このプロジェクトは、当社でサポートされていた私は、LO1417 (文部省、青少年、チェコ共和国のスポーツ)。

<ol>
	<li>Barbosa, I. C. R., Shikata, H., Zourelidou, M., Heilmann, M., Heilmann, I., Schwechheimer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+composition+and+a+polybasic+motif+determine+D6+PROTEIN+KINASE+polar+association+with+the+plasma+membrane+and+tropic+responses.">Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.</a> Development. 143 (24), 4687-4700 (2016).</li><li>Kato, M., Aoyama, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ca2+-binding+protein+PCaP2+located+on+the+plasma+membrane+is+involved+in+root+hair+development+as+a+possible+signal+transducer.">The Ca2+-binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer.</a> Plant J. 74 (4), 690-700 (2013).</li><li>Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence Microscopy: From Principles to Biological Applications. , (2013).</li><li>Janecki, A. J., Janecki, M., Akhter, S., Donowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+plasma+membrane+expression+of+a+fusion+protein+of+Na/H+exchanger+NHE3+and+green+fluorescence+protein+(GFP)+in+living+PS120+fibroblasts.">Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.</a> J Histochem Cytochem. 48 (11), 1479-1492 (2000).</li><li>Peitzsch, R. M., McLaughlin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+acylated+peptides+and+fatty+acids+to+phospholipid+vesicles:+pertinence+to+myristoylated+proteins.">Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.</a> Biochemistry. 32 (39), 10436-10443 (1993).</li><li>Vosolsob&#283;, S., Petr&#225;&#353;ek, J., Schwarzerov&#225;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+plasticity+of+plasma+membrane+interaction+in+DREPP+family+proteins.">Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.</a> Biochim Biophys Acta - Biomembr. 1859 (5), 686-697 (2017).</li><li>Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N. D., Satiat-Jeunemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM-dyes+as+experimental+probes+for+dissecting+vesicle+trafficking+in+living+plant+cells.">FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.</a> J Microsc. 214 (2), 159-173 (2004).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>R Development Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A language and environment for statistical computing. , (2008).</li><li>Logan, D. C., Domergue, O., Teyssendier de la Serve, B., Rossignol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+plasma+membrane+polypeptides+differentially+regulated+during+plant+development.">A new family of plasma membrane polypeptides differentially regulated during plant development.</a> Biochem Mol Biol Int. 43 (5), 1051-1062 (1997).</li><li>Ide, Y., Nagasaki, N., Tomioka, R., Suito, M., Kamiya, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+properties+of+a+novel,+hydrophilic+cation-binding+protein+associated+with+the+plasma+membrane.">Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.</a> J Exp Bot. 58 (5), 1173-1183 (2007).</li><li>Nagasaki-Takeuchi, N., Miyano, M., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasma+membrane-associated+protein+of+Arabidopsis+thaliana+AtPCaP1+binds+copper+ions+and+changes+its+higher+order+structure.">A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure.</a> J Biochem. 144 (4), 487-497 (2008).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDP25,+A+Novel+Calcium+Regulatory+Protein,+Mediates+Hypocotyl+Cell+Elongation+by+Destabilizing+Cortical+Microtubules+in+Arabidopsis.">MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules in Arabidopsis.</a> Plant Cell. 23 (12), 4411-4427 (2011).</li><li>Qin, T., Liu, X., Li, J., Sun, J., Song, L., Mao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+microtubule-destabilizing+protein+25+functions+in+pollen+tube+growth+by+severing+actin+filaments.">Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.</a> Plant Cell. 26 (1), 325-339 (2014).</li><li>Nagata, T., Nemoto, Y., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+BY-2+Cell+Line+as+the+&amp;#34;HeLa&amp;#34;+Cell+in+the+Cell+Biology+of+Higher+Plants.">Tobacco BY-2 Cell Line as the &amp;#34;HeLa&amp;#34; Cell in the Cell Biology of Higher Plants.</a> Int Rev Cytol. 132, 1-30 (1992).</li><li>Hellens, R. P., Anne Edwards, E., Leyland, N. R., Bean, S., Mullineaux, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGreen:+A+versatile+and+flexible+binary+Ti+vector+for+Agrobacterium-mediated+plant+transformation.">pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.</a> Plant Mol Biol. 42 (6), 819-832 (2000).</li><li>Vermeer, J. E. M., Thole, J. M., Goedhart, J., Nielsen, E., Munnik, T., Gadella, T. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+phosphatidylinositol+4-phosphate+dynamics+in+living+plant+cells.">Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.</a> Plant J. 57 (2), 356-372 (2009).</li><li>La&#328;kov&#225;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Dynamics+of+Plant+Plasma+Membrane+Proteins+with+Fluorescence+Recovery+and+Raster+Image+Correlation+Spectroscopy.">Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.</a> Microsc Microanal. 22 (2), 290-299 (2016).</li></ol>

著者が明らかに何もありません。

ここで説明する方法は、膜が末梢関連タンパク質に蛍光強度5の測定に基づく他のアプローチに比べて分割のより正確な推定を生成します。このメソッドの主要な改善は、光の回折と原形質膜と細胞質の信号の重ね合わせ、考慮します。ただし、これらのメソッドの結果は、平均細胞質膜位置に蛍光強度の比較に基づく簡単な法の結果との相関 (6図以前) のような信号、この手法の主な利点特に、膜信号が細胞質の信号よりも低い結果の関連性の推定を可能にする (信号分解による原因不明) の残留可変性の決定であります。タンパク質の分配の計算は 1 ポイントだけに基づいていないために、この方法は信号ノイズをより堅牢なも。
分析には、共焦点画像のみ 1 つ 2 つのチャネルが必要です。高速画像取得が重要な要件 ( FRAP アプローチ18長いタイム ・ ウィンドウのタンパク質拡散現象の測定に基づくと対照をなして説明メソッドはよりダイナミックな生体内でのアプローチに適用例えば、信号伝達の探求、抑制の試金)。メソッドは、すぐに大量の統計的評価の十分なデータを得ることに適しています。
メソッドは、単一の膜のみに限定されます。現在、隣接する細胞の 2 つの密接に隣接する膜からの信号の解析はサポートされていません。この場合、信号の継ぎ手は、成果物のリスクが高いとより厳しいです。
ただし、植物の懸濁液の細胞15、分析は元々、それは他のセルタイプの分析に適用することがあります。花粉管と根毛の植物生物学におけるこのメソッドの潜在的に非常によいターゲットを表します。植物表皮細胞の外部の膜は、細胞壁の FM 4-64 の分類されない蛍光が干渉は発生しません、以前の検証後分析できます。滑らかな膜で蛍光が干渉、酵母、真菌細胞は、動物細胞と同様の無料原生細胞で説明されているメソッドを使用して分析されるようただし、動物細胞や上皮細胞の刷子縁に線維芽細胞のリーディング エッジのタンパク質分布の分析は管理不可能します。柔軟な解析設定により FM 4-64 の染色が置き換えられます他の膜の可視化アプローチよう蛍光付けられた蛋白質。注意して固定セルのメソッドを使用できます。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.
One of the affiliations was updated from:
Department of Experimental Plant Biology, University of Science, Charles University
to:
Department of Experimental Plant Biology, Faculty of Science, Charles University

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.

Erratum: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

末梢に関連付けられている膜蛋白質は、細胞シグナル伝達経路の主要なコンポーネントです。過渡膜協会そして解離細胞膜と細胞質の間のシグナル伝達のために重要である彼らの基本的な役割の一つであります。脂質アンカー (N-ミリストイル化、アシル化、S またはプレニル化) または脂質結合ドメイン ( 、ホスファチジン酸ホスファチジルイノシトール隣酸塩との相互作用によって、細胞膜の末梢関連プラズマ膜タンパク質を取り付けることがなど)。
これらの蛋白質の特性は、プラズマ膜結合検査生体内で、例えば、蛍光タグ付きタンパク質が主要アミノ酸のサイト指示された突然変異誘発によって変更されたとき、または影響を与える様々 な阻害剤と扱われる脂質シグナリング。周辺膜蛋白質の分布主評価されている質的に、特に場合は、蛋白質の再分布が明らかなとき。提案手法は、蛋白質の再配布、部分的にしかと定量的評価が必要な状況に最適です。膜協会は共焦点から推定したときの頻繁に使用されるアプローチ レーザー走査型顕微鏡画像のない細胞膜と細胞質の1、2、蛍光強度の比が簡単なと正確になります。細胞膜での蛍光強度は、特定の蛍光顕微鏡法および光学素子の使用3の光回折特性による原形質膜と細胞質の信号の重ね合わせを反映しています。その結果、細胞内の信号は、膜領域にも含まれています。このため、膜信号選択4マスクとして FM 4-64 の染色パターンを使用できません。さらに、FM 4-64 常に体系的に最大の汚損によって定義された位置で膜信号の測定は実際膜信号の重ね合わせによる末梢関連プラズマ膜タンパク質を過大評価、膜と細胞質の化合物。末梢関連タンパク質の蛍光タグの観測信号の最大値も共同膜マーカー (すなわちFM 4-64 の styryl の染料)、最大でローカライズされていないが、細胞質にシフトします。別の制限は、FM 4-64 放出ピークは光回折3の波長依存性により GFP など緑の蛍光蛋白質のための放出のピークと比較してより広いという事実に基づく。
ここで説明したメソッド、タグ付きタンパク質の信号はそれぞれの細胞膜と細胞質シグナル仮説的分布を記述する 2 つの経験的な関数によって装着です。この信号の分解は蛍光付けられた蛋白質を表現する共焦点セクションを定期的に、2 チャンネルのソース画像で膜に垂直に細胞表面に適用される線形蛍光プロファイルに適用します。FM 4-64 染料ラベルの付いたセルです。
継ぎ手に使用される最初の関数では、セルの端に細胞質シグナルの回折について説明します。それは興味の細胞膜の末梢に関連付けられている蛋白質として同じ発色団によって付く細胞質のタンパク質マーカーを発現する細胞で測定した蛍光以前に取得したプロファイルから取得されます。プラズマ膜信号の回折を説明する 2 番目の関数は、FM 4-64 の蛍光から派生されます。この信号は、点光源の光の回折の近似モデリングに使用されているガウス関数で近似はまず。第二に、発光 FM 4-64 の有効なこのモデルは数学的に膜で興味の末梢に関連付けられている蛋白質のタグ付けに使用される色素の発光波長に関連するフォームに変換されます。両方の機能は、それぞれ最大の強度、FM 4-64 と細胞質蛋白質信号の最高値の 10% から平均が正規化されます。この信号の分解 (非線型最小正方形管継手法)、膜の比と検査された蛋白質の細胞質画分を推定できる簡単かつ正確に。膜表面濃度と細胞質の体積濃度を比較するため、分割係数の計算の実際の物理的な寸法はマイクロメータの範囲内。それは細胞膜から膜の周辺のように、同じ量のタンパク質がローカライズされて細胞質までの距離を定義します。この値は、パーティション分割を相当係数K2導入以前5。メソッドは非常に簡単で、ルーチンの共焦点レーザー走査型顕微鏡を使用して集録した唯一の共焦点セクションを必要として計算上要求されていません。解析コアは、ポータブルの R パッケージに実装されているし、追加の ImageJ マクロは直感的なダイアログから分析を実行するグラフィカル ユーザー インターフェイスを提供するために書かれました。ソフトウェアおよび方法の詳細な説明 (以前公開された6) http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/ で見つけることができます。
メソッドは単離細胞、プロトプ ラスト、組織に適した個々 の細胞の細胞膜には明確に区別できる、蛍光タグの構造を表現する末梢関連タンパク質を調べた。発色団 FM 4-64 の染色を使用する必要がありますと互換性があります。FM 4-64 発光赤;したがって、検査されたタンパク質は、青、緑、または黄色の放射 (例えばGFP、CFP、YFP) と蛍光タンパク質によるタグできます。蛋白質の分布の少ない人工より再現可能な観察を可能にするためは、生物材料の安定した変換をお勧めします。検査されたタンパク質が比較的均質な細胞質の分布を持っていることが必要です。小胞体または別の細胞内膜コンパートメントの蛋白質のローカリゼーションは、人工の結果を生成できます。
また、比較のため細胞質マーカーを表現する同じ生物学的材料を使用する必要があります。細胞は、廃止膜結合容量と無料 chomophore (周辺蛋白質、例えば、無料の GFP のタグ付けに使用されると同じ) することによって興味の蛋白質をタグに変換できます。膜結合容量廃止されることが、たとえば、膜結合ドメインのトリミングによってまたは重要なアミノ酸ボッスクカ (例えば N-ミリストイル化、アシル化、S またはプレニル化等のためのサイト) のサイト指示された突然変異誘発によって。
共焦点顕微鏡の細胞は色素 FM 4-64 のような膜マーカーによって表示しなければなりません。場合 FM 4-64 の染色 (蛍光が干渉している、貧しい染料の浸透などにより) 研究材料に適しては、原形質膜ラベル付けできますが、たとえば、適切な chomophore (タグ付き積分膜タンパク質によるmCherry、RFP、等)。マーカーでは、細胞内膜コンパートメント (膜) のごくわずかのローカリゼーションが不可欠です。
固定サンプルや抗体を操作する場合は、修正可能なアナログ FM 4-64FX または適切なターゲットに対する抗体によって分類する膜が使用できます。この場合、固定法は細胞質から細胞膜タンパク質の選択的損失につながることができますので、非常に慎重に結果を評価するために不可欠です。

<table><tbody><tr><td>FM 4-64</td><td>ThermoFisher Scientific</td><td>T13320</td><td>Plasma membrane dye</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D4540 Sigma</td><td>Dye solvent</td></tr><tr><td>Ordinary equipment (microscopic slides, pipettes, tips, tubes)</td><td></td><td></td><td>Equipment for cell labelling and microscopy</td></tr><tr><td>Confocal laser scanning microscope</td><td></td><td></td><td></td></tr><tr><td>Ordinary computer</td><td></td><td></td><td></td></tr></tbody></table>

determination of plasma membrane partitioning for peripherally-associated proteins

このメソッドは、あらゆる蛍光タグ末梢に関連付けられている蛋白質の膜間での蛍光強度のプロファイルを使用してパーティション分割膜による高速アプローチを提供します。測定された蛍光プロファイルは、細胞周囲に対して垂直線に沿って膜と細胞質の蛍光分布モデルによる取り付けられています。このモデルは、FM 4-64-ラベルの付いた原形質膜と細胞質の蛍光タグのマーカーを発現する参照細胞の蛍光強度の値から構成されます。さまざまなセルタイプおよび生物に応用することができます。ただし、非近隣の細胞の細胞膜だけを評価できます。この顕微鏡を用いた高速メソッドは実験、細胞膜関連マーカーの微妙な動的変更が予想されるとを定量化する必要があります、例えば、阻害剤治療、蛋白質の突然変異体バージョンの分析に適して信号伝達の観察。メソッドは、ユーザーフレンドリーなインターフェイスとして機能する ImageJ マクロと結合されたマルチプラット フォーム R パッケージに実装されます。

N-ミリストイルおよびホスファチジルイノシトール リン酸11,と静電相互作用を介して細胞膜に関連付けられている植物固有の末梢に関連付けられている膜タンパク質である DREPP10 12です。 DREPP 植物細胞内カルシウム シグナル伝達機械のコンポーネントとして記述され、骨格13,14ともやり取りします。提示実験で野生型タバコ DREPP2 における非突然変異体バージョン (Gly2 アラで代用) GFP 付けられた6とタバコによる 2 懸濁液細胞15 CaMV 35S プロモーター16の下で表されます。3 日齢 (希釈後 3 日) で測定したこれらの蛋白質の膜分配、FM 4-64-ラベル (8 μ M) のセルの文化6 (図 1 a) と (図 1 b) ホスホイノシチド 3-キナーゼの添加なし阻害剤ワートマンニン (10 μ M)17、上記プロトコルに従って。蛍光分布モデルの構築 (図 1) の細胞質マーカーとして使用されたトリミング バージョン DREPP2(Δ1-23) 結合ドメイン6膜を欠けています。
データが平方根変換を計算 (最小の負の値に対応する正の値が肯定的なデータのみを取得するすべてのデータに追加された) データを R9で双方向の分散分析により調べたと。突然変異と阻害剤の治療の効果が有意 (p < 10-16 x 2.2 * * *)。すべてのグループを比較したテューキー HSD テスト;すべてのグループは、DREPP2 ワートマンニンと非投与 DREPP2(G2A) (図 1) を除いて有意。
これらの結果は、タバコ DREPP2 タンパク質の細胞膜協会が協調性を持って機能している N-ミリストイルと静電相互作用の結果であることを明確に示します。N-ミリストイル サイト突然変異とワートマンニン治療の相互効果だけプラズマ膜から DREPP2 蛋白質の完全解離の原因。これらの結果は、以前に公開されたデータ6準拠しています。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57837/57837fig1.jpg"> 図 1: N-ミリストイル化サイト ・末梢関連プラズマ膜タンパク質カルシウム植物細胞内シグナル伝達に関与する DREPP の膜分配ワートマンニン治療の突然変異の効果。(A) 野生型フォーム DREPP2 GFP および突然変異体を発現するタバコ細胞の内側共焦点セクションを形成する DREPP2 (Gly2Ala)-GFP (グリーン チャンネル)。セル fm 4-64 色素 (マゼンタ チャンネル) を標識した、スケール バー 10 μ m。 同じセル行 (B) は 10 μ m を施したワートマンニン (WM)。(C) 細胞細胞質マーカー DREPP2(Δ1-23) を表現します。(D) すべてのサンプルの推定膜分割します。両方の効果の意義が二元によってテストされた (p < 10-16 x 2.2 * * * 両方の場合元のデータは平方根変換された)。文字は、テューキー HSD テストにより決定される、互いと大幅に異なるではないグループを示します。箱ひげ図のひげは、95% 信頼区間を示します。<a href="/files/ftp_upload/57837/57837fig1large.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>。

Watch this Scientific Journal Video about Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins at JoVE.com

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

ここでは、末梢関連タンパク質の蛍光タグ付きのプラズマ膜協会のレベルの定量的分析を実行するためのプロトコルを提案する.メソッドは、膜の計算の分解、細胞膜蛍光マーカーの付いた信号の細胞質成分に基づいています。

細胞膜の末梢関連タンパク質の分割の決定

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

determination-plasma-membrane-partitioning-for-peripherally

Nanyang Technological University

Research

JoVE Journal

Biology

8.3K ビュー.  Faculty of Science, Charles University. ここでは、末梢関連タンパク質の蛍光タグ付きのプラズマ膜協会のレベルの定量的分析を実行するためのプロトコルを提案する.メソッドは、膜の計算の分解、細胞膜蛍光マーカーの付いた信号の細胞質成分に基づいています。

ビデオ: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

透過型電子顕微鏡により膜相互作用タンパク質を視覚化し、分析するための方法

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

生化学

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

脳膜分画：<em&gt; Ex Vivo</em&gt; Subsynaptic Protein Localizationを評価するアプローチ

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

窒素キャビテーションと差動遠心分離培養細胞の周辺膜蛋白質の分布を監視することができます。

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal transduction; from sensing environmental signals to biological response; from metabolic regulation to developmental control. Accurate structural information of protein-protein interactions is crucial for understanding the molecular mechanisms of membrane protein complexes and for the design of highly specific molecules to modulate these proteins. Many in vivo and in vitro approaches have been developed for the detection and analysis of protein-protein interactions. Among them the structural biology approach is unique in that it can provide direct structural information of protein-protein interactions at the atomic level. However, current membrane protein structural biology is still largely limited to detergent-based methods. The major drawback of detergent-based methods is that they often dissociate or denature membrane protein complexes once their native lipid bilayer environment is removed by detergent molecules. We have been developing a&#160;native cell membrane nanoparticle system for membrane protein structural biology. Here, we demonstrate the use of this system in the analysis of protein-protein interactions on the cell membrane with a case study of the oligomeric state of AcrB.

Presented here is a protocol for the determination of oligomeric state of membrane proteins that utilizes a native cell membrane nanoparticle system in conjunction with electron microscopy.

膜蛋白質相互作用解析用のネイティブ細胞膜ナノ粒子システム

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

This article aims to present a protocol on how to build a spot variation Fluorescence Correlation Spectroscopy (svFCS) microscope to measure molecular diffusion at the plasma membrane of living cells.

生細胞原形質膜における分子拡散解析のためのスポット変異蛍光相関分光法

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

リバーシブルビオチン化によって原形質膜タンパク質エンドサイトーシス率を測定

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

生物学

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

PIP-on-a-chip：タンパク質 - ホスホイノシチド相互作用のラベルフリー研究

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

生物工学

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

脂質ラフトの決定は、蛍光相関分光法（FCS）による生細胞の蛍光タグ付きプローブの分割

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳類細胞における効率を再封止膜の高スループット測定

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

免疫・感染学

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

細胞膜の末梢関連タンパク質の分割の決定

要約

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