D.サムズはG.ハロルド＆レイラY.が慈善財団と科学と工学のコロンビア大学の取り組みをマザーズに感謝。 D.サムズとD.スルザーは、神経科学賞でマックナイトの技術革新のためにマックナイト財団に感謝 D.スルザー感謝NIDA、NIMH、および回Picowerとパーキンソン病の基礎。 H.チャンはNARSADに感謝。 RHエドワーズは、マイケルJフォックス財団、国立パーキンソン財団、NIDAとNIMHに感謝。 我々は、全反射顕微鏡のセットアップと技術サポートのために6 - OHDA注入やアドバイス、有用な議論のためのマークのSonders、画像解析のプログラミングのためのメレクシュウ、とJan Schmoranzerのためにロバートバークに感謝。

このメソッドは、で報告された研究で使用されて<a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator ら、Science 324（5933）。 1441-1444（2009） 。</a> 1。急性線条体スライスの準備<ol><li>急性線条体スライスを準備する前に、一つは酸素を人工脳脊髄液（ASCF）と前脳の抽出には少なくとも15分間氷でそれを冷やし必要があります。 ACSFの氷冷を維持し、解剖時の酸素。 ACSF（mMで）：NaClの125、KClを2.5、NaHCO 3の 26のCaCl 2 2.4、硫酸マグネシウム1.3、KH 2 PO 4 0.3、グルコース10、HEPES 5、pHが7.3から7.4、290から295 mOsm。 </li><li>麻酔なしで雄マウスを刎ねる。 </li><li>マウスから脳全体を抽出し、そしてクレイジー®接着剤を使用してビブラトーム上に配置ビブラトームトレイにマウントします。脳の抽出と取り付けは2分以内に行う必要があります。この時間の間に、ACSFの氷冷を維持し、組織の健康を保つために酸素をしてください。 </li><li>ブレグマ1.54の間に0.62 1〜250μmの厚さで冠状線条体の脳切片をカット。三全体のスライスは、針6の半分のスライスにカットすることができますが得られます。 </li><li>プローブ負荷の前に少なくとも1時間室温で酸素化されたACSFの脳切片をインキュベートします。スライスは良いFFN511ローディングを表示し、スライスの調製後4時間まで撮影のためにアクティブのまま。 </li></ol> 2。ローディングFFN511 <ol><li> FFN511ローディング液（ACSFで10μMFFN511）を準備し、また、ACSFで100μMADVASEP - 7を準備する。すべてのソリューションを新たに調製し、スライスにロードするために少なくとも15分前に酸素化されるべきである。 </li><li>個別に室温で30分間FFN511ローディングソリューションでスライスをインキュベートする。 </li><li>その後、室温2で30分間酸素100μMADVASEP - 7 ACSFでロードされたスライスをインキュベートすることにより、細胞外組織に結合した色素を取り除く。スライスは、現在のイメージングのための準備が整いました。 </li></ol> 3。脳スライスのイメージングFFN511 スライスのFFN511のイメージングは​​、多光子レーザー走査型顕微鏡を用いて行われる。我々は（我々は以前にツァイスLSM 510 NLO多光子レーザー走査型顕微鏡を使用していたし、いくつかの結果はツァイスの顕微鏡で得られた）プレーリーウルティマ多光子レーザー走査顕微鏡を使用しています。 <ol><li>録音室でFFN511負荷スライス（RC - 27L、ワーナー）を配置し、分あたり1-2 mlの流量で酸素化されたACSFで表面かん流する。その後、実験時の動きを最小限に抑えるために、スライスの上にナイロン弦とプラチナのハープを置きます。さらなる動きを最小限に抑えるために、スライスは、イメージングの前にチャンバー内で少なくとも10分間安定化させることができます。 </li><li>視覚化し、10倍の水浸対物レンズの下で明視野発光と背側線条体を探します。 </li><li>この地域の場所や位置バイポーラツイストタングステン電極刺激実験を行う場合。理想的な撮像領域を刺激バイポーラ電極の2つのヒントの間に300μmの内に位置しています。視野の中心にこの地域を置きます。 </li><li> 63x（0.9 NA）水浸紫外線の目標の下に画像FFN511標識線条体の端末。 FFN511はスペクトル物理学と線条体端末の最適な蛍光からマイタイのレーザーと760 nmで励起され、バンドパスフィルタ（480〜520 nm）を介して見られている。画像は512 × 512ピクセルの解像度での関心の75 × 75μmの領域と12ビット形式でキャプチャされています。刺激脱色実験については、z平面上の1μm以下で区切られたz軸のシフト、5-7画像のz -シリーズ、、を補正するためには、各期間に取得されます。 </li></ol> 4。 FFN511脱色 FFN511ラベルは、KClの高濃度（我々は70 mMのKClを使用）、（+）-アンフェタミン硫酸（AMPH、20μM）、または電気刺激によりいずれかを脱色することができます。における高カリウムのACSF液を脳のスライスに適用される塩化カリウム、脱色実験、、2分以内にFFN511ラベルdestains。レコードには、XYZ - Tの画像を時間をかけてFFN511脱色を追跡する。以下では、電気刺激に依存するドーパミン端末脱色について説明します。 <ol><li>時間をかけてFFN511脱色を追跡するために、XYZ - Tの画像が記録されます。スライスの最小限の動きを（z軸に4μm未満）を確実にしていないレーザーによる退色、Zスタックの制御の時系列画像は、刺激前に少なくとも5分間取得する必要があります。そうでなければ、レーザーパワーを調整する。 </li><li> 1、4または20 Hzの周波数で刺激を開始しながら、これらのコントロールのイメージに続いて、XYZ - Tのイメージングを続ける。 1、4または20 Hz（300μ× 1 mA）を少なくとも刺激は双極電極を使用してマスター- 8パルスジェネレータ（AMPI、エルサレム、イスラエル）によって引き起こされるアイソフレックスの刺激アイソレータによって局所的に線条体に適用されます。最小限にリリースサイトの脱分極の変化は、我々は、サイクリックボルタンメトリーによって決定される最大刺激強度の150％を適用し刺激プロトコルを使用している。 </li><li>画像解析のための適切なソフトウェアを使用してください。私たちの研究室では、我々は、時間3で涙点蛍光と変化を定量化するためにイメージJ（ウェインRosband、国立衛生研究所、ロックビル、MD）とIDLのカスタムで作成されたソフトウェア（リサーチシステムズ、ボルダー、CO）を使用します。 </li></ol> Represenative結果図1は、線条体スライスにFFN511読み込みが完了であることを示しています。 10倍と60倍対物レンズを用いて代表的なイメージは、図1に示されています。ドーパミン端末の選択的標識は、20μMのアンフェタミンによって脱色することができます。 図2は、高塩化カリウムによってFFN511ラベリングの脱色を示しています。 図3は、地元の電気刺激によってFFN511ラベリングの脱色を示しています。 <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="図1" /> 1 FFN511ラベルライブ皮質-線条体急性スライス内のドーパミンの端子図急性ライブ皮質-線条体スライスのFFN511によって（）ラベル：。線条体に豊富なラベリング（STR）、皮質（CTX）におけるスパースなラベリング、およびでないラベル脳梁（CC）。スケールバー：100μmのアンフェタミンによる線条体からFFN511の（B）脱色。。左のパネル：アンフェタミンの前に、右のパネル：20μMのアンフェタミンの20分後。スケールバー：10μmである。 <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="図2" /> 図2。高KClをしてFFN511ラベルの脱色。 FFN511標識はACSFで70 mMのKClの2分間のアプリケーション内で脱色されています。スケールバー：10μmの。 <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="図3" /> 図3：周波数依存性の線条体におけるFFN511ラベルの脱色。（）4 Hzで局所刺激は端末からの脱色となりました。刺激は、t = 0で始まった。スケールバー：5μmの（B）4 HzでFFN511の脱色は、Ca 2 + -及び周波数に依存する。。コントロールにはない刺激（3スライスから153涙点）を受信しません。塩化カドミウム（200μM）で脱色すると非刺激対照（5スライスから475涙点）と同一であった。各刺激の周波数のための脱色曲線は単一指数減衰関数と半減期により適合させた（T 1 / 2）τX 0.693（1 Hzと計算値：9スライスから765涙点、4 Hzの7のスライスから410涙点、 20 Hzの：6スライスから416涙点）。線条体の脳のスライス標本におけるエキソサイトーシスの間にFFN511脱色の2光子顕微鏡からのビデオ顕微鏡写真を参照してください。

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

このビデオでは、我々は光学的に蛍光ドーパミンの類似体を用いて神経伝達を可視化する方法を示しています。 FFN511は、我々が開発したFFNsの第1世代です。それは、科学に示すように（皮質のシナプス小胞に細胞質からモノアミン神経伝達物質を運ぶ神経細胞の小胞モノアミントランスポーター（VMAT2）、そして特にラベル線条体におけるドーパミンの端末と推定されるカテコールアミンおよび/またはセロトニンの端末をターゲットにして設計されましたが、紙）FFN511は比較的疎水性であるため、適切なローディング期間は特異性のための非常に重要です。我々はその40分より長くするためのインキュベーションは、線条体切片における豊富な非特異的染色をもたらす発見。特異性は、素直に機能的なシナプス小胞内にFFNの放出を引き起こすはずKClの高濃度を、使用して脱色によって決定することができる。 15分未満のためFFN511へのスライスの曝露は、端末の染料の不十分な負荷に起因する微弱な蛍光シグナルになります。このプロトコルでは、我々は細胞外組織に結合した色素を除去するために100μMADVASEP - 7を使用してください。この手順は必要ありませんが、それが省略された場合、ACSFで洗い流し時間は延長する必要があります。要約すると、選択した準備に応じて、最適なラベルを決定するために濃度およびローディング期間を変更する必要があります。

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<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
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<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
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<td>PELCO PrepEze 6-Well Holder</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>36157-1</td>
<td>For slice incubation</td>
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<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
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dopamine release at individual presynaptic terminals visualized with ffns

神経系は、シナプス小胞融合の間に神経伝達物質の放出を介してニューロン間の信号を送信する。直接個々のシナプス前終末から神経伝達物質の取り込みと放出を観察するために、我々は、シナプス小胞モノアミントランスポーターの基質として蛍光偽の神経伝達物質を設計した。線条体での画像のドーパミンの放出にこれらのプローブを使用して、我々は、シナプス可塑性に関連するいくつかの観測を行った。我々は、刺激ごとに神経伝達物質を放出するシナプス小胞の割合は、刺激周波数に依存していたことがわかった。動力学的に別個の&quot;リザーブ&quot;シナプス小胞の人口は、これらの実験条件下で観察されなかった。シナプス前終末の周波数に依存する不均一性は、シナプス前選択の周波数に依存するコーディングのためのメカニズムを示し、D2ドーパミン受容体に部分的に依存していたことが明らかになった。 ホイ張とNiko G.のGubernatorはこの作品にも同様に貢献しています。

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

光学的に蛍光ドーパミンの類似体を用いて神経伝達を測定するための新しい手段。

個々のシナプス前終末でのドーパミン放出はFFNsと可視化

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Research

JoVE Journal

Biology

24.7K ビュー.  Columbia University. 光学的に蛍光ドーパミンの類似体を用いて神経伝達を測定するための新しい手段。

ビデオ: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

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

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

パーキンソン病モデルマウス：6 - OHドーパミンの病変から行動試験に

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Cell Electrofusion Visualized with Fluorescence Microscopy

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

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

細胞の電気融合を蛍光顕微鏡で可視化

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

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - トランスクリプトーム全体の個々のヌクレオチド分解能タンパク質- RNA相互作用のマッピング

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

個々の準備ショウジョウバエ</emライブイメージングのための>エッグチェンバース

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

ワンナノメートルの精度で蛍光イメージング（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...