nmでCyPetの直接排出量は475nmで、その排出に比例していた。CyPet-SUMO1が50の濃度は、100、200、500、750 nMおよび1 mMで調製した、475および530 nmでの排出量α（ 図3-1）を決定するために414 nmで励起した後に測定した。 </li><li> βの定数比で475 nmで励起した場合に414 nmで励起下で530nmでYPetの直接排出量は530nmで、その排出に比例していた。YPetを50の濃度は、100、200、500、750で調製したND 1,000 nmで、サンプルをβ（ 図3-2）を決定するために414および475 nmの波長によって励起されたときに530nmの発光を測定した。 </li><li> CyPet-（プリSUMO1）YPetがSENP1によって消化されたとき、切断はCyPet-SUMO1とYPetとSUMO1尾をリリースしました。化合物は、414 nmで励起したとき、530nmの（ フロリダ州&#39;414分の530）での蛍光発光が減少しましたが、まだのように3つの部分に分けることができます： </li></ol><img alt="式（2）" src="/files/ftp_upload/4430/4430eq2.jpg" /> フロリダ&#39;414 nmで励起した場合に414分の530は消化後に530nmで総蛍光発光であり、 フロリダ州&#39;はどこに残っている絶対的なFRETシグナルですフレット 、 フロリダ&#39;CyPet（414分の475）のとき消化後に475nmのCyPet放出で414 nmの（ここCyPet発光は二つの部分からなります。未消化CyPet-（プリSUMO1）YPetと消化CyPet-SUMO1）で励起し、FL YPet（475分の530）CyPet-（プリSUMO1）YPetが消化されているかどうか。定数である475 nmで励起YPet排出、ある<ol start="6"><li> SENP1による消化後、残りは放出（ フロリダ州&#39;はFRET）であるFRET： </li></ol><img alt="式（3）" src="/files/ftp_upload/4430/4430eq3.jpg" /> ここで、Cは CyPet-（プリSUMO1）YPet と xの合計濃度であると、消化の濃度CyPet-（プリSUMO1）YPetです。 <ol start="7"><li>すべてのアイテムを組み合わせることにより、414 nmの励起下で530nmで（ フロリダ州&#39;414分の530）で検出された蛍光発光は、次のとおりです。 </li></ol><img alt="式（4）" src="/files/ftp_upload/4430/4430eq4.jpg" /> 4。酵素速度論的研究のためのFRETベースプロテアーゼアッセイ<ol><li> CyPet-（プリSUMO1）YPetは（20mMのトリス-HCl（pH7.4）、50mMのNaCl、0.1％の緩衝液中で37℃でSENP1の触媒ドメインとインキュベートしたV /80ミリリットルの全体積V）のTween-20および1mM DTTおよび384ウェルプレートに移した。 </li><li>ランは、15秒間隔で5分間蛍光マルチウェルプレートリーダーで414 nmで励起した後に475および530 nmでの蛍光発光を測定することにより行った。 </li><li>反応速度（v）のように基板（S）の量の変化と相関していた： </li></ol><img alt="式（5）" src="/files/ftp_upload/4430/4430eq5.jpg" /><ol start="4"><li>生成物の濃度は 、t = 0 [S] 0（元の基質濃度）として、0から指数関数的に増加した。 </li></ol><img alt="式（6）" src="/files/ftp_upload/4430/4430eq6.jpg" /><ol start="5"><li> T = 0のとき、元の速度（V 0）は、次のとおりです。 </li></ol><img alt="式（7）" src="/files/ftp_upload/4430/4430eq7.jpg" /><ol start="6"><li>濃SENP1のentrationを固定し、CyPet-（プリSUMO1）YPetの濃度がミカエリス·メンテン式によって必要とされる酵素の濃度よりも100倍以上まで増加した。 </li><li>蛍光測定値のすべてが定量的なFRET分析法により分析し、ミカエリス·メンテン式に合わせてグラフパッドプリズムVソフトウェアにプロットした。非線形回帰も、スプレッドシートプログラム（Microsoft Excelなど）のようなより一般的なソフトウェアパッケージの助けを借りて行うことができます。 </li></ol> 5。代表的な結果 SENP1による事前SUMO1の成熟は、プロセス中に475および530 nmでの蛍光信号の変化を監視することによって決定することができる。結果が示され、その基板用量依存的にSENP1による事前SUMO1消化の速度（ 図4）。プリSUMO1の成熟のための優れた活性SENP1展示の触媒ドメインというこれが示唆している。初期REACる速度が異なる基質濃度（ 表1）上記の分析により算出した。 K 猫 / K M比は 、一般に、1つの基板または異なる基質を持つ特定の酵素と異なる酵素の効率を比較するために使用されます、KとMとV maxは 、様々な初期速度をプロットすることにより、ミカエリス·メンテン式から得ることができ、対応する。CyPet-（プリSUMO1）YPet（ 図5）の異なる濃度に k 猫は、次のように得られた： <img alt="式（8）" src="/files/ftp_upload/4430/4430eq8.jpg" /> 上記の分析によると、計算されたK Mは 0.21±0.04μMでは、k catは ±0.28 s -1で 6.90であった、 と k 猫 / K M比 （0.55±3.2）であったx10の7 M -1 </sup<html> &gt; s -1で 。 <img alt="図1" src="/files/ftp_upload/4430/4430fig1.jpg" /> 図1 SENPのプリSUMOs成熟のためのFRETベースプロテアーゼアッセイのグラフ。 <img alt="図2" src="/files/ftp_upload/4430/4430fig2.jpg" /> 図2ドナー、アクセプターとFRETによる貢献として、蛍光シグナルの定量分析。 CyPet-414 nmで励起下（プリSUMO1）YPet。 フロリダCyPet（414分の530）からの発光スペクトルの解剖 CyPetの排出量は414 nmの励起下で530nmである、 フロリダFRET FRETによって誘発される414 nmの励起下で530nmでYPet放出、およびFL YPet（414分の530）で414 nmの励起下で530nmでYPetの放出である。 load/4430/4430fig3.jpg &quot;/&gt; 図3方向排出係数αとβの計算。 <img alt="図4" src="/files/ftp_upload/4430/4430fig4.jpg" /> 図4 SENP1の触媒ドメインの異なる比率で消化CyPet-（プリSUMO1）YPetの定量分析。反応は、最初の5分以内にモニターした。 <img alt="図5" src="/files/ftp_upload/4430/4430fig5.jpg" /> 図5 SENP1によってCyPet-（プリSUMO1）YPetの消化のミカエリス-メンテングラフィカル解析。データをプロットし、グラフパッドプリズムVおよび非線形回帰により分析した。 <table border="1"><tbody><tr><td> [S]（μM） </td><td> V 0（μm/ sで） </td></tr><tr><td> 0.115 </td><td> 0.0023±0.00005 </td></tR&gt; <tr><td> 0.214 </td><td> 0.0028±0.00004 </td></tr><tr><td> 0.407 </td><td> 0.0033±0.00007 </td></tr><tr><td> 0.594 </td><td> 0.0037±0.00013 </td></tr><tr><td> 0.725 </td><td> 0.0043±0.00012 </td></tr><tr><td> 1.471 </td><td> 0.0051±0.00036 </td></tr><tr><td> 1.899 </td><td> 0.0050±0.00031 </td></tr><tr><td> 2.300 </td><td> 0.0050±0.00062 </td></tr></tbody></table> 表1。SENP1による事前SUMO1の成熟の初期速度を決定。各基質濃度では、4つのサンプルは、消化を測定するために使用されていました。標準偏差は、これらの4つのサンプルのバリエーションから来ました。 <table border="1"><tbody><tr><td> K M（μM） </td><td> K 猫 （S-1） </td><td> K 猫 / K M（M -1•S-1） </td></tr><tr><td> 0.21±0.04 </td><td> 6.90±0.28 </td><td> 3.2±0.55×10 7 </td></tr></tbody></table> 表2定量によってSENP1による事前SUMO1の成熟の速度論的パラメータは、FRET分析。標準偏差は、各基質濃度で4つのサンプルから来ました。

<ol>
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特別な利害関係は宣言されません。

FRET技術はSENP1 9でプリSUMO1の成熟を ​​研究するために使用されています。 CFP-YFPは、ドナー·アクセプターへの排出量の比率であるFRETペアとレシオメトリック分析、として使用された速度論的性質を特徴づけるために使用されていました。しかし、伝統的なレシオメトリックでの自己蛍光がFRET分析ドナーとアクセプターのは考慮されていません。比は、直接消化された基質の量と?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> PCR II TOPOキット</td><td>インビトロジェン</td><td> K466040 </td></tr><tr><td> 2xYT </td><td>リサーチ製品Internationa.l株式会社</td><td> X15600 </td></tr><tr><td>のNi-NTAアガロース</td><td>サーモサイエンティフィック</td><td> 88222 </td></tr><tr><td>クマシープラス（ブラッドフォード）アッセイリージェント</td><td>サーモサイエンティフィック</td><td> 23238 </td></tr><tr><td> 384ウェルプレート（ガラスボトム） </td><td>グライナー</td><td> 781892 </td></tr><tr><td> FlexStation II 384プレートリーダー</td><td>分子素​​子</td><td></td></tr></tbody></table>

quantitative fret (f&#246;rster resonance energy transfer) analysis for senp1 protease kinetics determination

ユビキチンまたはユビキチン様タンパク質（Ubls）を持つタンパク質の可逆的翻訳後修飾は、広く動的にタンパク質の活性を調節し、多くの生物学的プロセスで多様な役割を持っているために使用されます。たとえば、SUMOは共有結合で細胞周期調節、細胞の生存と死、DNA損傷応答、ストレス応答1月5日を含む多くの細胞プロセスにおいて重要な役割を持つ大規模な番号またはタンパク質を変更します。 SUMO特異的プロテアーゼとしてSENP、SUMO前駆体の成熟にエンドペプチダーゼとして、あるいはその標的タンパク質からSUMOを削除し、SUMO化サイクル1,3,6,7をリフレッシュするイソペプチダーゼとして機能します。 酵素の触媒効率や特異性は、最高の速度定数は、k 猫 / K Mの比であることを特徴とする。いくつかの研究では、SUMO-SENPペアの速度論的パラメータは、RAD、ポリアクリルアミドゲルベースの​​ウェスタンブロットを含む様々な方法によって決定された基板8から13というラベルの付いたioactive標識基質、蛍光化合物またはタンパク質。しかし、 &quot;ネイティブ&quot;のタンパク質を使用しましたが、ポリアクリルアミドゲルベースの​​技術は、面倒で技術的に厳しいですが、その容易に詳細な定量分析には役立たない。 （7 -アミノ-4 - carbamoylmetylcoumarin）[ACC]またはAMC（7 -アミノ-4 -メチルクマリン）蛍光体でテトラペプチドまたはタンパク質を用いた研究から得られたK 猫 / K M天然の基質よりも低い二桁までのいずれかであったまたは明らかにiso-およびSENPsのエンドペプチダーゼの活動を区別することはできません。 最近では、プロテアーゼアッセイと脱ユビキチン化酵素（ダビング）またはSENPsを勉強するために使用されたベースがFRETは、シアン蛍光タンパク質（CFP）と黄色蛍光タンパク質（YFP）9,10,14,15のFRETペア。プロテアーゼAC用信号モニタのFRETのドナー発光に対するアクセプター発光の比率は、定量的なパラメータとして使用されましたtivity決定。しかし、この方法では、アクセプターとドナーの自己蛍光によるアクセプターとドナーの発光波長での信号のクロスコンタミネーションを無視し、したがって、正確ではありませんでした。 我々はSENP1によってプリSUMO1成熟の速度論的パラメータを決定するための新規高感度かつ定量的なFRETベースプロテアーゼアッセイを開発しました。工学的には、大幅に改善FRET効率と蛍光量子収率とペアCyPetとYPetのFRET CyPet-（プリSUMO1）YPet基板16を生成するために使用された。私たちは、差別やドナーとアクセプターが寄与するとそれぞれ、アクセプターおよび発光波長でFRETが絶対的な蛍光シグナルを定量化した。 K 猫 / K Mの値は（3.2±0.55）として得た一般的な酵素の速度論的パラメータと一致しているプレSUMO1、向かってSENP1のx10の7 M -1 s -1で 。したがって、この方法は有効であり、使用することができます同様に他のプロテアーゼを特徴付けるためのSAの一般的なアプローチ。

Watch this Scientific Journal Video about Quantitative FRET FÃ¶rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination at JoVE.com

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

FRETの定量分析（フェルスター共鳴エネルギー転移）信号を含む新規な方法は、酵素反応速度論を研究するために記述されている。<em&gt; K<sub&gt; M</sub</em&gt;と<em&gt; K<sub&gt;猫</sub</em&gt;は（ユビキチン類似因子）プリSUMO1へSENP1（SUMO / Sentrin特異的プロテアーゼ1）の触媒ドメインの加水分解が得られた。この量的-FRETベースプロテアーゼ動態検査の一般的な原理は、他​​のプロテアーゼに適用することができます。

SENP1プロテアーゼキネティクス決定のための定量的なFRET（フェルスター共鳴エネルギー移動）の分析

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

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Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Unit 1

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18.9K ビュー.  University of California, Riverside. FRETの定量分析（フェルスター共鳴エネルギー転移）信号を含む新規な方法は、酵素反応速度論を研究するために記述されている。 K M

ビデオ: Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

マイクロパターン細胞を用いた薬物の効果の改善可視化と定量分析

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

生物工学

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

用接着周波数アッセイその場で</em細胞間のインタフェースにおけるクロス接合分子間相互作用の>速度論解析

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

のオンデマンド定量分析のためのマイクロ流体ベースの走電性<em&gt;線虫（Caenorhabditis elegans）</em&gt; &#39;ロコモーション

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

アデニリルシクラーゼの薬物誘発性感作：アッセイは、小分子およびsiRNAスクリーニング用途のために合理化·小型化

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

発光共鳴エネルギーは、哺乳動物細胞で発現膜タンパク質の立体構造変化を研究するために転送

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

TCR-のpMHCが結合を測定します<em&gt;その場で</em&gt; FRETベースの顕微鏡アッセイを使用して

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

FRETフローサイトメトリーでProteopathic播種活動の高感度検出

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A Protocol for Using F&amp;#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

原子力LINCコンプレックス全体に機械的な力を測定するために、蛍光共鳴エネルギー移動（FRET）-forceのバイオセンサーを使用するためのプロトコル

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

単層カーボンナノチューブ上のバイオミメティックポリマーとエンジニアリング分子認識

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...