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1 細胞の成長および外因性抗原添加 ビオチンはタンパク質ラベリングを使用して <ol><li>準備 bOVA キット次の製造元 &#39; s プロトコル。 。
	注: 通常、bOVA 含まれています 1 M OVA あたり 2 M ビオチン平均します</li>。
	<li>55 mM 2-メルカプトエタノール、10 mM HEPES (pH 7.5) と 5 の 37 ° C で 10% ウシ胎仔血清 (以下 RPMI) 100 U/mL ペニシリン-ストレプトマイシン 0.1 mM 必須でないアミノ酸 2 mM L グルタミン 1 mM ピルビン酸ナトリウム添加 RPMI 1640 年に成長 DC2.4 細胞加湿のインキュベーターで % CO 2 (以下、言及せず細胞に孵化するこの条件で)。10% 牛胎児血清、3.7 g/L NaHCO 3 (以下 DMEM) RPMI 代わりに DMEM を使用する可能です</li>。
	<li>A 浄化、前日 RPMI 1 × 10 5 セル/ml 培養プレートに、セルを分割しました。コンフルエントの状態で細胞を保つことは避けてください。DC2.4 細胞ことができます高半コンフルエントの状態まで外因性の抗原を組み込むが急速に過剰合流状態に到達した後の能力を失う</li>。
	<li>前に、DC2.4 細胞は半合流、1 x 10 6 セル/mL の bOVA の 250 μ g/mL を追加し、2-4 h. インキュベート 
	注意: 下流の実験の間にすべてのバッファーと試薬が保管 4 ° C で特に明記しない限り</li>。
</ol> 2。ミクロソームの準備 <ol><li>新しい 50 mL の円錐管に組織プレートから穏やかなピペッティングによる DC2.4 細胞を収穫します</li>。
	<li>1,000 x g で 4 ° C、5 分間遠心吸引により bOVA の中を慎重に取り外します.</li>
	<li>40 mL の PBS バッファー (1.37 mM NaCl、8.1 mM Na 2 HPO 4、2.68 ミリメートル KCl、1.47 mM KH 2 PO 4) 5 分、4 ° C の 1,000 × g で遠心分離によって、2 回 DC2.4 細胞を洗浄し、吸引するたびに上澄みを廃棄します</li>。
	手順 2.3 で 1-2 でペレットを再懸濁します <li>mL (1/20-1/10 培地量) プロテアーゼ阻害剤のカクテルと転送再懸濁 DC2.4 の 1/1,000 ボリュームと均質化媒体 (0.25 M ショ糖, 1 mM EDTA、10 mM HEPES NaOH [Ph7.4]) の細胞に、冷たい Dounce ホモジナイザー</li>。
	<li>中断再懸濁 DC2.4 細胞優しく冷たい Dounce ホモジナイザーと 10-20 ストロークで 。
	注: 停止手順の間に氷で冷却は Dounce ホモジナイザー</li>
	<li>セル中断の懸濁液を新しい 15 mL の円錐管に転送します</li>。
	<li>10 mL 合計に 8-9 mL 均質媒体を追加し、2,000 g x と 4 で 10 分の円錐形の管を遠心分離機 ° C</li>
	<li>上清を新しい 15 mL の円錐管を切れ目のない細胞や核を削除するに転送し、g x 2,000 と 4 で 10 分間のために再度円錐管を遠心分離機 ° C</li>
	<li>新しい遠心チューブに 100,000 g x と 4 の 45 分の遠心上清を転送 ° C</li>
	<li>、上清を吸引し、ピペッティングを上下に数回下流実験用微粒体の一部分をするソリューションによってプロテアーゼ阻害剤カクテルの 1/1,000 ボリュームと均質媒体の 1-2 mL で慎重にペレットを再懸濁します</li>。
	<li>は、新しい 5 mL 丸底チューブに微粒体の一部分を移す 。
	注: この手順の後ことはメーカーによれば iodixanol 密度勾配遠心による客観的ミクロソームを豊かに &#39; s プロトコル、非特異的ミクロソームを削除します。外因性の抗原のピーク画分を収集し、浄化 (ステップ 3) の次のステップを受ける</li>。
</ol> 3。ボーヴァとミクロソームの浄化を受けて ERAD ステップ 2.11 5 mL 中の微粒体の一部分に <ol><li>追加 1/100 新鮮な量 SA 磁気ビーズ ラウンド底部チューブ 。
	注: この手順の前にそれは事前非特異的ミクロソームの汚染を減らすために制御磁気ビーズによる微粒体の一部分を消去することです</li>。
	<li>優しく混ぜるし、4 で 30 分間ゆっくりと丸底チューブを回転させて ° C</li>
	<li>丸底チューブに 4 mL 合計に均質媒体の 2-3 mL を追加し、そっと混ぜる</li>。
	<li>マグネット スタンドの丸底チューブを置き、4 ° C で 10 分間インキュベート小胞にバインドされているビーズは磁石に引き付けられる、ので茶色のマイクロ ビーズが磁石に最も近い管壁に徐々 に蓄積されます</li>。
	<li>チューブとマグネット スタンドに残って慎重に培養上清によって破棄非連結の小胞を削除する吸引</li>。
	<li>磁気によって均質媒体の 5 mL で 2 回小胞にバインドされた磁気ビーズ洗浄 4 ° C で 10 分間立って、慎重に吸引のたびに滅菌蒸留水します 。
	注: 磁気スタンド、10 分間 2,000 × g で遠心により精製なし 4 ° C もあり</li>。
	<li>100 μ L の均質化の中で慎重に小胞を上下に数回ソリューションをピペッティングによってバインドされる磁気ビーズを再懸濁します</li>。
	<li>は、下流の実験用精製微粒体として新しいチューブに再懸濁の小胞を転送します 。
	注: 通常、1 x 10 の 7 セルから 5-20 μ g 蛋白質を含んでいる 50 μ L ミクロゾーム画分に分離できます</li>。
</ol> 4。精製ミクロソームの解析 <ol>磁気再懸濁します <li>ビーズから小胞にステップ 3.8 TNE バッファーの 100-200 μ L (20 mM トリス塩酸 pH 7.4 では、150 mM の NaCl、0.5 M の EDTA、1% ノニデット P-40) プロテアーゼ阻害剤カクテルの 1/1,000 ボリュームと均質媒体の代わりにします</li>。
	<li>ライセート手順 4.1 から新しい 1.5 mL のマイクロ チューブに転送します</li>。
	<li>は、メーカーごとの BCA アッセイ キットを使用してステップ 4.2 のタンパク質濃度を測定 &#39; s プロトコル 。
	注: 通常、5-10 μ L ステップ 4.2 から溶解液は、タンパク質の濃度を測定するのに十分です</li>。
	<li>ステップ 4.2 (銀製の汚損のための蛋白質の 2 μ g と西部にしみが付くことのための蛋白質の 10 μ g) から新しいチューブにライセートを転送します</li>。
	<li>X SDS ゲル読み込みバッファー 1 で 95 ° C そして沸騰のタンパク質で熱ブロック、チューブを入れて (100 mM トリス塩酸 pH 6.8, 200 mM 4 %sds ジチオトレイトール 0.2% ブロモフェノールの青、20% グリセロール) 5 分</li>
	<li>G x 21,500 と 4 で 10 分間のチューブを遠心分離機 ° C は、不溶性画分を削除する新しいマイクロ チューブに培養上清を収集します</li>。
	<li>4.6 (2 μ g) 標準的な SD ページの手順で解決の蛋白質を分析します</li>。
	<li>可視化銀の銀染色などによる汚損でタンパク質のバンドが次のメーカー キット &#39; s プロトコル</li>。
	<li>は、標準的な SD のページおよび西部のしみが付くことによって 4.6 (10 μ g) の手順で解決の蛋白質を分析します。メーカーごとに試薬 (SA-HRP) を使用 &#39; s プロトコルし、透視によって視覚化</li>。
</ol> 5。体外ボーヴァを使用して精製したミクロソームの ERAD ユビキチン化の再構成 <ol><li>転送、精製ミクロゾーム (5-10 μ g のタンパク質) ステップ 3.8 網状赤血球ライセート (RL) の 50% のボリュームの反応バッファー (50 mM トリス pH 7.4 では、ATP、3 mM 0.5 mM MgCl x 12) と新しいチューブ 12:02 フラグ タグ ユビキチン。この実験の最終巻は 20-40 μ L.</li>
	<li>加温 37 で 2 時間チューブ ° C 
	注: ウェスタンブロッテイングにより検出する手順 5.12 でユビキチン bOVA の量が小さすぎる場合は MG132 の 10 μ M またはプロテアソーム、ユビキチン bOVA の処理を抑制するラクタシスチンの 2 μ M 追加します</li>。
	<li>氷にチューブを配置することによって反応を停止</li>
	<li>プロテアーゼ阻害剤カクテルの 1/1,000 ボリュームを 500 μ L の TNE バッファーを追加して、ミクロソームを解決します</li>。
	<li>21,50 で 10 分間遠心したマイクロ チューブg と 4 x 0 ° C は 体外 ユビキチン化アッセイ前に、DC2.4 でユビキチン ボーヴァを含む不溶性画分を削除する新しいチューブに培養上清を収集します</li>。
	<li>上清を新しいチューブに転送し、新しい SA 磁気ビーズ (通常 500 μ L の培養上清を 5 μ L) の 1/100 ボリュームを追加します</li>。
	<li>優しく混ぜるし、4 で 30 分間ゆっくりとチューブを回転 ° C</li>
	<li>21,500 g x と 4 で 10 分間のチューブを遠心分離機 ° c. 破棄ボーヴァとユビキチン ボーヴァを回復する吸引による SA 磁気ビーズのバインドします</li>。
	<li>21,500 g x と 4 で 10 分間遠心分離によって収集 SA-磁性体ビーズ TNE バッファーの 1 mL を 2 回を洗う ° c. 破棄清吸引するたびにします</li>。
	<li>SA 磁気ビーズによって浄化された蛋白質を解決する熱ブロック 95 ° C で 5 分間 SDS ゲル x 1 の SA 磁気ビーズを沸騰します</li>。
	<li>G x 21,500 と 4 で 10 分間のチューブを遠心分離機 ° C は、不溶性画分を削除する新しいチューブに培養上清を収集します</li>。
	<li>は、標準的な SD のページおよび西部のしみが付くことによって、タンパク質に結合 SA 磁気ビーズを分析します。抗体 (抗フラグ、アンチ-マルチ-Ub と抗マウス IgG HRP) および試薬 (SA-HRP) の使用は、メーカーごと、&#39; s プロトコル透視により可視化されたと</li>。
</ol>

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<li>Varabyova, A., Stojanovski, D., Chacinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+protein+homeostasis%5BTitle%5D)+AND+%22IUBMB+Life%22%5BJournal%5D)">Mitochondrial protein homeostasis.</a> IUBMB Life. 65 (3), 191-201 (2013).</li>
<li>Shen, Z., Reznikoff, G., Dranoff, G., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cloned+dendritic+cells+can+present+exogenous+antigens+on+both+MHC+class+I+and+class+II+molecules%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules.</a> J Immunol. 158 (6), 2723-2730 (1997).</li>
<li>Imai, J., Otani, M., Sakai, T., Hatta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+the+subcellular+compartment+in+which+exogenous+antigens+undergo+endoplasmic+reticulum-associated+degradation+from+dendritic+cells%5BTitle%5D)+AND+%22Heliyon%22%5BJournal%5D)">Purification of the subcellular compartment in which exogenous antigens undergo endoplasmic reticulum-associated degradation from dendritic cells.</a> Heliyon. , (2016).</li>
<li>Kovacsovics-Bankowski, M., Rock, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+phagosome-to-cytosol+pathway+for+exogenous+antigens+presented+on+MHC+class+I+molecules%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules.</a> Science. 267 (5195), 243-246 (1995).</li>
<li>Ackerman, A. L., Giodini, A., Cresswell, P. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S1074761306004262">A role for the endoplasmic reticulum protein retro translocation machinery during cross presentation by dendritic cells.</a> Immunity. 25 (4), 607-617 (2006).</li>
<li>Ackerman, A. L., Kyritsis, C., Tampé, R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+phagosomes+in+dendritic+cells+form+a+cellular+compartment+sufficient+for+cross+presentation+of+exogenous+antigens%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.</a> Proc Natl Acad Sci U S A. 100 (22), 12889-12894 (2003).</li>
<li>Saveanu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IRAP+identifies+an+endosomal+compartment+required+for+MHC+class+I+cross-presentation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">IRAP identifies an endosomal compartment required for MHC class I cross-presentation.</a> Science. 325 (5937), 213-217 (2009).</li>
<li>Zehner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8%28%2B%29+T+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.</a> Immunity. 42 (5), 850-863 (2015).</li>
</ol>

著者が明らかに何もありません。

CP の以前の研究で法人の外因性の抗原は蛍光顕微鏡観察16,30,31,32後期エンドソーム小胞体の制限区域に蓄積。ERAD のようなトランスポートおよび外因性抗原の処理は ER または後期エンドソームのこれらの専門分野の運ばれている推定されて細胞内コンパートメントは蔗糖あるいは iodixanol 密度勾配...

MHC 私分子すべての有核細胞の表面に細胞質1ユビキチン-プロテアソーム システムによって処理される内因性抗原由来抗原ペプチドを発現しています。処理後、抗原ペプチドは、ペプチド輸送体タップで小胞体 (ER) の内腔に運ばれます。小胞体内腔に特定シャペロンのシリーズは、ペプチドの読み込みと、フォールディング MHC I の複合体を支援します。分子のこのシリーズは、小胞体、ペプチドを MHC に私2の読み込みのため中央のコンパートメントであることを示すペプチド負荷複合体 (PLC) と呼ばれます。読み込み、ペプチッド MHC 後私分子細胞表面に運ばれ、キーとして役割を果たす適応免疫系の自己のマーカー、および CD8 を有効に+細胞傷害性 T リンパ球 (Ctl) がん細胞や感染性病原体を検出する抗原によって3非自己蛋白質由来ペプチド。
抗原提示細胞 (APC)、外因性の抗原の抗原性ペプチドもいる4,5,6,7,8 経由でCP、主に運ばれる私 MHC に提示で Dc9,10,11。CP が不可欠であるナイーブ CD8+ T の活性化のための両方セルとメモリ CD8 の不活化による+ T 細胞の免疫寛容の維持、抗感染症、抗腫瘍 Ctl12,13に自己演技ナイーブ T 細胞14,15。CP は、CP の分子メカニズムがまだ詳細に記載する適応免疫系の多くの重要な役割を果たしています。外因性の抗原が小胞体と、エンドソームの両方にローカライズされ、外因性の抗原がエンドソームから ERAD のような処理とペプチド16 の読み込みの小胞体に輸送されることを示唆している、ERAD で処理された CP の以前の研究で明らかに.しかし、蓄積された証拠は、小胞体ではなく、ER (図 1)17,18 の特徴がまたある非古典的エンドサイトーシス コンパートメントではなく CP のペプチド読み込みの実施を示します ,,1920,21。アミノペプチダーゼの高活性で抗原ペプチド前駆体の劣化を避けるためにこれらの非古典的エンドサイトーシス コンパートメント (図 1) の近位領域に細胞質、処理およびペプチドの CP で読み込みで22が発生します。これらのエンドサイトーシスのコンパートメントのキャラクタリゼーションが物議を醸すが、この区画で外因性の抗原以外の既存の特定の分子があります。
ERAD です具体的には小胞体からタンパク質を除去する細胞経路です。ERAD 経路の誤って折りたたまれたタンパク質は細胞質に小胞体膜を経由、ユビキチン-プロテアソーム システム23,24,25によって処理される逆行性。これらの分子が通過 Sec61 複合体とデルリン26ER と複雑なトムの複雑なティムなど、透過装置を複合体と呼ばれる分子装置、タンパク質などの大きな分子は脂質二重膜を輸送の際、ミトコンドリア27。外追加抗原は、小胞体膜を通じて転送されます、彼らは Sec61 複合体などの translocons との複合体の脂質二重膜を貫通する必要があります。ここで説明する方法は、エンドサイトーシスのコンパートメントのためのマーカーとしてこれらの膜貫通分子を利用してターゲットを絞った小胞を精製しました。
ここで説明する方法は、外因性抗原として DC のような細胞ライン DC2.428とビオチン化アルブミン (bOVA) を使用して新しい小胞浄化プロトコルです。エンドサイトーシスのコンパートメントにより精製されたストレプトアビジン (SA)-磁気ビーズ メーカーとして膜貫通ボーヴァを使用します。この精製のミクロゾームで外因 bOVA 膜画分でまだ維持されたが、ミクロソームとし、ユビキチンの外側に運搬・処理を追加いくつかの in vitro29。この精製ミクロゾーム ERAD とペプチド複合体での読み込みのエンドサイトーシスのコンパートメントに固有のタンパク質だけでなく、小胞体のタンパク質に含まれるCP29将来エンドサイトーシス コンパートメントを細胞内コンパートメントには示唆しています。このプロトコルは、外因性の抗原の種類に依存しない、また他 DC サブセットおよび他の細胞型、マクロファージ、B 細胞、内皮細胞など熟練した CP の Dc の正確な分子機構を明らかにするために適当。

<table><tbody><tr><td>RPMI 1640</td><td>gibco by life technologies</td><td>11875-093</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Equitech bio</td><td>SFB30</td><td></td></tr><tr><td>Sodium pyruvate</td><td>gibco by life technologies</td><td>11360-070</td><td></td></tr><tr><td>MEM non-essential amino acids</td><td>gibco by life technologies</td><td>11140-050</td><td></td></tr><tr><td>HEPES</td><td>gibco by life technologies</td><td>15630-080</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>gibco by life technologies</td><td>21985-023</td><td></td></tr><tr><td>L-glutamine</td><td>gibco by life technologies</td><td>25030-164</td><td></td></tr><tr><td>Penisicillin-Sreptomycin</td><td>gibco by life technologies</td><td>15140-122</td><td></td></tr><tr><td>DMEM</td><td>gibco by life technologies</td><td>12100-46</td><td></td></tr><tr><td>OVA</td><td>SIGMA</td><td>A5503</td><td></td></tr><tr><td>Biotin-protein labelling kit</td><td>Thermo Fisher Scientific</td><td>F6347</td><td></td></tr><tr><td>MG-132</td><td>Santa Cruz Biotechnology</td><td>201270</td><td></td></tr><tr><td>lactacystin</td><td>SIGMA</td><td>L6785</td><td></td></tr><tr><td>Dounce homogenizer</td><td>IUCHI</td><td>131703</td><td></td></tr><tr><td>protease inhibitor cocktails</td><td>SIGMA</td><td>P8340</td><td></td></tr><tr><td>iodixanol</td><td>Cosmo bio</td><td>1114542</td><td></td></tr><tr><td>SA-magnetic beads</td><td>New England Biolabs</td><td>201270</td><td></td></tr><tr><td>control magnetic beads</td><td>Chemagen</td><td>M-PVA012</td><td></td></tr><tr><td>magnetic stand</td><td>BD Biosciences</td><td>552311</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Thermo Fisher Scientific</td><td>23225</td><td></td></tr><tr><td>silver staining kits</td><td>Cosmo bio</td><td>423413</td><td></td></tr><tr><td>Reticulocyte Lysate</td><td>Promega</td><td>1730714</td><td></td></tr><tr><td>Flag-tagged ubiquitin</td><td>SIGMA</td><td>U5382</td><td></td></tr><tr><td>anti-ovalbumin (OVA,mouse)</td><td>Antibody Shop</td><td>HYB 094-06</td><td></td></tr><tr><td>ant-multi-ubiquitin (mouse)</td><td>MBL</td><td>D058&amp;minus;3</td><td></td></tr><tr><td>anti-Flag (mouse)</td><td>SIGMA</td><td>F3165</td><td></td></tr><tr><td>trypsin</td><td>SIGMA</td><td>85450C</td><td></td></tr></tbody></table>

purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation

樹状細胞 (Dc) は、主要組織適合抗原 (MHC) クラスに内面化された外因性抗原の提示や画像処理能力が高く、私分子として知られているクロス プレゼンテーション (CP)。CP は+ T ナイーブ CD8 の刺激だけでなく、重要な役割を果たしている細胞とメモリー CD8+ T 細胞の感染と動圧の不活性化にも腫瘍免疫ナイーブ T 細胞、T 細胞アネルギーや T 細胞削除。CP の重要な分子機構になって、蓄積された証拠を示すことが外因性の抗原は小胞体関連分解 (ERAD) 非古典からエクスポート後エンドサイトーシスを介して処理されます。コンパートメント。最近まで、外因性の抗原以外の特定の分子マーカーがなかったので、これらのエンドサイトーシスのコンパートメントの特徴は限られていた。ここで説明する方法は、これらのエンドサイトーシスのコンパートメントの浄化では、新しい小胞の分離プロトコルです。この精製微粒体を使用して、我々 再構成 ERAD のようなトランスポート、ユビキチン化、および外因性抗原の体外、処理ユビキチン-プロテアソーム システムがこれからの輸出後外因性抗原を処理することを示唆しています。細胞内コンパートメント。このプロトコルは、CP の分子機構を明らかにする他の細胞型にさらに適用できます。

CP の分子機構を解明するには、外因性の抗原が ERAD のようなトランスポートと処理を受ける細胞のコンパートメントを識別するために必要です。蛍光顕微鏡や電子顕微鏡による観察は、外因性の抗原は累積1918,16,17、、、細胞内コンパートメントを識?...

Watch this Scientific Journal Video about Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation at JoVE.com

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

ここで説明する方法は、外因性の抗原クロス プレゼンテーションで小胞体関連分解によって処理される細胞コンパートメントの浄化は、新しい小胞の分離プロトコルです。

外因性の抗原クロス プレゼンテーション内の小胞体関連分解膜コンパートメントの精製

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

purification-membrane-compartment-for-endoplasmic-reticulum

Research

JoVE Journal

Immunology and Infection

8.2K ビュー.  Takasaki University of Health and Welfare. ここで説明する方法は、外因性の抗原クロス プレゼンテーションで小胞体関連分解によって処理される細胞コンパートメントの浄化は、新しい小胞の分離プロトコルです。

ビデオ: Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

医学

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

神経科学

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

完全精製成分を用いたMsp1抽出活性の再構成

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

生化学

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

自発的な形成と小胞体のボトムアップ モデル人工脂質ナノチューブ ネットワークの転位

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

生物工学

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

ウサギ筋肉からの筋小胞体Ca2+-ATPaseの精製

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

生物学

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

培養細胞における小胞体サブドメインの可視化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

タンパク質分解中のシクロヘキシミドチェイス分析<em&gt;サッカロマイセス・セレビシエ</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

The Endoplasmic Reticulum

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary protein and lipid synthesis factory for most cell organelles, such as the Golgi apparatus, lysosomes, secretory vesicles, and the plasma membrane. Despite being the most extensive and functionally complex subcellular organelle, ER was the last to be discovered. After years of deliberation, Keith Porter and George Palade in the year 1954, produced the first high-resolution electron microscope images to affirm the presence of ER in the eukaryotic cell.
The ER membranes were identified as sites for protein synthesis and crucial for intracellular transport using radio-labeled and fluorescent-labeled amino acids. The isolation of ER is a tricky task as it forms an intricate mesh with other subcellular organelles. However, on cell homogenization, the disrupted ER membranes reseal into small closed vesicles called microsomes. These vesicles form a microsystem capable of sustaining all ER-related functions like protein and lipid synthesis, calcium signaling, and glycosylation. Subcellular fractionation is the best and often-used technique for the purification of these membranes. When separating using a sucrose gradient, the rough ER microsomes sediments at a higher density than smooth ER microsomes.
The ER network in a cell is dynamic. It is constantly shape-shifting along with the cytoskeleton to bolster mechanical support for the cell structure. Despite the distinct cisternae and tubule morphologies of the ER membrane, interconversion between the two is possible and is governed by the expression of membrane proteins. The ER network rearranges by tubule growth, retraction, and fusion of adjacent ER-ER membranes.
As mentioned above, the ER network is required for optimal overall cellular health. Disruption of ER morphology is linked to pathological conditions, including neurological disorders like Alzheimer&#39;s disease, hereditary spastic paraplegia, and viral infections like hepatitis C virus, and dengue virus.

小胞体

The endoplasmic reticulum or ER makes up for more than half of the membranes in a cell and accounts for 10% of total cell volume. It is also the primary ...

Education

JoVE Core

細胞生物学

ユニット １

小胞体とペルオキシソームにおける膜貫通輸送

KEY TERMS AND CONCEPTS

ER Retrieval Pathway

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
The ER uses many checkpoints to prevent the entry of incorrectly folded or a resident protein as cargo onto a transport vesicle. These mechanisms include cargo selectivity of the receptors, aggregation of functionally similar proteins, and retention sequences on resident proteins.
Biochemical studies characterizing the cargo-sorting receptors have shown that they are ligand-specific and bind only at specific pH (pH 5.0 in yeast and mammals). Functionally similar ER-resident can proteins aggregate to form large complexes that cannot be loaded onto transport vesicles, thus avoiding the accidental escape of these proteins from the organelle. Additionally, the membrane-spanning domain of several ER and Golgi proteins contain a retention sequence that marks these proteins for staying back in the ER, so receptors do not bind them.
If an ER-resident protein escapes these checkpoints and gets loaded onto a Golgi-bound vesicle, a specific amino acid sequence called a retrieval signal helps the protein get recognized and transported back to the ER. The receptors involved in retrieval mechanisms recognize the retrieval signals at the carboxyl terminus of soluble proteins of the ER and in the cytoplasmic domain of some ER and Golgi membrane proteins. The receptors selectively capture such proteins and package them in vesicles that transport them back to the ER.
Two well-characterized retrieval signals are the carboxy-terminal tetrapeptide KDEL and KKXX found in many ER lumen resident proteins. BiP, a molecular chaperone, plays a vital role in preventing the aggregation of misfolded proteins. Interaction between BiP and unfolded proteins is mediated by a substrate-binding domain and a nucleotide-binding domain for ATPase activity. KDEL receptor in the post-ER compartments such as ERGIC and Golgi binds with the carboxyl-terminal retrieval signal of BiP and returns BiP to the ER via COPI vesicles.

ER検索経路

In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct ...

外因性の抗原クロスプレゼンテーション内の小胞体関連分解膜コンパートメントの精製

要約

さらに動画を探す

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

完全精製成分を用いたMsp1抽出活性の再構成

自発的な形成と小胞体のボトムアップモデル人工脂質ナノチューブネットワークの転位

ウサギ筋肉からの筋小胞体^Ca2+-ATPaseの精製

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

培養細胞における小胞体サブドメインの可視化

タンパク質分解中のシクロヘキシミドチェイス分析

小胞体

ER検索経路

外因性の抗原クロス プレゼンテーション内の小胞体関連分解膜コンパートメントの精製

要約

さらに動画を探す

アミロイド前駆体タンパク質細胞内ドメインの精製と集約

ミトコンドリア関連のER膜（MAMS）とスフィンゴ糖脂質マイクロドメインエンリッチド（GEMS）：マウス脳から単離

完全精製成分を用いたMsp1抽出活性の再構成

自発的な形成と小胞体のボトムアップ モデル人工脂質ナノチューブ ネットワークの転位

ウサギ筋肉からの筋小胞体Ca2+-ATPaseの精製

哺乳動物細胞における小胞体ローカライズされたmRNAの可視化

培養細胞における小胞体サブドメインの可視化

タンパク質分解中のシクロヘキシミドチェイス分析

小胞体

ER検索経路

外因性の抗原クロスプレゼンテーション内の小胞体関連分解膜コンパートメントの精製

自発的な形成と小胞体のボトムアップモデル人工脂質ナノチューブネットワークの転位

ウサギ筋肉からの筋小胞体^Ca2+-ATPaseの精製