我々は、神経科学研究のためのタフツセンター、P30 NS047243（ジャクソン）、ボストン大学メディカルキャンパス、リンク医学Corporationの学際的医学研究のためのエヴァンスセンターでサポートされているミトコンドリアアフィニティ研究協力（mtARC）と、この作業を支援するためのツァイスに感謝します。

1。イメージングプレートの準備<ol><li> 10パーセント標準のウシ胎児血清を含むRPMI培地で培養INS1細胞、1％ペニシリン-ストレプトマイシン、2mMのL-グルタミン、50μM2 -メルカプトエタノール、5mMのNaHCO 3を 、2 mMのHEPES、2mMのピルビン酸、11 mMグルコース80％コンフルエントに。 </li><li> 0.05％トリプシンおよびプレートそれらのポリ-D-リジンコートしたカバースリップ底イメージングプレート（30〜40％コンフルエント）上でINS1細胞培養をトリプシン処理。 </li><li> 24時間（MOI = 5）プレート60-80％のコンフルエント（〜2日）に到達し、ミトコンドリアのマトリックスが標的に追加する（COXVIII）アデノウイルスは、PA-GFPを可能にします。交換媒体、細胞、イメージングの前に別の2日間に成長することができます。 </li><li>イメージングの日に、イメージングプレートに7から15 nMのTMREを追加し、少なくとも45分間平衡化します。 </li><li>インキュベーターのこの時間のターン（ステージ上のインキュベータは、ここで使用されている）の間に、顕微鏡°Cで約1時間37に平衡化することができます。 5％のCO 2をオンにします。 </LI&gt; </ol> 2。ツァイスLSM 710共焦点顕微鏡用撮像パラメータ<ol><li>顕微鏡で平衡化した後、取得タブの下に、パネルを設定し、撮像を開き、 &quot;ショーマニュアルツール&quot;をクリックし、 &quot;チャネルモード&quot;を選択し、すべての &quot;フレーム&quot;を追跡する切り替えます。 </li><li>光パスパネルでは、LSMおよびチャネルモードを選択します。標本のアイコンから作業して上向きに、690 +、およびMBS 488 &quot;リア&quot;、MBSを選択します。パネルの下部にある、明るいフィールドまたはDICチャネルの可視化を有効にするには、 &quot;T-PMT&quot;を選択します。 </li><li> GFPの色素のために490から540 nmへとTMRE色素のために580から700 nmまでの範囲を設定することにより、光パスのパラメータを調整して終了します。 </li><li>アクイジション·モードでは、512×512ピクセルのスキャンフィールド、平均4倍を使用して、（キャリブレーションの目的のために一貫性を維持したい場合があります）ズーム倍率を選択します。 </li></ol> 3。撮像パラメータの最適化<ol><li> FOCは、プランアポクロマート100倍（1.4 NA）の対物レンズを使用して、毒性からミトコンドリア保護するためにハロゲンライトではなく、蛍光灯、とあなたの細胞上に私達。 </li><li> PA-GFPは最大限に開いたピンホールを用いて細胞を発現し、明るい緑色であるセルを見つけるためにスキャンするための画面が表示されます。 </li><li> PA-GFPを有効にして、信号が検出器を飽和させないことを保証する撮像パラメータを最適化するために必要な2光子レーザーの最も低消費電力を見つける。 PA-GFPシグナルはいくつかのZシリーズ用TMRE信号と共局在することを確認してください。 TMRE信号の損失は、ミトコンドリアの脱分極または毒性を示し、これらの特性を示す細胞を解析に使用すべきではありません。 </li><li> PA-GFP発現細胞を見つけて、ズームの倍率を指定します。 </li><li> 6スライス（特定のZ-フォーカスが各位置で設定されていない限り、この範囲がすべての10個の細胞を満足する必要があります）収集するためにZシリーズの範囲を設定します。 </li><li>各セル（セル1、セル2など）のイメージング法を保存します。 </li></ol> 4。自動化を調整するDプログラムの部分と、あなたが1時間ミトコンドリア融合アッセイのためにウィル·フォロー10セルを指定します<ol><li> multitimeウィンドウの左側のパネルで、 &quot;省エネルギー&quot;パネルを選択し、ファイルが保存される場所を指定します。 </li><li> &quot;取得&quot;パネルで、スキャン設定で、セル1のために保存、取得メソッドをロードおよびzスタックのチェックボックスをオンにします。また、 &quot;ZスタックのZ-中央マーク&quot;を選択します。 </li><li> &quot;ブロック&quot;パネルで、 &quot;それぞれの場所で単一のブロック&quot;を選択してください。測定する間隔ごとに &quot;追加のブロック&quot;をクリックしてください。 </li><li> &quot;タイミング&quot;パネルで、 &quot;最初の場所だけでブロックするまでの待機間隔&quot;の &quot;間隔を待つ&quot;と入力し、 &quot;0&quot;を選択してください。ブロック2-4は、このセクションの &quot;15分&quot;になります。 </li><li>場所パネルのと、 &quot;場所との間の位置をロードするためにフォーカスを移動&quot;を選択し &quot;&#39;次のLOC&#39;をクリックするか、 &#39;は、LOCに移動する&quot;ときに設定スキャン負荷の下で &quot;編集場所のリスト&quot;を選択し、 &quot;複数&quot;を選択して、 &quot;すべてをクリア場所は &quot;ステージを電動。 </li><li> UNDER &quot;ブリーチ&quot;パネルは、 &quot;漂白剤&quot;ボックスをクリックし、 &quot;設定&quot;で設定ファイルを指定するプルダウンメニューなどの主要なソフトウェアのウィンドウで指定された。そして、 &quot;漂白&quot;ウィンドウで、適切な光活性化の方法を保存します。地域のパネルでは、この特定のセルのROIを選択し、ウィンドウの &quot;ROIのリストに現在の領域を追加する&quot;を選択してmultitimeこのROIを追加します。 &quot;ROI&quot;のプルダウンメニュー内の同じ場所を選択してください。 </li></ol>タイミングが正しく機能するためには、各セルは15分間隔を持っているため、2つの方法が保存する必要があります。ブロックのリストには、一つ目は &quot;本当の&quot;光活性化の構成を持って、残りは二光子レーザーを使用していません &quot;モック&quot;の設定があります。最初のブロックはまた遅延（BKIntv = 0）を持っていないブロックのみとなります。したがって、このメソッドは、光活性化のスキャンに続いて1ベースラインスキャンで始まります。ブロックの残りの部分は、900秒BkIntvを持っており、各時点でタイミングの一貫性を維持するためにだけ一度に2つのスキャンが0秒、があります。 <ol start="7"><li>時間の経過後にされる10個の細胞のそれぞれについて、このシーケンスを実行します。 表現するPAGFP見つけるのイメージング法をセルを保存する 場所パネルのマークステージ位置は、撮像方法を指定 主なROIパネルを選択し、特定のROIをパネルに保存し、またROIボックスにそれを読み込む光活性化ブリーチように関心領域を： ROIを消去し、1スキャンズームをリセット 次のセルを見つけるとズームを設定 次の9セルのためのプロセスを繰り返します。 </li></ol>各ステージの場所とすべてのブロックが関連付けられている適切な撮像法（スキャン設定）を持っていることを確認してください。また、残りは &quot;モック&quot;メソッドが含まれていながら、それぞれの場所で最初のブロックは、適切な光活性化法に対応していることを確認してください。最後に、 &quot;実行&quot;を選択してください。 5。 PA-GFPシグナル強度を解析する<ol><li&gt;の光活性化GFP-信号が赤TMRE信号に局在している細胞については、PAGFP画像（ここではmetamorphを使用します）のバックグラウンドを減算します。 </li><li>その後、データをExcelにエクスポートし、各時点で、Z-スタックの平均強度を計算します。無信号を持たない光学切片を破棄します。 </li><li>オリジナルの光活性化信号の割合を計算することによって、各Z-スタック内の信号の希釈を測定します。 </li></ol>各データポイントは、各時点の重複Z-スタック情報、その結果、2回スキャンされ、記録されます。 Z-スタックの値が一致していることを確認してください。そうでない場合は、これは問題（例えば、フォーカス、細胞運動）の指標である。 6。代表的な結果融合イベントが活性化および非活性化されたPA-GFPミトコンドリアの間で発生した場合には、ミトコンドリアのマトリックス内PAGFPは、非標識マトリックスと混合し、より大きな領域の上に希釈してなり、信号を減少させる強度（ 図1A）。ミトコンドリア融合の平衡に達するまで、INS1細胞では、信号強度の有意な減少は、（約1時間）、15分ごとに発生します。 図1Bのセルでは、PA-GFPとTMRE信号のほぼ完全な局在を示すことに注意してください。これらのアッセイでTMREの非常に低濃度（15 nM）をPAGFPの光活性化を標的とするための、また、細胞の健康を監視するために使用されています。脱分極ミトコンドリアが豊富な細胞がPAGFPとTMREの不完全な局在があります、これは死にかけている状態で、光毒性、または細胞のいずれかを示しているため、分析すべきではありません。 ミトコンドリアの融合のための平衡時間は、通常、ミトコンドリアの体積の約15％がアクティブになっているINS1細胞における1時間です。時には、小さな領域が点灯している場合であっても、ミトコンドリアのほとんどは、さらなる融合を検出することは困難である場合には、高度にネットワーク化されているために光活性化になる。他の細胞型は、異なる平衡化時間を示すことが、ミトコンドリアのダイナミクスを特徴づけるために短い間隔で、長い期間にわたってテストする必要があります。ミトコンドリアの融合を阻害するために、細胞をlipotoxic環境内に配置することができます。それは以前に0.4 mMのは、断片はミトコンドリアパルミチン酸、及びミトコンドリアの融合9を阻害することが実証されている。この効果は、ミトコンドリアが断片化され、 図2に見られることができますが、mtPAGFPの信号強度は、通常の条件（ 図1）の下でできるだけ変更されません。したがって、PA-GFPシグナルの希釈は、ミトコンドリアの融合ではなく、核分裂によるものである可能性があります。他の細胞タイプでは、我々はこのようなミトコンドリアの融合14が必要であるOPA1を黙らせるようにミトコンドリアの断片化を誘導する他の既知の方法を使用して、お勧めします。 <img alt="図1" src="/files/ftp_upload/3991/3991fig1.jpg" /> 図1。 </stroNG&gt;ミトコンドリア融合アッセイ中の光活性化後のPA-GFP信号の典型的な希釈。A. PA-GFPは、徐々に暗くこれらの投影画像に見られるように増加する領域の上にタンパク質の希釈につながるミトコンドリアの融合イベントのためになります15分ごとに定量化6光のセクション。PA-GFPとB. TMREは、ミトコンドリア脱分極アクティブではないことを示しています。 <a href="http://www.jove.com/files/ftp_upload/3991/3991fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図2" src="/files/ftp_upload/3991/3991fig2.jpg" /> 図2ミトコンドリア融合が0.4mMのパルミチン酸で阻害は、PA-GFPの希釈を減少させます。時間の経過とともに変化しない信号強度で短いミトコンドリアを示す6光学切片のA.投影 。TMREとPA-GFPのBの局在はミトコンドリアではないことを示していますド偏光。 <a href="http://www.jove.com/files/ftp_upload/3991/3991fig2large.jpg" target="_blank">拡大図を見るにはここをクリック</a> 。

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買収は、光活性化後、15分ごとに発生した場合、このメソッドは、一度におよそ10細胞のイメージングを可能にします。細胞の正確な数は1つがどのように迅速に培養皿内でmtPAGFP発現する細胞を見つけて、マークすることができ、どのように迅速に1つのすべてのソフトウェアのパラメータを設定することができますによって異なります。指定されたZ-スタックマージンはすべてのセルに対して適用されるので自動化がスムーズに実行できるように細胞の均一な層を使用する必要があります。 この初期の光活性化領域のサイズは、平衡時間を制御します。ミトコンドリアの融合を測定することができるようにするには、それが残りのネットワークへの信号の広がりは経時的にモニターすることができるようなネットワークの唯一の10-20％、光活性化することが重要である。ネットワークのあまりに多くが光活性化されている場合は、それは完全な融合が速すぎると発生する可能性があり、イベントがキャプチャされません。 細心の注意を払う必要があり二光子レーザーのレーザパワーだけでなく、ミトコンドリアの脱分極を引き起こす毒性を避けるためにTMRE濃度を調整します。 mtPAGFP信号はTMRE信号と共局在することを保証することは、毒性と一般的な細胞の健康8,15を評価するのに役立ちます。 epifluoerescent光照射は避けるべきである。 mtPAGFPを発現する細胞を捜している間、低消費電力488nmの励起でスキャンしながら、ピンホールを最大限に開いている必要があります。 PA-GFPは1時間かけて信号を測定することではなく、セルのいずれかが8トリッキーなことができoversaturateないように十分な光活性化するために2つの光子レーザパワーを調整します。一度自動化されたプログラムが開始されたしかし、時間は、この最適化のステップに費やさなければならない、それは複数のセルを選択するには、それを停止し、再開するのは面倒です。 品質管理のため、微分干渉コントラスト（DIC）の買収は、セルにフォーカスを監視するための画像（または透過光）は非常にすることができます親切にも、スキャン中に浸油に形成された気泡を検出するための良い方法、これは時々ステージの動きから発生します。 このmtPAGFPメソッドを使用すると、ラベルが付いていないされているものに光活性化ミトコンドリアからのミトコンドリアのマトリックスタンパク質の単方向の動きにデータを収集しますが、それは他のプロセスを研究するためにこのテクニックを利用することが考えられる。の融合は、可溶性マトリックスタンパク質15の混合とは異なる時間スケールで発生し、ABC-私のために示されているようにたとえば、特定の蛍光色素は、核融合イベント時に、その特定の動きを観察し、膜タンパク質に結合することができます。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> COXIII-アデノPA-GFP </td><td>博士リッピンコット - シュワルツ</td><td></td></tr><tr><td> TMRE </td><td>インビトロジェン</td><td> T669 </td></tr><tr><td>ツァイスLSM 710共焦点</td><td>ツァイス</td><td></td></tr></tbody></table>

a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

ミトコンドリアの融合は、時間をかけてネットワークを介してミトコンドリアのマトリックスphotoconvertedをターゲットとしたGFPの平衡を追跡することによって測定した。これまでのところ、1つだけのセルが同時に時間の長い運動解析に供することができます。我々はこれにより、データ収集プロセスをスピードアップ、同時に複数のセルを測定する手法を提案する。

並列共焦点顕微鏡を使用して複数のセルの動力学的データの取得の高速化、高解像度、MTPA-GFPベースのミトコンドリア融合アッセイ

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

a-faster-high-resolution-mtpa-gfp-based-mitochondrial-fusion-assay

Research

JoVE Journal

Biology

16.4K ビュー.  Tufts School of Medicine. ミトコンドリアの融合は、時間をかけてネットワークを介してミトコンドリアのマトリックスphotoconvertedをターゲットとしたGFPの平衡を追跡することによって測定した。これまでのところ、1つだけのセルが同時に時間の長い運動解析に供することができます。我々はこれにより、データ収集プロセスをスピードアップ、同時に複数のセルを測定する手法?...

ビデオ: A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

生細胞におけるストア作動性カルシウムエントリの蛍光ベースの測定：培養癌細胞から骨格筋線維に

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

生物学

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

酵素による細胞融合アッセイを使用して、SNARE媒介膜融合の解析

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

共焦点および多光子顕微鏡を用いて5蛍光タンパク質によってマーク造血幹細胞および前駆細胞の生体内でクローン追跡で

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

透過処理と無傷の細胞でミトコンドリア機能を評価するために高解像度呼吸計測

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

有糸分裂の多機能の細胞周期蛋白質の役割を研究する有糸分裂細胞同期と高分解能共焦点顕微鏡を組み合わせた

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

天然化合物の存在下でそのまま β 細胞のミトコンドリア機能を測定するための高解像度の呼吸

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

部位特異的DNA切断のための並列高スループット単一分子運動アッセイ

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

高分解能肺活量測定法を用いた初代ヒト網膜色素上皮細胞における生体エネルギーのリアルタイム解析

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

ミトコンドリア膜電位およびスーパーオキシドレベルの定量化

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

著者スポットライト:HeLa細胞のライブイメージングを使用したミトコンドリア膜電位およびスーパーオキシドレベルの蛍光ベースの定量化  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

HeLa細胞におけるライブイメージングを用いたミトコンドリア膜電位およびスーパーオキシドレベルの蛍光ベースの定量

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...