This work was supported in part by NIH grants (1R01CA137494, R01CA132115, R01CA086072 to R.G.P.), the Kimmel Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.), a grant from the Breast Cancer Research Foundation, generous grants from the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.) and a grant from the Pennsylvania Department of Health (R.G.P.). In part this work was supported by an American Italian Cancer Foundation postdoctoral fellowship (G.D.) and Bioimaging Shared Resource of the Sidney Kimmel Cancer Center (NCI 5 P30 CA-56036).The Department specifically disclaims responsibility for an analysis, interpretations or conclusions. There are no conflicts of interest associated with this manuscript.

両発現ベクターEYFP-パーキンとpDsRed2-水戸での線維芽細胞の1エレクトロポ<ol><li> 100 mg / mlでDMEM（ダルベッコ改変イーグル培地）、10％ウシ胎児血清、2ミリモル/リットルのL-グルタミン、100 U / mlペニシリンを添加した培地を用いて10センチメートルの組織培養プレートに不死化マウス胚性線維芽細胞成長、および37℃、5％CO 2を含む加湿雰囲気中ストレプトマイシン。 <ol><li> 80％の細胞集密度で、無菌吸引によって完全DMEM培地を捨て、滅菌1×リン酸緩衝溶液（PBS）10 mLを加え（塩化ナトリウム80gを、塩化カリウム2.0gを、のNa 2 HPO 4 14.4 gのKH 2.4gの7.4）2 PO 4、1 LのH 2 O、PHを蒸留しました。 </li><li>無菌吸引によってPBSを捨て、トリプシンEDTA、0.25％の1ミリリットルを追加します。 （ - 3分2）細胞が分離されるまで、37℃でプレートをインキュベートします。 _、完全DMEM培地の4ミリリットルを加え、細胞を再懸濁し、10を取り出します6;血球計カウントのための細胞懸濁液リットル。 注：16角の正方形の一組のセルの数は、細胞×10 4 / mlの数に相当するように、血球計数器を設計します。 </li></ol></li><li>エレクトロポレーションプロセスの24時間前に10cmの組織培養皿上にシード1×10 6細胞。 </li><li>無菌吸引によって完全DMEM培地を捨て、滅菌PBSの10ミリリットルを追加します。無菌吸引によってPBSを捨て、トリプシンEDTA、0.25％の1ミリリットルを追加します。 （ - 3分2）細胞が分離されるまで、37℃でプレートをインキュベートします。完全DMEM培地4mlを加え、15 mlチューブに細胞を再懸濁。 </li><li>冷蔵遠心分離（4℃）を用いて5分間250×gで細胞をスピンダウン。無菌吸引によって上清を捨て、滅菌PBS 1 mlにペレットを再懸濁。 4℃で5分間、250×gで細胞をスピンダウン。 </li><li>無菌吸引によって上清を捨て、そして100μlのOを追加エレクトロポレーションのためのF溶液ミックス細胞ペレットに（82補足ソリューション1のエレクトロポレーションソリューションV + 18マイクロリットルのμl）を、穏やかにピペッティングしてペレットを再懸濁（詳細については、ユーザーのガイドを参照してください）​​。 EYFP-パーキン（励起/発光527分の514）2μgのを追加し、細胞懸濁液で1μgのpDsRed2-水戸（励起/発光620分の565）。 </li><li>使い捨てのパスツールを使用して、無菌のキュベットへの解決策を（両方のツールは、キットで提供されています）転送し、予め設定されたプログラムNIH / 3T3 U-030（シングルパルス、電圧200 V、容量960μF、パルス時間20ミリ秒を使用して、エレクトロポパルス番号：1）。 </li><li>電気穿孔直後に、予め温めておいた新鮮な完全DMEM培地500μlのを追加し、6センチメートル組織培養プレートライブイメージンググレード上の細胞に接種し、37℃で5％CO 2を含む加湿雰囲気中でそれらをインキュベート24時間。 </li></ol> 2.タイムラプスビデオ顕微鏡<ol><li&gt;は使用前に37℃での顕微鏡のチャンバーの温度を設定します。 </li><li>この時点で、実験手順を続行するために特定のライブイメージング媒体を準備します。次のようにライブイメージング媒体を準備します。混合DMEMフェノール不含の10％ウシ胎児血清、2ミリモル/リットルのL-グルタミン、100 U / mlペニシリン、および100mg / mlストレプトマイシンを補充しました。水浴中で37℃でライブイメージング媒体を事前に温めます。 </li><li>無菌吸引によってエレクトロポレーションした細胞の培地を捨て、予め温めておいたライブイメージング媒体の1ミリリットルを追加し、P1000ピペットを用いて、37℃で30分間静置します。 </li><li>プレートのインキュベーション期間中に流入、5％CO 2顕微鏡のチャンバ内にすでに37℃で安定した温度で実施されます。 </li><li>主要な動きや振動を避けて、顕微鏡のチャンバ内へのエレクトロポレーションした細胞でプレートを置きます。 </li><li>ソフトウェア・インターフェースを使用して、両方のfluoreを検出するために顕微鏡を設定します同時トランスフェクトするベクター、EYFP-パーキン（510 nmおよび放射範囲520〜550 nmの励起範囲495、緑）、pDsRed2-水戸（励起最大558 nmおよび放出によって、最大583 nmでのコードされた融合タンパク質からのscence信号、赤）。 <ol><li>タイムラプスビデオ顕微鏡ソフトウェアを開きます。上部のメニューでpDsRed2-水戸のためのEYFP-パーキンおよびローダミンのためのFITCを選択します。上部のメニューで倍率（20X）を選択します。 </li></ol></li><li>ソフトウェアインターフェイスを使用して、両方の同時トランスフェクトするベクターを発現する単一のセルを探し、位置を登録します。 10細胞の最小値は、すべての実験条件（実験群）のために収集されるまで、この手順を繰り返します。 <ol><li>メニュー「アプリ」を選択し、多次元の取得をクリックしてください。多次元取得ウィンドウでは、このような買収の数、それぞれ取得し、記録セルの位置との間の時間間隔として必要なパラメータを選択します。取得」をクリックします。「取得プロセスを開始します。 </li></ol></li><li> EYFP-パーキンと15分の合計の間隔で5分ごとに画像を収集DsRed2の-水戸蛍光信号の両方の基底取得を開始します。 </li><li>最終的な作業濃度の2倍FCCPの濃度で予熱したライブイメージング媒体を準備する（0.1から10μM、細胞型に依存します）。 </li><li>取得処理を中断し、P1000ピペットを用いて顕微鏡のチャンバ内のプレートにFCCPで予め温めておいたライブイメージング媒体の優しく1ミリリットルを追加します。 </li><li>以前に記録された位置を使用して、3時間の全期間のために、説明したように取得プロセスを再起動します。それらを分析し、買収が終わったときmitophagicプロセスのビデオを作成するために取得したすべての画像を保存します。 </li></ol> 3.分析<ol><li>パソコン上で「.TIFF」の形式で取得したすべての画像を収集</li><li>グラフィックソフトで画像を開きます。ウェアおよび時間的間隔を定義するには（画像は5分ごとに取得される）ミトコンドリア膜にパーキンの動員を示す第1の画像にFCCPとミトコンドリア誘起脱分極から発生しました。実験群内の各セルのために、この分析を繰り返します。 </li><li>実験グループ名を持つスプレッドシート上の列の最初のセルにラベルを付けます。各実験群について、10セルの最小のパーキン募集のための時間的間隔を測定します。 Parkinの募集のための計算された時間的間隔の平均を作成し、各実験群の標準偏差定義（平均値±SDを、N = 10）。 <ol><li>列に単一の計算された時間的間隔を整理します。すべての列は、n =実験群の10の測定値が含まれています。 </li><li>数式パレットメニューから「平均」機能を選択します。列のデータを選択します。関数式ビルダのメニューから「標準偏差」を選択します。セレ列内のデータのct。 </li></ol></li><li>統計的手法を用いて、実験群間マイトファジーを動的に有意な差を定義するために適切な統計値を適用し、実験群を比較します。 （例えば、二つのグループ= スチューデントのt検定 、3つまたはそれ以上の基= ANOVAプラスポストホック検定） </li></ol>

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The authors have nothing to disclose.

タイムラプス顕微鏡は、長期間にわたって携帯ダイナミクスの観察に時間的に1つの観測からのライブセルイメージングを拡張する技術として定義することができます。それは観察者がリアルタイムで単一の蛍光標識タンパク質を同定し、単一生細胞内でそのダイナミックに追従することができますので、この方法は、単純な共焦点または生細胞顕微鏡と区別されます。実際には、共焦点顕微鏡は、簡単に蛍光抗体を用いた免疫標識タンパク質を同定することができますが、それは、細胞およびそのライブ環境での分子事象を観察することはできません。 タイムラプスビデオ顕微鏡は、主にHEK293 16などの異なる種類の細胞の分子プロセスを監視、研究、一次ニューロン17およびHela細胞18で使用されるだけでなく、妊娠や予測異数性19,20として臨床応用を有しています。タイムラプス顕微鏡はembryolog提供します臨床妊娠のために胚発生についての追加情報をISTS、この情報を転送するための実行可能な胚を選択する能力を改善するために使用されることが可能です。タイムラプス顕微鏡の使用のさらなる利点は、以下の胚の取り扱いおよび実験室19内の損失や汚染の危険性を低減するであろう中間の観測のための彼らの料理を用意しています。 細胞は、マイトファジーを介して、損傷や機能不全ミトコンドリアを分解することにより、健康なミトコンドリアの恒常性を維持することができます。不良マイトファジーは23とガン24を老化、パーキンソン病21、アポトーシス22にリンクされています。マイトファジーは、ユビキチンリガーゼパーキンとPINK1 21によって駆動されます。健康ミトコンドリアの状態を維持するために、このプロセスの重要性を、我々はそれが重要なタイムラプスビデオ顕微鏡を使用して、このイベントのダイナミックを研究することがわかりました。提案されたプロトコールは、ミトコンドリアの恒常性は、遺伝子発現やタンパク質の機能の変化を含む様々な細胞条件下でどのように影響されるかについての証拠を提供します。 本研究では、一時的にエレクトロポレーションによって線維芽細胞をトランスフェクトしました。エレクトロポレーション法は、膜のリン脂質二重層を妨害することによって細胞膜の透過性を増加させるために、細胞懸濁液に数マイクロ秒持続する最適化された電圧の電気パルスを印加します。このアクションは、一時的な孔の形成をもたらします。膜透過性は、DNAが細胞25内に導入されることを可能にします。我々は、一過性の両方のプラスミドEYFP-パーキンとpDsRed2-水戸の高いトランスフェクション効率を有するために、線維芽細胞をトランスフェクトするために、このプロセスを選択しました。実際には、エレクトロポレーション法は、化学的トランスフェクションの26よりも約10倍有効です。画像取得処理は、両方エクステによって侵害される可能性がありますrnal因子;温度変動、振動、湿度、内部要因。 pHは、細胞の運動性。エレクトロポレーションおよび蛍光追跡は、この実験プロトコルの成功のために最も重要であると考えられて2ステップです。トランスフェクション効率は、他の方法よりも高い場合であっても、エレクトロポレーションは、エレクトロポレーションした細胞の〜20％を死滅させます。もう一つの大きな問題は、監視ステップの間に画像の取得の品質です。細胞環境は動的であるので、焦点面は、取得プロセスの全体にわたってフォーカスを維持するために連続的な調整が必要です。 ミトコンドリアの損傷は、膜、ミトコンドリア脱分極および酸化的ストレスによって引き起こされ得ます。これらの要因はmitophagic応答を誘導します。ミトコンドリアの脱分極は、選択マイトファジーを誘導することができることを示す証拠は、ROS産生とミトコンドリア損傷27-30につながる光損傷実験から来ています。ミトコンドリアのMEMを誘導するためにブレーン脱分極とmitophagic応答、FCCPはプロトノフォア10として使用されています。 FCCPは、プロトンが膜の脱分極を誘導する脂質二重層を横断することを可能にするイオノフォアです。このアプローチを用いて、我々は、マイトファジーを誘導し、分子マーカーの動的に続きました。 それは細胞毒性であることと、細胞死を促進することができるようFCCPの濃度は、非常に重要です。このアプローチは、生細胞の観察に依存しているので、FCCPの最終濃度は、マイトファジーを誘導することなく、細胞死に至るメカニズムをトリガするために、細胞型依存的に調節されなければなりません。 全体的に、我々は蛍光マーカーとしてEYFP-パーキンを使用して、マイトファジーの動的、以下、ミトコンドリア機能に新たな洞察を提供することを考えています。マイトファジーを介してミトコンドリアの選択的な除去は、ミトコンドリア質、細胞の生存および恒常性の維持に重要なプロセスです。このライブイメージングアプローチは、彼なりますlpが健康でストレス条件下でmitophagicプロセスを理解します。 現代的なアプローチは、さらに、携帯ダイナミクスの映画を作る超えタイムラプス顕微鏡観察を拡張しています。ますます、この境界は、サイトメトリー技術は、細胞および細胞内構造物31の動的活動を監視し、測定するためのイメージング技術と一体化されているようにぼやけています。

Macro autophagy is an intracellular process that involves the catabolic degradation of both damaged and dysfunctional cellular components, such as organelles and proteins for the purpose of either recycling or energy production. To initiate this metabolic process, the cell engulfs the damaged cellular components into a double-membrane structure, known as an autophagosome, which fuses with a lysosome and its content is degraded and recycled 1,2. There are two major types of autophagy, the non-selective and selective. The non-selective autophagy process occurs when the cell is under nutrient deprivation conditions and needs to scavenge for both essential nutrients and energy. However, selective autophagy occurs to mediate the removal of both dysfunctional/damaged organelles and proteins that otherwise could be toxic. One of the most studied selective autophagy process is the removal of mitochondria, termed mitophagy 1,3-5.
Mitochondria are the central organelles for cell metabolism and the primary source of adenosine triphosphate (ATP) via oxidative phosphorylation through the electron transport chain, fatty acid oxidation, and tricarboxylic acid (TCA) cycle. Moreover, mitochondria regulate reactive oxygen species (ROS) production and release proteins that participate in cell death pathways 6-8.
PTEN-induced putative kinase 1 (PINK1) and Parkin RBR E3 ubiquitin ligase (Parkin) are the key proteins implicated in the mitophagy process. Parkin can protect against cell death by keeping the cell healthy through mitochondrial quality control9. Upon the loss of mitochondrial membrane potential, cytosolic Parkin is recruited to the mitochondria by PINK1. This recruitment triggers the sequential events of mitophagy 10. There is a broad range of evidence that mitophagy is a fundamental mitochondria quality control process and abnormalities in this process drive disease 7. For instance, autosomal recessive Parkinson's disease has been associated with mutations in the genes that encode for Parkin and PINK1 (PARK2 and PINK1, respectively) 11. The quality control of mitochondrial health is essential for the removal of mitochondria that contribute to the accumulation of ROS12. Excessive presence of intracellular ROS can lead to damage of both nuclear and mitochondrial DNA (DNA and mt DNA, respectively).
Herein, we show a time-lapse video microscopy approach to follow the aggregation of Parkin after the induction of Parkin-mediated mitophagy in immortalized mouse embryonic fibroblasts via in vitro administration of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupling agent. FCCP disrupts ATP synthesis by short circuiting protons across the outer mitochondria membrane and hence uncoupling oxidative phosphorylation from the electron transport chain 13. Triggering the depolarization of the mitochondrial membrane leads to the disruption of mitochondria and selective Parkin-dependent removal. Therefore, transfecting the cells of interest with an expression vector encoding Parkin fused with a fluorescent marker (enhanced yellow fluorescent protein, EYFP) can be used as a fluorescent tag to follow the recruitment of Parkin during the mitophagic process. In order to visualize the mitochondria, we co-transfected pDsRed2-Mito, which encodes red fluorescent protein (DsRed2) that contains a mitochondrial targeting sequence of cytochrome c oxidase subunit VIII (Mito). pDsRed2-Mito is designed for fluorescent labeling of mitochondria14. The time required for Parkin translocation into the mitochondrial membrane can be measured and gives an indirect measure of cellular health. For example, we can say that if a cell line knocked-out for a particular gene of interest shows either a faster or slower recruitment of Parkin after the induction of mitophagy by FCCP, that gene product would be a key player in order to keep the metabolic rates of the cell at the physiological status and prevent the development of diseases. Therefore, the time-lapse video microscopy provides a very powerful tool for both basic and clinical research applications in following the dynamic of labeled proteins during their molecular processes and understanding how these processes are affected during a pathological condition.

<table border="0" cellpadding="0" cellspacing="0" width="1736">
	<tbody>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">10-013-CV</td>
			<td style="width:369px;">Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PHENOL-FREE DMEM</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">17-205-CV</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">FETAL BOVINE SERUM</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">F2442</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">L-GLUTAMINE</td>
			<td style="width:357px;">Gibco</td>
			<td style="width:276px;">25030</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">PENICILLIN/ STREPTOMYCIN</td>
			<td style="width:357px;">Corning Life Science</td>
			<td style="width:276px;">30-002-CI</td>
			<td>Pre-warm at +37 C before use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">EYFP-PARKIN EXPRESSION VECTOR</td>
			<td style="width:357px;">Addgene</td>
			<td style="width:276px;">23955</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">pDsRed2-Mito EXPRESSION VECTOR</td>
			<td style="width:357px;">Clontech</td>
			<td style="width:276px;">632421</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">NUCLEOFECTOR 2B DEVICE</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">AAD-1001S</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">NUCLEOFECTOR FOR KIT R NIH/3T3</td>
			<td style="width:357px;">LONZA</td>
			<td style="width:276px;">VCA-1001</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">ZEISS AXIOVERT 200M INVERTED MICROSCOPE</td>
			<td style="width:357px;">CARL ZEISS</td>
			<td style="width:276px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Carbonyl Cyanide 4-(trifluoromethoxy)-Phenylhydrazone (FCCP)</td>
			<td style="width:357px;">Sigma-Aldrich</td>
			<td style="width:276px;">C2920</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:735px;">MetaMorph</td>
			<td style="width:357px;">Molecular Devices</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:735px;">ImageJ</td>
			<td style="width:357px;">National Institute of Health</td>
			<td style="width:276px;">Experimental Builder</td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse video microscopy for assessment of eyfp-parkin aggregation as a marker for cellular mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

ここで、我々は、タイムラプスビデオ顕微鏡は、単一のセル内の蛍光標識タンパク質の分子のイベントを追跡するために使用することができる強力な技術である方法を示しています。代表的な結果はまた、この技術は、高品質な画像の取得を可能にする方法を示します。分子過程の画像が得られると、我々は、さまざまな方法でそれらを分析する機会を持ちます。ここでは、選択的に損傷したミトコンドリアを除去する細胞の恒常性を維持する重要な分子過程で誘導さマイトファジープロセスの開始の間の時間の間隔を分析します。 この技術は、幅広いアプリケーションを設立します。それを研究する必要があるプロセスに関与する重要な分子の標識化を介して、細胞遊走またはミクロ分子事象としてマクロ細胞プロセスを監視するためにも使用することができます。 O：キープtogether.withinページ= &quot;1&quot;&gt; パーキン媒介マイトファジー 損傷したと機能不全ミトコンドリアプロセスの選択パーキン媒介除去が脱共役剤FCCPにより誘発される膜の脱分極はミトコンドリア外膜にPINK1を募集し、選択パーキン媒介マイトファジーをトリガー図1に要約されています。続いて、パーキンは、損傷したミトコンドリアに動員し、PINK1と相互作用しています。この複合体パーキン-PINK1は、損傷した細胞小器官のユビキチン化およびリソソームと融合オートファゴソーム、中への取り込みにつながります。マイトファジーと呼ばれるこのプロセスは、健康な細胞環境を維持し、機能的ミトコンドリア8,15の再生を可能にするために重要な代謝過程であると考えられています。 実験プロトコル 「FO：キープtogether.within-ページ=「NT 1 &quot;&gt; 図2は、実験プロトコルおよびFCCP誘発性マイトファジープロセスの前と後にEYFP-パーキンの募集を下記のために使用される顕微鏡のセットアップの概略図を示します。 無偏光化ミトコンドリア膜へのパーキンリクルート 図3Aに、我々は前パーキン分布を示し、FCCPの投与後。基礎時点（BT5、BT10とBT15分）の間、EYFP-パーキン（緑）が均一に細胞全体に拡散され、ミトコンドリアネットワーク（レッド）が十分に相互接続されたように思われます。 FCCPの投与後（FT = 0分）（ 図3B）、我々は、ミトコンドリア分画（FT = 5分）を観察します。しかし、EYFP-パーキンはまだ均質細胞質外にかかわらず、拡散されます。マイトファジーは、55分後FCCP投与で刺激されますEYFP-パーキンは、マイトファジープロセス（FT = 55）をトリガするために、損傷ミトコンドリア膜に動員されます。 <img alt="図1" src="/files/ftp_upload/53657/53657fig1.jpg" /> 図1.パーキン媒介マイトファジーの模式図。膜電位（Ψm）の減少は、損傷または機能不全のミトコンドリアで発生し、脱共役剤FCCPによって誘導することができます。膜電位の損失は、ミトコンドリア外膜にPINK1（レッド）を募集し、選択パーキン媒介マイトファジーをトリガーします。パーキン（ブルー）を直接PINK1と相互作用することにより損傷したミトコンドリアに動員されます。パーキンの存在は、損傷した細胞小器官とリソソームと融合オートファゴソーム、中へのその取り込みのユビキチン化（イエロー）につながる。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>。 <img alt="図2" src="/files/ftp_upload/53657/53657fig2.jpg" /> 図2.顕微鏡セットアップおよび実験プロトコルの表現。プロトコルセクションに記載された実験イベントの（A）の概要。報告された実験のイベントは、オペレータが一時的にタイムラプスビデオ顕微鏡法のための（B）のセットアップを使用してFCCP誘発性マイトファジープロセスを追跡するために、エレクトロポレーションによってEYFP-パーキンとpDsRed2-水戸で細胞を同時トランスフェクトすることができます。 BT =基礎時点; FT = FCCP時点。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53657/53657fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53657/53657fig3.jpg" /> パーキンRecruitmeを表示図3.線維芽細胞<html> NT FCCPにより誘導されるミトコンドリア膜脱分極。EYFP-パーキン（緑）転後のミトコンドリア（赤）に基礎時点（BT）の間にタイムラプスビデオ顕微鏡によって監視され、FCCP管理（FT）を投稿しました。 BT = 0から、EYFP-パーキンとミトコンドリアの蛍光は、5分ごとに検出されました。 （A）EYFP-パーキンは、均一に全体の基礎期間全体に拡散され、ミトコンドリアのネットワークは、無傷（白矢印）です。 FCCPにより、膜の脱分極に（B）ミトコンドリア分画は、FT = 55分（白矢印）で開始し、損傷ミトコンドリア膜のFT = 5分（白矢印）およびEYFP-パーキンの動員で記録されています。白いボックスは、パネル（5X）の拡大面積を表します。この実験プロトコルの適用に関する詳細については8を参照するために参照してください。 （スケールバー：10μm）を。&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。

Watch this Scientific Journal Video about Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy at JoVE.com

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

携帯マイトファジーのマーカーとしてEYFP-パーキン集計の評価のためのタイムラプスビデオ顕微鏡

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

time-lapse-video-microscopy-for-assessment-eyfp-parkin-aggregation-as

Nanyang Technological University

Research

JoVE Journal

Biology

7.1K ビュー.  Thomas Jefferson University, SKCC. Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

ビデオ: Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

タイムラプス顕微鏡で分解アクチンの監視

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

における酸素欠乏によって誘導された仮死状態を可視化するために時間の経過顕微​​鏡の使用<em&gt; Cエレガンス</em&gt;胚

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

SOX10中の顔面の開発の可視化：楓トランスジェニックゼブラフィッシュラインタイムラプス共焦点顕微鏡を用いて

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

線虫の加齢とともに固有の蛋白質の集合の変化を研究するためのメソッド

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

共焦点顕微鏡を明らかに細胞表面受容体画像相関分光法による凝集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

シロイヌナズナの取付汎用性の高い方法は、生体のタイムラプス イメージングの葉します。

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...