この作品は、ドイツ学術振興（DFG）BL567/3-1とフリードリヒ·バウアー·財団の補助金によって支えられてきた。我々は、Tag-RFP-T2を私達に提供するためロジャーY.ツィン、カリフォルニア大学サンディ​​エゴ校ハワードヒューズ医学研究所研究所に感謝したいと思います。私たちは、ありがたいことに私たちにレンチウイルスプラスミドFUGWを提供し、psPAX2/pMD2.Gためデイヴィッドボルティモア、カリフォルニア工科大学、パサデナ、そしてディディエTrono、ジュネーブ、ジュネーブ、大学を認める。

<ol>
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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

TED方法の利点と欠点は、最近の刊行物34,35で広く議論されている。上述した他の方法に比べて、TEDは、細胞を破砕し、灌流の問題を回避する。さらに、二つの側面が強調される必要がある。 ERカルシウムイメージングのためのTEDの原理はTEDベクター構築の助けとの低親和性合成AM-エステルの（2）効率的かつ優先的なリリースで、ERの内腔に積極的にカルボキシルエステラーゼの（1）?...

ERの生理的なカルシウム応答を解決するために、我々は、ERに合成炭酸カルシウム感受性色素の捕捉を改善するための新しい戦略を開発した。方法は、細胞外カルシウムの存在下で自由ERカルシウムの直接、無停止リアルタイム監視を可能にします。 ERカルシウムの機能とシグナリングカルシウム信号は、異なる細胞型、 例えば、筋肉細胞、ニューロンおよび学習および記憶1,2におけるシナプス伝達の関与に筋収縮を媒介からグリア細胞とその機能の範囲に見出される。カルシウムは遺伝子転写、細胞増殖、神経細胞の興奮性、細胞死および他の細胞シグナル伝達事象1-7の調節に関与しているため、遊離カルシウム濃度の変化は、高い科学的関心である。これらのすべての細胞内カルシウムシグナルは、細胞内カルシウム機能的に接続され、貢献店舗ダイナミクス8-10。 すべてのカルシウムシグナルの間で共通の特徴は、主にERやミトコンドリア、細胞外空間、細胞質と細胞小器官の間でのカルシウムの流れです。これは、異なるシグナル伝達成分によって感知されるこれらの細胞小器官内のカルシウム濃度のダイナミックな変化を引き起こす。一般的には100〜800μmの間のERの範囲内カルシウム濃度は、細胞質ゾルのカルシウム濃度は、100nMの近隣にあり、細胞外空間内濃度は1〜2 mMの程度である。従って、細胞質ゾルのカルシウム2,9,10に向かって流れに対して高い化学的駆動力が存在する。 最も一般的に調査し、ER由来のカルシウムシグナルは、その後ホスホリパーゼC（PLC）を活性化するGタンパク質共役受容体（GPCR）の刺激に依存します。順番にPLCはイノシトール1,4,5 -三リン酸（IP 3）1を生成します。その視聴にIP 3の結合時ER-膜におけるeptor（IP 3 -録音、 図1）は 、カルシウムイオンは、ER内腔から放出される。歴史的には、ERからIP 3媒介カルシウム放出は初めてだった-たとえ間接的に- Streb ら腺膵臓細胞で測定された1983年11インチ 。本書は、初めてアセチルコリン、ホスホリパーゼC、およびIP 3が関与するシグナル伝達カスケードを示唆した。カルシウム放出のこの方法は、一般的にIP 3誘発性カルシウム放出（IICR）（ 図1）と呼ばれます。受容体チロシンキナーゼリンクIP 3標高12後シグナリングERカルシウムの増殖因子や神経栄養因子の作用によるホスホリパーゼCγのキナーゼ依存的活性化。 IICRに加えて、カルシウム上昇は電位依存性カルシウムチャネル（CA V）、およびリアノジン再によるその後のカルシウム誘発性カルシウム放出（CICR）を経由して、たとえば、イオンチャネル型カルシウム流入によって媒介され得るceptors（RYR）。 IICRとCICRは生理的にストア作動性カルシウム流入（SOCE）にリンクされています。 SOCEはERのカルシウム放出するためのセンサであるSTIM（間質相互作用分子）の作用を有している。 STIMは、一過性受容体電位チャネル（トリプトファン）13、往来カルシウムチャネル14、さらには電位依存性カルシウムチャネル15（ 図1）を介して細胞外のカルシウム流入を刺激することが示されている。 ERカルシウムの損失が動的に積極的にポンプカルシウムバックERにサーコ-小胞体カルシウムATPアーゼ（SERCA）の作用によって救出されています。などタプシガルギンなどの薬物とのSERCAをブロックすると、細胞質コンパートメントにERカルシウムの継続的損失を発表。このERカルシウム&quot;リーク&quot;などSec61タンパク質複合体16,17（ 図1）のように小胞体膜内の細孔複合体によって引き起こされる。 1998年には、Berridgeは、モデル、 &quot;ニューロンモデル内ニューロン&quot;、whicを発表hはニューロンのカルシウム5を統合するERの原則生理的役割を示唆している。このモデルは、神経細胞の原形質膜5の細胞内の&quot;画像&quot;を形成する連続的なER膜システムの存在を考慮する。このバイナリ真核生物の膜システムは、ニューロンにおける高速と低速のカルシウムシグナルの時間的·空間的統合のための基本的な前提条件であると主張しました。同じニューロンの異なる棘や樹状突起に同時にまたはその後のどちらか起こるカルシウムシグナルは、細胞の細胞体または彼らは5,18を総括しているERを介して核に付与されています。そして、その合計がシグナル伝達カスケードのニューロンの興奮性、遺伝子転写の調節や統合に影響を与えることがある。したがって、ERは、カルシウムシグナルの統合をサポートしています。この概念の一つの前提条件は、いくつかの研究で主張されていると、少なくとも体性-Dのために証明されている単一のセル内のERの継続ですendriticエリアと短距離軸索投射19-21。長い軸索投射内ER継続性があるかどうかは議論の問題です。 ER膜上の遊離カルシウムの流れを測定するための戦略カルシウムシグナルは、最も頻繁に細胞質ゾル22,23に監視されています。したがって、容易のCa 2 +を細胞外から6,24又は細胞内貯蔵から細胞質内に流入されているかどうかを区別することができない。この制限を克服するために、直接ERカルシウムイメージングのための方法論的な戦略が開発されている。要約すると、以下の戦略が使用されます。（1）ER-ターゲット遺伝子組み換えタンパク質の指標25-27タンパク質ベースの低親和性のCa 2 +インジケーターはタンパク質を感知カルシウムとの組み合わせで生物発光タンパク質イクオリンやGFPを使用しています。これらの遺伝子組み換えのCa 2 +インジケータ（GECIs）ができますシグナルペプチドの助けを借りて、ERを対象とすることが、積極的に保持し、検索のモチーフを使用してERに保持されます。カメレオン原則上の共通ERのCa 2 +指標ベースとカメレオンYC4.3 26,28です。カメレオンスプリットYC7.3ER 29と、カメレオンD1 30。 （2）インジケーター色素のAM-エステル低親和性のCa 2 + 指標31,32。AM-デリバティブの直接エステラーゼ系色素ローディング （MAG-たFura2-AM、MAG-Fluo4-AMまたはFluo5N-AM）が合格親油性、カルシウムと小文字を区別しない状態での生体膜。その後、細胞質ゾルで同様ERで、内因性エステラーゼAM-エステル基の開裂とは、細胞質内のアクティブな染料ある程度の後ろとERに残して、Ca 2 +のインジケータを解放。細胞質カルシウム濃度が十分にデ以下に留まるようしたがって、このアプローチは限り、ERの高いカルシウム濃度の条件下で有用である特に特徴的なカルシウムシグナル（低μMに例えば nM）を間に低親和性指標の護制限、。 （3） 細胞膜透過性32との組み合わせでAM-エステルローディング。すべて残り細胞質のCa 2 +指示薬は、人工細胞内バッファに&quot;マイルド&quot;洗剤（ 例えばサポニン）少量の細胞膜透過処理によって除去される。したがって、細胞内膜を直接形質膜における&quot;細孔&quot;を介して、細胞内のバッファ内のIP 3を、例えば 、刺激されてもよい。 （4）ER内腔や細胞質ゾル 32,33 中のCa 2の細胞全体の構成や同時測定+下の細胞質ゾルの透析。セルが最初に低親和性のCa 2 +指示薬（ 例えば MAG-たFura2-がロードされAM、）、レシオメトリックUV光。その後、パッチピペット、残っCYTの助けを借りてosolic低親和性のCa 2 +インジケーターは高親和性のCa 2 +指示薬（ 例えばフルオ-3、可視光）を含む緩衝液を用いて細胞質ゾルの外透析されています。この戦略は、細胞質およびER派生信号の同時記録が可能。 （5） 目標-エステラーゼ誘発性染料ローディング 8,34。カルボキシルエステラーゼ（CES）は、ERの内腔に標的化および低親和性のCa 2 +指標のAM-エステル体を効率的に捕捉するための高エステラーゼ活性を提供する。 標的と-エステラーゼ誘導し染料ロード（TED） ER内腔に低親和性のCa 2 +指標のターゲッティング改善するには、TEDが開発されました。 TEDは、発現構築物によって達成されるER標的マウスカルボキシルエステラーゼ（CES2）（ 図2）の過剰発現を必要とします。換えCES-コンストラクトを発現する細胞は、中のカルシウムのAM-エステルフォームでインキュベートするdicator色素（Fluo5N-AM、 図2）。次に、ERで、染料は、したがって、ER内腔8に高濃度で染料をトラップ、高エステラーゼ活性によりCa 2 +の敏感な、膜不浸透性のCa 2 +指示薬複合体（Fluo5N/Ca 2 +）に変換されます、34。方法はSERCA 17の遮断後にインスタンスに対して直接&quot;リークチャネル&quot;を介して、代謝、プリンまたはグルタミン酸受容体8,34を介して、例えばIICR-経路を介して、小胞体のカルシウム放出を調査し、ERカルシウム枯渇を可視化するために特に便利です、34。我々の経験に低親和性のCa 2 +指示薬Fluo5N-AMは、現在TED染料ローディング戦略で使用する利用可能な最善の指標である。 Fluo5N-AMは、Ca 2 +のための低親和性を持っている（解離定数K D〜90μM、 図2）、ほぼ非蛍光そのAM-形であるが、calciu際高い蛍光発光を提供していますMバインディング8,34。 Fluo5N/Ca 2 +錯体は、そのようなFITC、アレクサ488、またはeGFPのような標準的な染料に対応〜490ナノメートルの標準光源で励起することができる。細胞質カルシウム信号は殆ど低μMの範囲の濃度に到達しない為、ほとんど細胞質内Fluo5N 35によって検出される。 TEDのパフォーマンスを向上させるには、いくつかの組換えベクター構築物（ 図3）を開発した。もともと、CES2のコード配列（のRefSeqアクセッション番号NM_145603、CES2c）と最高のTEDのパフォーマンスに基づいてTEDベクトルはCES構築物の安定発現で観察されています。新しいTEDベクターは、赤色蛍光タンパク質TagRFP-T2 36に融合CES2のコア要素を表現する。これらのベクターは、それらが形質導入細胞識別し、Fluo5N/Ca 2の変化を正規化するための内部コントロールとして赤色蛍光を使用するために使用することができるという利点を有し+蛍光。赤色蛍光はまた刺激条件でERとERダイナミクスの変化の構造分布を可視化する可能性を提供しています。

<table border="1">
	 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(S)-3,5-DHPG (Dihydroxyphenylglycine)</td>
 <td>Tocris</td>
 <td>0805</td>
 <td></td>
 </tr>
 <tr>
 <td>5';-ATP-Na2, ATP disodium salt hydrate</td>
 <td>Sigma</td>
 <td>A26209-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 supplement,50x</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbamoylcholine chloride (Charbachol)</td>
 <td>Sigma</td>
 <td>C4382-1g</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyclopiazonic acid (CPA)</td>
 <td>Ascent scientific</td>
 <td>Asc-300</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM with Glutamax</td>
 <td>Invitrogen-Gibco</td>
 <td>31966-047</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS without Ca/Mg 1x</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Invitrogen-Gibco</td>
 <td>10270-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-5N-AM, 10 x 50 &mu;g</td>
 <td>Invitrogen</td>
 <td>F14204</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Ascent scientific</td>
 <td>Asc-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Ham's F12 nutrients mixture</td>
 <td>Invitrogen-Gibco</td>
 <td>21765-029</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS</td>
 <td>PAA Laboratories</td>
 <td>H15-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>SHD3250YK</td>
 <td>Linearis</td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Ca2+ salt</td>
 <td>Ascent scientific</td>
 <td>Asc-116</td>
 <td></td>
 </tr>
 <tr>
 <td>murine EGF </td>
 <td>PreproTech</td>
 <td>315-09</td>
 <td></td>
 </tr>
 <tr>
 <td>N2 supplement 100x</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen-Gibco</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127, low UV absorbance,2g</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine hydrobromide PORN</td>
 <td>Sigma</td>
 <td>P8638</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide</td>
 <td>Sigma</td>
 <td>P7280</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-Lysin</td>
 <td>Sigma</td>
 <td>P2636</td>
 <td></td>
 </tr>
 <tr>
 <td>Thapsigargin</td>
 <td>Calbiochem</td>
 <td>586005-1mg</td>
 <td></td>
 </tr>
 <tr>
 <td>TryLE Express</td>
 <td>Invitrogen</td>
 <td>12605-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Worthington</td>
 <td>LS-003707</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsininhibitor from Glycine MAX (soybean)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Confocal system: inverted laser scanning microscope: FV1000 with IX81</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: scan head FV10-SPD with spectraldetector</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Heater controller TC-344B</td>
 <td>Warner Instruments</td>
 <td>64-0101</td>
 <td>to control in line solution heater</td>
 </tr>
 <tr>
 <td>NIH ImageJ software</td>
 <td>WS Rasband, ImageJ, US National Institutes of Health, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997--2006</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UAPON20XW340 NA 0,7 WD 0,35</td>
 <td>Olympus</td>
 <td>N2709100</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLFLN40XO</td>
 <td>Olympus</td>
 <td>N1478700</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLSAPO60XO/1.35, WD=0.15</td>
 <td>Olympus</td>
 <td>N1480700</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, inverse</td>
 <td>custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, RC-49FS</td>
 <td>Warner Instruments</td>
 <td>W4 64-1709 </td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion tube: PT-49</td>
 <td>Warner Instruments</td>
 <td>64-1730</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Peristaltic pump MiniPlus 3 (4 channels)</td>
 <td>Gilson</td>
 <td>n.a. </td>
 <td>perfusion pump</td>
 </tr>
 <tr>
 <td>Single inline solution heater SH-27B</td>
 <td>Warner Instruments</td>
 <td>64-0102</td>
 <td>controlled by heater controller</td>
 </tr>
 <tr>
 <td>Suction tubes: ST3R</td>
 <td>Warner Instruments</td>
 <td>64-1407</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Heating insert: Tempocontrol 37-2 digital 2-channel</td>
 <td>Pecon</td>
 <td>n.a. </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

カルシウム動態の可視化は、細胞生理学におけるカルシウムの役割を理解することが重要である。カルシウム動態を調べるために、合成蛍光Ca 2 +のウインカが人気となっている。ここでは、TED（=ターゲット·エステラーゼ誘導される色素ロード）、のCa 2 +放出異なる種類の細胞の小胞体内腔におけるインジケーター色素を改善するための方法を示しています。今日まで、TEDは、細胞株、グリア細胞、およびin vitroでのニューロンに使用した。ベクターコンストラクトを用いた小胞体内腔に高いカルボキシルエステラーゼ活性の標的効率的組換えでTED拠点その急行カルボキシルエステラーゼ（CES）。最新のTEDベクトルは、このように同時2カラー撮像を可能にし、赤色蛍光タンパク質に融合CES2のコア要素を含んでいる。対応するER構造が赤で表示されている間、ER内遊離カルシウムのダイナミクスは、一つの色に結像される。プロシージャの先頭で、細胞をレンチウイルスで形質導入されています。その後、感染した細胞再は、最終的に生細胞のイメージングを可能にするためにカバースリップ上に播種した。その後、生きている細胞は、インスタンスFluo5N-AM、MAG-Fluo4-AM、またはMAG-たFura2-AMのため、低親和性のCa 2 +指標のアセトキシメチルエステル（AM-エステル）フォームでインキュベートする。のCa 2 +指標と親水性蛍光色素/ Ca 2 +の錯体のAM形からの疎水性側鎖を切断オフER内エステラーゼ活性が形成され、ER内腔に閉じ込められている。色素ローディング後、細胞を倒立型共焦点レーザー走査顕微鏡で分析される。細胞は継続的にリンガーのようなソリューションで灌流し、ERのカルシウムダイナミクスタイムラプスイメージングによる直接可視化されています。 ERカルシウムストアの補充は、蛍光強度の増加を生じるのに対し、ERからのカルシウム放出は、関心のある領域における蛍光強度の減少によって識別される。最終的に、経時的に蛍光強度の変化はΔF/ F 0の計算によって決定される。

このプロトコルは、無料のERカルシウムの直接イメージングのための非破壊的なアプローチを提供します。低親和性合成のCa 2 +指標が解放され、標的ER、組換えエステラーゼ酵素活性の助けを借りて、ERの内腔に閉じ込められている。 ER内腔へのCa 2 +指示薬色素の改良された負荷は、ERカルシウムストアダイナミクスの直接かつ高速イメージングを可能にします。 <p class="jove...

Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

標的と-エステラーゼ誘導し染料ロード（TED）は、蛍光イメージングによる細胞内カルシウムストアのダイナミクスの解析をサポートしています。それは合成の低親和性のCaローカルマスキングを改善し、小胞体（ER）に換えカルボキシルエステラーゼの標的に対するメソッド拠点<sup&gt; 2 +</supER内腔&gt;インジケーター色素。

ターゲット-エステラーゼ誘起染料の読み込み（TED）とERカルシウムの直接イメージング

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

direct-imaging-er-calcium-with-targeted-esterase-induced-dye-loading

Research

JoVE Journal

Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

19.1K ビュー.  University of Wuerzburg. 標的と-エステラーゼ誘導し染料ロード（TED）は、蛍光イメージングによる細胞内カルシウムストアのダイナミクスの解析をサポートしています。それは合成の低親和性のCaローカルマスキングを改善し、小胞体（ER）に換えカルボキシルエステラーゼの標的に対するメソッド拠点 2 +インジケーター色素。

ビデオ: Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

カルシウムインジケータ付きのロードショウジョウバエの神経終末

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

生物学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

レイヤーin vivoでの2光子カルシウムイメージングマウスの2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

AMフラ-2を用いて皮質ニューロンのカルシウムイメージング

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

アセンブリ、読み込み、および分析用超遠心器のサンプルセルの整列

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別 2 + ER膜のリークチャネルとその調節機序

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

ワクチン接種のための発現およびウイルス様粒子の精製

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

目標とするCaを用いたER / SRカルシウム放出のモニタリング<sup&gt; 2+</sup&gt;センサーキャッチャー<sup&gt; +</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

中間径フィラメントと微小管 2次元直接確率光再構成顕微鏡イメージング

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Aggresome のような誘発構造で退色の黄色蛍光タンパク質タグ p62 後の蛍光回復

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

マウス膵島細胞の亜集団におけるカルシウムダイナミクスのイメージング

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...