この作業は、プロジェクトZ2 - 9229とプログラムP2 - 0249とスロベニアの研究機関によってサポートされていました。このビデオでは、&quot;エレクトロポレーションベースの技術と治療&quot;リュブリャナ大学で電気工学の学部で一年おきに主催科学的なワークショップや大学院、、スロベニアの補助材料を表します。

パートI：予備的な手順<ol><li>この実験ではチャイニーズハムスター卵巣細胞株（CHO - K1）が使用されます。細胞は8％ウシ胎児血清を添加したHAM - F12培地中で〜0.7x10 5細胞/ ml、0.15 mg / mlの時のLab - Tek社IIチャンバー（2ウェルを、4cm 2のそれぞれ）（Nalge Nunc社、ドイツ）でメッキされていますL -グルタミン、16 mg / mlのゲンタマイシン（Sigma - Aldrich社、シュタインハイム、ドイツからのすべて）、および200ユニット/ mlのCrystacillin（Pliva、ザグレブ、クロアチア）、および37℃、5％CO 2でインキュベート℃のまた、細胞はまた、ポリリジンなどの細胞接着剤を塗布＃1ガラスカバースリップ（厚さ0.13〜0.16ミリメートル）、にめっきすることができる。 </li><li>それらの培地で細胞をインキュベートする。それでもほぼ球形であるが、しっかりとそれらの膜の小さな部分と表面に付着した2〜4時間の収量細胞続くインキュベーション。また、インキュベーションの16〜20時間後、細胞は完全に表面に付着し、より複雑な形を持っているが、それらのほとんどは、まだ割っていませんされています。 </li><li>元インビトロジェンバイアル中の染料の5mgにDMSOの843μL（シグマ - アルドリッチ、シュタインハイム、ドイツ）を追加することにより、ジ- 8 - ANEPPS（Invitrogen社、ユージン、オレゴン州、米国）の10 mMのストック溶液を調製。ストック溶液は、数ヶ月間、4℃の冷蔵庫に保管することができます。 DMSOの結晶が溶解するまで実験を開始する前に、ソリューションのウォームアップを行います。 </li><li>いくつかの細胞株は、細胞膜への色素の取り込みを容易にするために、プルロニックの使用が必要になる場合があります。プルロニックは、20％DMSO（F - 127、Invitrogen社、ユージン、オレゴン州、米国）のストック溶液、またはDMSO中でプルロニックを溶解して調製することができる、同じ濃度のストック溶液で購入することができます。プルロニックのストック溶液を室温で保存することができます。 </li></ol>パートII：ジ- 8 - ANEPPSを細胞にロード<ol><li> 10mMのジ- 8 - ANEPPSと最小必須培地のSMEMのスピナーの変更（カルシウム枯渇版）（ミディアムM8167またはM4767、Sigma - Aldrichは、シュタインハイム、ドイツの1 mlの20％プルロニック2.5μlの3μlを混ぜて1.5mlのエッペンドルフチュー​​ブに）。これは約30μMジ- 8 - ANEPPSとプルロニックの0.05％を含む&quot;ローディングソリューション&quot;を得られます。他の細胞型の場合は、それらのネイティブの培養培地は、SMEMの代わりに使用できます。 </li><li>ローディング溶液でのLab - Tek社チャンバー内の培養液を交換してください。 4℃で10分間冷蔵庫にチャンバーを転送℃にこの温度で​​は、形質膜を介して色素のインターナリゼーションが大きく阻害される。 </li><li>染色後、優しく純粋なSMEMに過剰な色素を2〜3回洗浄する。 </li><li>チャンバー内のSMEM 1.5mlを残す。 </li></ol>パートIII：実験と画像取得<ol><li>細胞は、油浸対物レンズ（X63、NA 1.4）、モノクロ（ポリクロームIV、Visitron、ドイツ）と冷却CCDカメラ（VisiCamを装備した蛍光顕微鏡を（我々の場合ツァイスAxioVert 200、ツァイス、ドイツ）を用いて観察される1280年、Visitron、ドイツ）。画像はMetaFluor 7.1.1で買収し、MetaMorph 7.1.1（両方Molecular Devices社、ダウニングタウン、ペンシルバニア州、米国）で処理されますが、他の同じような買収のソフトウェアも使用することができますされています。 490nmでの集録ソフトウェアの励起波長を設定し、例えばANEPPSに適した発光フィルター、605 nmの中心のバンドパスフィルタを（605/55m、ダイクロイック565 DCXR、クロマ、ロッキンガム、バーモント州、米国）を選択します。モノクロメータが使用できない場合は、490 nmの中心の励起フィルターを代わりに使用することができます。 </li><li>顕微鏡ステージ上に細胞をチャンバーを置く、チャンバーの下部に電極を配置し、パルス発生器に接続します。培地には、電極をカバーする必要があります。 </li><li>細胞の生存を維持し、発熱を低減するために、パルスが十分に低い振幅と持続時間が短いはずです。この実験では35 V振幅と50 msの持続時間（エレクトロポレーションを防ぐのに十分な低さ）の方形パルスは直流電圧の供給とカスタムメイドのマイクロプロセッサー制御のスイッチャーのデバイスを使用して生成されます。また、そのようなS88（グラス、ウェストワーウィ​​ック、ロードアイランド、米国）などのアンプや商業刺激装置に接続されている33210A（アジレント、サンタクララ、カリフォルニア州、米国）などの商業パルスジェネレータは、、、使用することができます。パルスは、電極と誘導ΔΦの間に約88 V / cmの電場を作る、0.8mmの直径とそれらの間に4 mmの距離で2つの並列のPt / Irのワイヤ電極に配信されます。この実験で使用される電極間の正確な電界分布が他の場所[1]に記載されています。 </li><li>目的の細胞を見つける。単一の電気パルスまたはパルスのシーケンスを適用します。すぐにパルス（制御画像）1つ前とパルス（パルスの画像）中に1つ：各パルスの場合は、二つの蛍光画像を取得。ジ- 8 - ANEPPSの低い応答のために、蛍光の変化は、肉眼で識別し、明らかにONLになることは困難である処理の後にyを。パルスの配信は、買収のソフトウェアの特徴である画像取得、と同期する必要があります。加熱を退色し、可能な回避するために、照明は、パルスの持続時間に制限することができます。 </li></ol>第IV部：画像処理と解析<ol><li> MetaMorphソフトで画像を開きます。各パルスの場合、制御とパルスのイメージの両方でバックグラウンドを引く。 </li><li>セルを選択し、それが膜に対応するように関心領域を設定します。制御とパルス画像内のこの領域に沿って蛍光強度を測定し、スプレッドシートに値を転送する。 </li><li>各パルスは、パルスデータから制御データを減算し、相対的な蛍光の変化を得るための制御データで結果を割ります。パルスシーケンスが適用されている場合は、各パルスに決定相対的な蛍光の変化の値は、より信頼性の高い測定を得るために平均化することができます。 </li><li>検量線を使用してΔΦに相対的な蛍光の変化を変換します。この曲線の大まかな推定は、文献から得たものですが、より高い精度のために、それはそれぞれ特定のセットアップのために測定する必要がありますすることができます。細胞と本実験で使用したセットアップのために蛍光で6％の減少は、ΔΦ[2]で100 mVの増加に対応しています。 </li><li>最後に、このようなエクセル（マイクロソフト社、レドモンド、ワシントン州、米国）、シグマプロット（SYSTAT Software社、サンノゼ、カリフォルニア州、米国）などのグラフィック系ソフトウェアパッケージの相対的な弧の長さの関数として電圧をプロットする、または原点（OriginLabの（株）、ノーサンプトン、マサチューセッツ、米国）。曲線はまた、移動平均フィルタのような適切なフィルターを用いて平滑化することができます。 </li></ol>

<ol>
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Eng. 34, 642-652 (2006).</li><li>Huang, C. J., Harootunian, A., Maher, M. P., Quan, C., Raj, C. D., McCormack, K., Numann, R., Negulescu, P. A., Gonzalez, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+voltage-gated+sodium-channel+blockers+by+electrical+stimulation+and+fluorescence+detection+of+membrane+potential.">Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.</a> Nat. Biotechnol. 24, 439-446 (2006).</li><li>Bedlack, R. S., Wei, M., Fox, S. H., Gross, E., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+electric+potentials+in+soma+and+neurite+membranes.">Distinct electric potentials in soma and neurite membranes.</a> Neuron. 13, 1187-1193 (1994).</li><li>Cheng, D. K. 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誘導膜（膜貫通）電圧、ΔΦ、の測定値は、電位依存性膜チャネル、活動電位の伝播、心筋細胞の刺激、または細胞膜のエレクトロポレーション[3、4、5、6の研究など、様々な実験の設定、に重要になることがあります、7]。単純細胞の形状で、ΔΦ解析的に計算することができます。例えば、球状の細胞のために、ΔΦは、電圧が電界強度とセルのサイズに比例であることを示すと膜に沿って余弦関数を次シュの方程式、で与えられる[8、9]。より複雑な細胞の形状の場合は、ΔΦ余弦から大幅に逸脱することができ、電位差染料[12、13、14、15]を使用して、[2、10、11]、または実験的にコンピュータを使用して、どちらかの数値的に決定する必要があります。 広くこの目的で使用される電位差染料の一つは、ジ- 8 - ANEPPS（ジ- 8 -ブチル - アミノ-ナフチル-ピリジニウム-プロピルスルホン酸エチレン）、励起と発光の膜電位に依存するスペクトルを持つ高速な色素であり、その細胞膜上にΔΦのバリエーションの非侵襲的観察を可能にし、その値を測定する。このビデオでは、我々は、ジ- 8 - ANEPPSを使用して、ΔΦの決定のための実験的なアプローチを示しています。 染料は、コネチカット大学の教授レスリーロウズら[13、14]によって開発され、高速応答色素のクラスに属していた。ジ- 8 - ANEPPSは水に非蛍光であり、それは細胞膜の脂質二重層に組み込まれてときに強く蛍光性になる。分子内電荷分布と色素の蛍光のスペクトルプロファイルと強度の対応する変化の変化ΔΦの結果の変化。ジ- 8 - ANEPPSの蛍光強度は、ΔΦの変化に比例して変化する。色素の反応は、-280 mVから250までmVの[4、16]電圧に対して線形である。このような努力にも[17]が報告されたが染料、不均一な膜染色の蛍光は比較的小さな変化、および染料内在化は、膜電位、例えば、その休憩成分の絶対測定のためのジ- 8 - ANEPPSはあまり適していません。それは、しかしながら、そのような興奮性細胞[4,5]で外部電界にさ​​らされる非興奮性の細胞の誘導膜電位の発症[12、13]、または活動電位のような膜電位の大きな変化を、測定に適しています。ここで適用されていないものの、ジ- 8 - ANEPPSも[18]または応答の感度を増加させると上記の効果を軽減発光[19]、蛍光励起のレシオメトリック測定によりΔΦの決定を可能にする。ジ- 8 - ANEPPSの汚れ膜として、それはまた、細胞膜マーカー[2]としてはっきりと使用することができます。 染料の欠点の一つは、それが退色する傾向があることですので、強い光に長時間暴露は避けるべきであること。染料のキャリブレーションは、外部媒体のいずれか（I）カリウムイオノフォアバリノマイシンと異なるカリウム濃度のセットで実行される[2,18]、または（ii）パッチクランプ電圧クランプモードで[17]。 最後に、より複雑な形状の球状の細胞と細胞のΔΦの測定と、ビデオは、セルの振幅の図形とΔΦの空間分布の影響を示しています。したがって、球状の細胞のためのΔΦより複雑な細胞の形状のためにΔΦの空間分布がより複雑である一方、シュヴァン方程式との合意で、コサインに近い[20] ...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>di-8-ANEPPS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3167</td>
<td>potentiometric fluorescent dye</td>
</tr>
<tr>
<td>pluronic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>potassium ionophore</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>SMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M8167 or M4767</td>
<td>Spinner modification of the Minimum Essential Medium</td>
</tr>
<tr>
<td>Ham-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N4888</td>
<td>culture medium</td>
</tr>
<tr>
<td>fetal calf serum</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>&nbsp;</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Lab-Tek II</td>
<td>&nbsp;</td>
<td>Nalge Nunc</td>
<td>155379</td>
<td>chamber</td>
</tr>
<tr>
<td>DC voltage supply</td>
<td>&nbsp;</td>
<td>Elektro-Automatik</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microprocessor-controlled switcher</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>electrodes</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

measuring the induced membrane voltage with di-8-anepps

外部電場にセルの配置は、膜を通過する電圧で、その結果、細胞膜の近傍に細胞の局所電荷の再分配の内側と外側を引き起こします。この電圧は、誘起膜電位（も誘導される膜貫通電圧、または誘導膜電位の差）と呼ばれるとΔΦで表されるが、唯一の外部磁界が存在する限り存在する。安静時の電圧が膜上に存在する場合、誘起電圧は、その上に（追加）重ね書きします。例えばジ- 8 - ANEPPSとして電位差蛍光色素、のいずれかを使用して、それは細胞膜上にΔΦの変動を観察し、非侵襲的にその値を測定することが可能です。 ΔΦの変化に比例した蛍光強度の変化と、細胞膜の脂質二重層に結合するとジ- 8 - ANEPPSは、強い蛍光となります。このビデオでは、測定するためのプロトコルΔΦ用いたジ- 8 - ANEPPSを示し、また、振幅とΔΦの空間分布に及ぼすセル形状の影響を示しています。

Watch this Scientific Journal Video about Measuring the Induced Membrane Voltage with Di-8-ANEPPS at JoVE.com

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

外部電界が細胞の膜上に電圧を誘導する、誘起膜電位は（ΔΦ）と呼ばれる。電位差染料ジ- 8 - ANEPPSを使用して、それは非侵襲的にΔΦを測定することが可能です。このビデオは、di - 8 - ANEPPSを使用してΔΦを測定するためのプロトコルを示しています。

ジ- 8 - ANEPPSで誘起膜電位の測定

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

measuring-the-induced-membrane-voltage-with-di-8-anepps

Research

JoVE Journal

Biology

17.3K ビュー.  University of Ljubljana. 外部電界が細胞の膜上に電圧を誘導する、誘起膜電位は（ΔΦ）と呼ばれる。電位差染料ジ- 8 - ANEPPSを使用して、それは非侵襲的にΔΦを測定することが可能です。このビデオは、di - 8 - ANEPPSを使用してΔΦを測定するためのプロトコルを示しています。

ビデオ: Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

ルシフェラーゼベースのレポーター遺伝子アッセイで膜の生合成を研究して

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

生物学

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

アストロサイトの細胞膜とグローバル細胞内カルシウム動態の近くの測定

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

リバーシブルビオチン化によって原形質膜タンパク質エンドサイトーシス率を測定

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

脂質中間相を用いた構造決定のための膜タンパク質の結晶化

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

脂質中間相における膜タンパク質の結晶化

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

膜タンパク質の構造動力学を調べるその場で部位特異的蛍光標識を持つ

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ザ·<em&gt;アフリカツメガエル</em蛍光測定で&gt;卵母細胞のカットオープンワセリンギャップ電圧クランプ法

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

pTIRFMによる画像形成細胞膜変形は

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

ハイドラ内グルタチオンによって誘発される摂食応答を測定する

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

ラット腸間膜に微小血管透過性の調節を調査するために、微小灌流法

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...