この作品は、NIH R01 EB014869 によって支えられました。

1. イメージング用細胞を準備
注: 任意の付着性のセルのタイプの直接力プローブ (DFP) を使用できます。ここでは、NIH 3T3 マウス線維芽細胞は、このプロトコルのモデル細胞ラインとして使用されます。
<ol><li>文化 NIH 3T3 線維芽細胞でダルベッコ変更されたワシの媒体 (DMEM) 1% と 10% ドナー牛血清ペニシリン-ストレプトマイシン 35 ミリメートルのガラスの下に目的の confluency まで皿します。37 ° C と 5% の CO2の細胞を維持します。<ol>
<li>必ずイメージングのための NIH 3T3 細胞を播種する前に 5 μ g/ml のフィブロネクチン (または類似の ECM 蛋白質) のすべての 35 mm ガラス底培養皿をコートしてください。 注: セルする必要が完全に普及して実験用皿に付着。DFP メソッドが動作するための密度の面で制約があります。</li>
	</ol>
</li>
	<li>実験直前にセルを洗浄して完全な培地で一回の洗浄が続く PBS で 2 回。</li>
	<li>ガラス底皿に完全な成長培地 3 mL を追加します。</li>
</ol>2. 顕微鏡と画像の取得
注: 倒立蛍光顕微鏡 (または同等) 側の腕は、メーカーの推奨事項に従ってインストール マイクロマニピュレーターと。顕微鏡は、37 ° c、温度および 5% の CO2レベルを維持するために環境チャンバーを装備する必要があります。マイクロマニピュレーターとマイクロインジェクター顕微鏡に接続も必要です。油浸漬 40 x/1.3 NA または 60 x/1.49 NA (または同等の目標) は実験のためにお勧めします。顕微鏡は、除振台にマウントする必要があります。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> 図 1.核変形と顕微鏡の焦点 A. 最大の核変形と核変形の緩和。最大の核の変形を計算する前に核の形態の背面のエッジが変形核の翻訳を修正する最初一致しました。ピペット先端部が抜け出る瞬間に核の形は引く前に初期の原子核の形にかぶせてありました。2 つの図形間の領域の違いを測定した Δ1。最大の核の変形は ΔA1 元の核の面積で割った値として定義されました。同様に、2 番目のパラメーター、Δ2、元の原子核の形でマイクロ ピペット剥離後最終的な定常状態の原子核の形を重ねることによって定義されるかもしれません。B. 焦点平面にセル、平面 B マイクロ ピペット チップを見つけるまで焦点の平面を移動します。画像の中に、マイクロ ピペットは右 (オレンジ色の矢印の方向) に翻訳しました。この図は、Neelamらから変更されています。15.<a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<ol><li>製造元のプロトコルごとマイクロインジェクターを入れます。</li>
	<li>対物レンズの上に液浸オイルの一滴を適用、浸漬オイル ドロッパーを使用します。</li>
	<li>料理を皿ホルダーにしっかりとクランプし、ステージ上に皿ホルダーをロードします。 注: セルは、実験を通じて CO2を 37 ° C、5% に保たれなければなりません。</li>
	<li>焦点 (平面 A図 1 b) にセルを持って目的の高さを調整します。</li>
	<li>目的のセルを検索する顕微鏡ステージに移動します。</li>
	<li>ピペット ホルダーをトップの位置に移動するマイクロマニピュレーターにジョイスティックを回転させます。0.5 μ m 径のヒント マイクロ ピペット ピペット ホルダーに読み込みます。<ol>
<li>ピペットへの細胞接着を避けるためには、あらかじめ室温で 0.3 mg/ml の 1 h の PLL g ペグ ソリューション マイクロ ピペット チップを扱います。任意の吸引圧力なし核にマイクロ ピペットを触れるし、核からピペットを翻訳密着性をテストします。密着性の不在は、核変形と翻訳の完全な欠如から識別できます。 注: パッケージを開く製造の提案に従ってください。</li>
	</ol>
</li>
	<li>細かい制御を調整することによって面 A と面 B のセルの上部の客観的焦点面を上げる (図 1 b、ステップ 2.4 を参照)。</li>
	<li>粗制御に変えればエンブリオバイオプシーに対応を設定します。ゆっくりピペットが焦点に完全にくるまをマイクロ ピペットのシルエット見て平面 B にマイクロ ピペットをもたらします。</li>
	<li>マイクロ ピペットのチップは、フォーカス、マニピュレーターを微調整に設定します。</li>
	<li>(平面 A、図 1 b) 細胞の赤道面に目標を下げるし、(図 1 b、破線ピペット) 平面の上約 15 μ m のマイクロ ピペットを下げます。</li>
	<li>圧が得られる; マイクロインジェクター on 補正圧力 (Pc)圧力を安定させるために数秒を待ちます。 注: 最適な圧セット ポイントはセルの種類および実験の特定の目的に依存します。ほとんどの場合、300 hPa は良い出発点になります。</li>
	<li>マイクロマニピュレーター パネルのクリーン設定を使用して、マイクロ ピペット チップから出てくる空気の泡を確認するためにチェックして、ピペットが詰まっていないことを確認します。</li>
	<li>先端は核の表面を軽く触れるまでマイクロ ピペットを徐々 に下げることによって、先端をセルに挿入します。 注: マイクロ ピペットを下げるとマイクロ ピペット チップのシルエットが焦点になるよう明確ななります。マイクロ ピペットに触れる核、前に上げる目的のフォーカス、核 (同じ x と y 座標、z 面の高い) とマイクロ ピペット チップを合わせます。フォーカスを核 (平面 A、図 1 b) の赤道面に戻り、マイクロ ピペット チップを徐々 に下げます。</li>
	<li>それにより大気中にマイクロ ピペット チューブの端を開くマイクロインジェクション システムから圧力供給管を切断してマイクロ ピペット チップと核膜のシールを作成します。この手順は、核の表面にPcに等しい負圧を作成します。</li>
	<li>顕微鏡画像コレクション ソフトウェアが画像を取得します。Avi ファイルの取得 (ビデオの) または nd 取得 (イメージ) をコレクションの画像ソフトで設定します。 注: 任意のイメージング ソフトウェアを取得、リアルタイムのビデオ画像や短い時間間隔でタイムラプス映像買収を設定します。</li>
	<li>対応するイメージング チャンネルに切り替え (すなわちGFP、RFP、等)、イメージングを開始。</li>
	<li>(右側、図 1 b) にセルの体からマイクロ ピペット チップを翻訳核は、マイクロ ピペットから外れるまで引っ張ります。 注: は、(ビューのフィールドの右側) に肯定的な x 方向のヒントを引き出します。引き率のプログラムやコンピューターによって制御されるジョイスティックを手動で移動することがでくことができます。引っ張って相関が見つかっていない率と核変形15を強制的に主に弾性応答を示唆しています。</li>
</ol>3. データ分析
<ol><li>利用できる基本的な画像処理ソフトウェアで画像解析を実行します。長さひずみ (Ɛ) または領域のひずみ (図 1 a) のいずれかによって核変形の程度を定量化することができます。式 1 を使用して長さひずみを定量化、L と L0はそれぞれ最大変形と初期位置で核の長さを表します。 <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(関係式 1)</li>
</ol>

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著者が明らかに何もありません。

彼らはいずれかの隔離された核 (核は細胞骨格から分離) を必要とするためピペット吸引16など、最新の方法のための挑戦は、細胞骨格に核の機械的統合を測定または核浮遊細胞 (細胞外の力、牽引力などは欠席)。力は、軸ひずみを膜17,18; に付着性のセルに適用することで、核に適用されています。ただし、この手法は、核の力が?...

癌などの疾患は、核の形状と構造1,2, '' 核3,4の軟化に伴う一般的に変化を含みます。原子力機械変形抵抗は、隔離された核5に力を適用することで一般的に特徴づけられています。
細胞核は細胞骨格に Nucleoskeleton リンカーと細胞骨格 (バイオス) 複雑な6,7,8,9分子接続されます。その結果、核は、細胞骨格と、細胞-基質間の癒着、細胞外マトリックスを機械的に統合されています。機械的に付着性細胞の中の核を探るこの機械的統合への洞察力を提供できます。マイクロ ピペット吸引10、11、および原子間力顕微鏡12,13,14生きた細胞中の核を操作するメソッドが含まれます。最近生活付着性のセル15に核に直接機械的な力を適用する直接力プローブ (DFP) について述べる。
ここでは、我々 は付着性細胞の核に直接知られているナノ機械的な力を適用する顕微鏡施設で利用可能なインジェクション システムを使用する手順を概説します。Femtotip (0.5 μ m 径マイクロ ピペット チップ) がマウントされ、チューブでのインジェクション システムに接続されています。まで核の表面に隣接して、培養皿の表面に対して 45 ° の角度で配置されている、先端が低下します。チューブは切断し、核の表面に負の吸引圧を作成し、核の表面に対してマイクロ ピペット先端をシール大気開放します。マイクロ ピペット チップの翻訳を通じて核は変形し、最終的に (力の大きさ) によって、マイクロ ピペットを切り離します。この剥離は、核および細胞、によって出される、復元 (耐熱) 力、ピペットによって適用される吸引力の等しい場合に発生します。核の変位を測定することによって分析を行うことが長さひずみ (式 1)、または領域のひずみ (図 1 a)。

<table border="0" cellpadding="0" cellspacing="0" style="width:843px;" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FluoroDish</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">SYTO 59</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11341</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Femtotips&#160;</td>
			<td style="width:71px;">Eppendorf</td>
			<td align="right">930000043</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">InjectMan NI2</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>discontinued, current equivalent model: InjectMan 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FemtoJet</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>Current model FemtoJet 4i</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plan Fluor oil immersion 40x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Apo TIRF oil immersion 60x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Donor Bovine Serum (DBS)</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">16030074</td>
			<td>NIH 3T3 serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dulbecco&#39;s Modification of Eagle&#39;s (DMEM)</td>
			<td style="width:71px;">Mediatech cellgro</td>
			<td style="width:119px;">MT10013CVRF</td>
			<td>NIH 3T3 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Mediatech</td>
			<td style="width:119px;">MT30004CIRF</td>
			<td>NIH 3T3 medium supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Immersion Oil Type LDF Non-Fluorescing</td>
			<td style="width:71px;">Nikon</td>
			<td align="right" style="width:119px;">77007</td>
			<td>Immersion oil for objective lens&#160;</td>
		</tr>
	</tbody>
</table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

核の機械的性質は、細胞で生成された機械的な力への応答を決定します。核が細胞骨格の分子に連続しているため付着細胞での力学的挙動を調べるための方法が必要です。ここでは、付着性細胞の核に直接力を適用するためのツールとして直接力プローブ (DFP) について述べる。我々 は、吸込みを伴う核の表面に細いピペットを添付します。マイクロ ピペットは、翻訳を変形させ、核を引き起こす核から変換されます。復元力が吸引力に等しい、核はデタッチし、弾性を緩和します。吸引圧が正確に知られているために、核の表面に働く力が知られています。このメソッドは、ナノの力が変形し、付着細胞で核を翻訳するのに十分なされ、力に抵抗する核を可能にする骨格の要素を明らかにしました。DFP を使用して、細胞内の核の力学特性に細胞や原子力部品の貢献を分析できます。

図 2 aは、NIH 3T3 マウス線維芽細胞核の強制を示しています。マイクロ ピペット チップの右側に翻訳核変形、最終的にマイクロ ピペット チップから切り離します。吸引力 (図 2 b) とともに増加する核の長さのひずみが見られます。核 (端を引っ張ってマイクロ ピペット) の前面の端を形成する核の突起とトレーリング エッ?...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

核と細胞骨格との間の機械的統合を測定するための直接力プローブ

このプロトコルでは生きている細胞の核に直接制御力を適用するマイクロ ピペット法について述べる。このアッセイでは、生活、付着性の細胞で核の力学特性の尋問をことができます。

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

a-direct-force-probe-for-measuring-mechanical-integration-between

Research

JoVE Journal

Biochemistry

15.0K ビュー.  University of Florida. このプロトコルでは生きている細胞の核に直接制御力を適用するマイクロ ピペット法について述べる。このアッセイでは、生活、付着性の細胞で核の力学特性の尋問をことができます。

ビデオ: A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

HCN1とTRIP8bの間のタンパク質 - タンパク質相互作用の小分子阻害剤を同定するための方法

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

生化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

原子間力顕微鏡を用いた単一タンパク質分子の力分光法

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

標的タンパク質の翻訳後修飾の内因性プロファイルを識別するために包括的な免疫沈降濃縮システムを活用

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

植物の鉄含有量を測定するための比色定量法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

リーシュ マニア、ひと細胞およびマウス精巣におけるプロファイリングをポリソーム

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

力の単一分子分光法のための蛋白質の共有結合固定

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

原子間力顕微鏡による細胞外小胞のイメージング

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

フォトニックチップを用いたハイスループット全内部反射蛍光と直接確率光学再構成顕微鏡

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

低分子とdNTPase SAMHD1の相互作用を調べるためのハイスループット酵素共役活性アッセイ

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...