I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

苗の調製<ol><li>種子を滅菌します。 <ol><li> 1μlの10％トリトンX-100〜500μlの滅菌水：500μLのNaClO（5.0％有効塩素）を加えることによって滅菌溶液を調製します。 </li><li>場所の約 10 形質転換A. 1.5 mlチューブに、GFP-PATROL1 8を運ぶ シロイヌナズナの種子。 </li><li> 1ミリリットルの70％エタノール溶液を追加し、5回反転させてよく混ぜます。 1分間のままにしておきます。 </li><li>種子は、チューブの底に沈む確認します。クリーンクリーンベンチでは、静かにマイクロピペットを用いて70％のエタノールを除去し、1ミリリットルに滅菌溶液を添加。 5回反転させることによって十分に混合し、5分間放置します。 </li><li>種子を洗ってください。それでもクリーンベンチに無菌状態の下で働いて、静かにマイクロピペットを用いて溶液を除去し、1ミリリットルに滅菌水を追加します。この5回繰り返します。滅菌種子は、2日間4℃で滅菌水に格納されてもよいです。 </li></ol></li><li>だから、0.5％ジェランガム固化1/2ムラシゲ・スクーグ培地プレート（pHは5.8）9の滅菌種子ワットサージカルテープの二つの層を用いてプレート上にふたをテープで固定します。 </li><li>冷蔵室、O / Nで、4℃で暗所でプレートをインキュベートします。 </li><li> 100マイクロモルメートル-2秒 -1白いライトを使用して12時間/ 12時間の明暗サイクルで23.5℃に設定し、成長チャンバ内にプレートを移し、7日間インキュベートします。長い1mm程度の子葉と苗木は、その後収穫することができます。 </li></ol> 2.スカイドロップ子葉片の取り付け注：VAEM観察用の試料の調製における重要な要因は、試料とカバーガラスの間に気泡の混入を回避されます。気泡が大きく屈折率の差を生じさせることによってVAEMの画質を低下させます。我々は、「空のドロップ」と呼ばれている単純な実装方法は、気泡を回避するために使用することができA.間のシロイヌナズナ子葉とカバーガラス。これは、観測の直前に行​​われる必要があります。 <ol><li> 76：スライドガラス（サイズの中央に基礎緩衝液30μl[5 mMの2-（N-モルホリノ） -エタン酸トリス（MES-トリス）pH6.5で、50mMの塩化カリウム、100μMのCaCl 2]を配置します×26ミリメートル、厚さ：1.0〜1.2ミリメートル）。 </li><li>解剖ハサミを使用して、7日齢の苗から子葉を削除します。観察側が基底バッファ降下（ 図1、ステップ1）上に向けて子葉をフロート。 </li><li>カバーガラスの中心に基礎緩衝液30μlを置き（サイズ：18×18ミリメートル、厚さ：0.12〜0.17ミリメートル）（ 図1、ステップ2）。逆さまに静かにカバーガラスを回します。表面張力が脱落バッファ低下を防ぐことができます。一方の縁部にピンセットでカバーガラスを保持します。バッファ降下は約子葉の上にあるように、スライドガラス上に反対側の端を置きます。 </li><li>それは子葉試料（ 図1、ステップ3）の真上になるように、スライド上の静止カバーガラスのエッジ、まだピンセットで反対側の縁部を保持して、カバーガラスの下にバッファのドロップの位置を調整します。カバーガラスを手放します。気泡のない準備で得られた試​​料のドロップをマウントします。 </li><li>糸くずの出ない組織を使用して余分なバッファを拭き取ってください。すぐに準備を観察し（ステップ3を参照）。 注：サンプルをシールする必要はありません。 </li></ol> 3. VAEM観察ムービー取得倒立顕微鏡はTIRFユニットと1.49の開口数とTIRF対物レンズを搭載しています。注：以下のように本研究で使用するTIRF顕微鏡システム9について説明します 。レーザー入射角のコンピュータ制御は、制御ボックスが使用されます。緑色蛍光タンパク質（GFP）は、488nmの光励起半導体レーザで励起され、そしてt彼は、蛍光が葉緑体の自己蛍光を防ぐために、510から550 nmのバンドパスフィルタを介して検出されます。ファイバ出力電力の測定された最大値は13.0から13.5ミリワットです。検出のために、電子増倍電荷結合素子（EM-CCD）カメラヘッドシステムとCマウントカメラ倍率変更手段が使用されます。 <ol><li>製造元の指示に従ってを中心と集光レーザーを調整します。 注：この手順は、正確なVAEM観測のために重要です。それは強くレーザー経路の調整手順の定期的なチェックは、あなたの顕微鏡に応じて、実行することをお勧めします。 <ol><li>顕微鏡室の天井に対物レンズのない光路で照射中心位置を決定します。中央の位置をマークするには、天井に色付きの円シールを置く（ 図2、ステップ1、2）。 </li><li>対物レンズ（ 図2、ステップ3、4）で天井を照らします。私を移動します中心位置（ 図2、ステップ5）に地域をlluminated。レーザー（ 図2、ステップ6）フォーカス。微調整の中心に集光されたレーザ（ 図2、ステップ7）の位置。 </li></ol></li><li>顕微鏡のステージ上の試料をセットし、明視野照明を使用して観察するためのセルを選択します。 </li><li>蛍光タンパク質は、細胞で観察することができることを確認し、落射蛍光照明（ 図3A）を用いて細胞表面でのz -軸位置を設定します。 </li><li> VAEM観測を行います。 <ol><li>コントローラボックスで徐々にレーザービームの入射角を傾け。同時に、慎重にライブ映像を監視します。最初は、イメージが（ 図3B）ぼかされます。 </li><li>レーザー角が増加するにつれて、VAEM画像は最終的には鮮明な画像（ 図3CおよびD）を製造する 、より少ない不鮮明になるであろう。この時点で、increaを停止レーザー角度を歌います。蛍光信号が失われた場合、より浅い角度に減少します。 </li><li>微調整のレーザー入射角は、より良好な画像を得ることができます。 Z -軸の位置の微調整は、画像を改善することができます。必要に応じて、光学パラメータ（レーザー出力、波長フィルタセット）を調整し、画像センサパラメータ[画像サイズ、露出時間、ゲイン、デジタイザと電子増倍（EM）ゲイン]。 注：以下のように上述のTIRF顕微鏡装置の場合には、代表的なパラメータです。ちょうどカメラの前でレンズの倍率：2倍。レーザー出力：1.0 mWの。画像サイズ：512×512ピクセル。露光時間：100ミリ秒。ゲイン：5×を。デジタイザ：11 MHzの。 EMゲイン：100。 </li></ol></li><li>商用顕微鏡ソフトウェアを使用して、複数ページのTIFF画像ファイルとして映画を取得します。ここでは、映画は、気孔細胞の表面上の点滅GFP標識されたドットです。 注：代表画像取得条件はFOLの通りであります安値。取得モード：ストリームは、RAMに。フレームの数：600;マルチページTIFFファイルサイズ：〜302メガバイト</li></ol>フィジーソフトウェアを使用したGFP標識ドット滞留時間の定量化のための4カイモグラフ分析<ol><li>著者の命令（http://fiji.sc/Fiji）を使用して、フィジー（ &#39;フィジーはちょうどImageJのである&#39;）ソフトウェア10をインストールします 。 </li><li>フィジーを実行し、フィジーメニュー「 ファイルを開く」を使用して取得したマルチページTIFFファイルを開きます。 </li><li>フィジーツールバーメニュー「 直線選択ツール」を使用して 、目的の部位に線を配置します。必要に応じて、「 セグメント化されたライン選択ツール 」または「 フリーハンドライン選択ツール」を使用します。ここに提示した結果では、100ピクセル（8.0ミクロンに相当する）の直線を子会社細胞（ 図4A）の上に置きました。 </li><li>フィジーメニューの 「画像・スタックダイナミックResliceを使用してカイモグラフイメージを作ります</eM&gt; &quot;（http://fiji.sc/Dynamic_Reslice）（ 図4B）。カイモグラフイメージで必要に応じて、「垂直フリップ 」とチェックボックスウィンドウで「90度回転」も（ここで提示された結果では使用されません）があり、x軸とy軸線位置に対応する（100画素を示しそれぞれ8.0μm）とし、観測時間（600ピクセルが60秒に相当）、。カイモグラフイメージが満足できない場合は、複数ページの画像上の線の位置と種類は（ストレート、セグメント化され、フリーハンド）のよう。変更することができますラインの変更、カイモグラフイメージが動的に変化します。フィジーメニュー「ファイルとして保存-TIFF」を使用して、TIFFファイルとしてカイモグラフ画像を保存します。 </li><li>時間軸の向きにブロブの長さを測定します。 <ol><li>ノイズリダクションを実行するには、フィジーメニュー「 プロセス・フィルタ-ぼかし（ガウス）」を使用してカイモグラフ画像にガウスフィルタを適用します。 「 シグマ（ラッドIUS）」パラメータが調整可能（1ピクセル半径が提示された結果のために使用される）です。 </li><li>セグメントフィジーメニューを使用して、しきい値による信号領域、「イメージ・調整・しきい値を 」。 注：選択可能ないくつかの閾値化アルゴリズムがあります。提示された結果では、「円」のアルゴリズムは、（ 図4C）を選択しました。 </li><li>メニューを使用してブロブの-軸の長さを時間（y）を測定するための準備」 を分析・セットの測定を 」。 「 セット測定」ウィンドウの「 境界の矩形」を確認してください。理想的には、領域セットは、ノイズを排除するために、可能な限り小さくすべきです。ここでは、最小面積は50ピクセルに設定しました。時間（y）を測定し、メニュー「 分析・解析粒子」を使用してブロブの長さを-axis。結果の値を取得し、測定領域の信頼性を証明するために、「 結果の表示」をチェックし、「Managerへの追加&lt;/ em&gt;の &quot;ボックス。 </li><li> 「 高さ 」の欄の値は、（y）が測定されたブロブ（ 図4D）のための長さを-axis時間です。結果テーブルメニュー「ファイルとして保存」を使用して値を保存し、計算し、統計パッケージ、および/ ​​または表計算ソフト（ 図4E）を使用してブロブ画像期間のデータセットを処理します。 </li></ol></li></ol>

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著者は、開示することは何もありません。

このビデオの記事では、プロトコルは、 シロイヌナズナの気孔複合体上のGFP-PATROL1ドットの動的挙動を監視し、測定するために与えられています。ここに示されているように、VAEM観測は、植物の細胞表面のライブイメージングのための強力なツールです。高感度EM-CCDはVAEM光学系では比較的弱い励起レーザの使用を可能にするため、GFP-PATROL1の監視のためにここに使用した実験条件下で...

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX-73</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TIRF unit</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">IX3-RFAEVAW</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TIRF objective lens&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">UAPON 100 &#215; OTIRF&#160;</td>
			<td>NA = 1.49</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser angle control box</td>
			<td style="width:71px;">Chuo Seiki</td>
			<td style="width:119px;">QT-AK</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Optically pumped semiconductor laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">SapphireTM LP USB 488-20 CDRH Laser</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">510&#8211;550 nm band-pass filter</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-FBNA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EM CCD camera</td>
			<td style="width:71px;">Hamamatsu Photonics</td>
			<td style="width:119px;">ImagEM C9100-13</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C-mount camera magnification change unit&#160;</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">U-TVCAC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaMorph software</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;">MetaMorph version 7.7.11.0</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">TIRF microscopy manual</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">AX7385</td>
			<td style="width:167px;">Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Immersion oil</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;">Immersion Oil Typr-F</td>
			<td>ne = 1.518 (23 degrees)</td>
		</tr>
	</tbody>
</table>

real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

このビデオの記事では、Aの GFP-PATROL1のVAEM観測のためのプロトコルシロイヌナズナ子葉気孔複雑な細胞が提供されています。空ドロップ実装はA.のVAEM調製物中の気泡の発生を減らすことができ、簡単な製造方法でありますシロイヌナズナ子葉（ 図1）。エントリーレーザーおよび/またはVAEMのための検体のz位置決めOvertiltingは不明画像を提供します。そ?...

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

このプロトコルの目的は、気孔複合体の細胞皮質内に、GFPタグPATROL1、膜輸送タンパク質のドットを点滅示す可変角度落射蛍光顕微鏡で植物細胞表面上の蛍光標識タンパク質のダイナミクスを監視する方法を示すことですシロイヌナズナ 。

可変角度落射蛍光顕微鏡を用いて植物細胞表面ダイナミクスのリアルタイムイメージング

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Research

JoVE Journal

Biology

8.9K ビュー.  The University of Tokyo. このプロトコルの目的は、気孔複合体の細胞皮質内に、GFPタグPATROL1、膜輸送タンパク質のドットを点滅示す可変角度落射蛍光顕微鏡で植物細胞表面上の蛍光標識タンパク質のダイナミクスを監視する方法を示すことですシロイヌナズナ 。 

ビデオ: Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

蛍光を経由してF -アクチンダイナミクスの生細胞イメージングでは、顕微鏡のスペックル（FSM）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

生物学

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

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

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

アセンブリ、読み込み、および分析用超遠心器のサンプルセルの整列

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

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

低親和性細胞外受容体 - リガンド相互作用の検出のためのスケーラブルな親和性ベースの細胞外相互作用のスクリーニング（AVEXIS）

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

タンデム高圧凍結および透過型電子顕微鏡のための植物組織の急速凍結置換

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

膜タンパク質のリアルタイム測定：受容体相互作用は、表面プラズモン共鳴を使用した（SPR）

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

における代謝プロファイルのリアルタイム分析<em&gt;エクスビボ</em&gt;マウス腸陰窩オルガノイド培養

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

蛍光顕微鏡でストロミュール頻度を可視化

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

ゲルの表面上に成長する植物の根の光シート蛍光顕微鏡

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

フラッシュとフリーズ：電子顕微鏡でメンブレンダイナミクスをキャプチャするために新しい技術

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

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 ...