Name: purification of the membrane compartment for endoplasmic reticulum-associated degradation of exogenous antigens in cross-presentation
Uploaded: 2017-08-21T14:00:00.000+00:00
Duration: 12 min 48 s
Description: 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

この作品は、高崎保健福祉大学のによってサポートされます。

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>

<ol>
	<li>van Endert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+ligands+for+MHC+class+I+molecules.">Providing ligands for MHC class I molecules.</a> Cell Mol Life Sci. 68 (9), 1467-1469 (2011).</li><li>Janeway, C., Travers, P., Walport, M., Shlomchik, 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> Immunobiology: The Immune System in Health and Disease. , (2001).</li><li>McDevitt, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+the+role+of+the+major+histocompatibility+complex+in+the+immune+response.">Discovering the role of the major histocompatibility complex in the immune response.</a> Annu Rev Immunol. 18 (1), 1-17 (2000).</li><li>Bedoui, S., 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=Cross-presentation+of+viral+and+self+antigens+by+skin-derived+CD103++dendritic+cells.">Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells.</a> Nat Immunol. 10 (5), 488-495 (2009).</li><li>Segura, E., Villadangos, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+by+dendritic+cells+in+vivo.">Antigen presentation by dendritic cells in vivo.</a> Curr Opin Immunol. 21 (1), 105-110 (2009).</li><li>Cheong, C., 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=Microbial+stimulation+fully+differentiates+monocytes+to+DC-SIGN/CD209(+)+dendritic+cells+for+immune+T+cell+areas.">Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas.</a> Cell. 143 (3), 416-429 (2010).</li><li>Henri, S., 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=CD207++CD103++dermal+dendritic+cells+cross-present+keratinocyte-derived+antigens+irrespective+of+the+presence+of+Langerhans+cells.">CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.</a> J Exp Med. 207 (2), 189-206 (2010).</li><li>Shortman, K., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD8++dendritic+cell+subset.">The CD8+ dendritic cell subset.</a> Immunol. Rev. 234 (1), 18-31 (2010).</li><li>Carbone, F. R., Kurts, C., Bennett, S. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation:+a+general+mechanism+for+CTL+immunity+and+tolerance.">Cross-presentation: a general mechanism for CTL immunity and tolerance.</a> Immunol Today. 19 (8), 368-373 (1998).</li><li>Nair-Gupta, P., Blander, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+view+of+the+intracellular+mechanisms+regulating+cross-presentation.">An updated view of the intracellular mechanisms regulating cross-presentation.</a> Front Immunol. , (2013).</li><li>Joffre, O. P., Segura, E., Savina, A., Amigorena, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+by+dendritic+cells.">Cross-presentation by dendritic cells.</a> Nat Rev Immunol. 12 (8), 557-569 (2012).</li><li>Kurts, C., 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=Constitutive+class+I-restricted+exogenous+presentation+of+self+antigens+in+vivo.">Constitutive class I-restricted exogenous presentation of self antigens in vivo.</a> J Exp Med. 184 (3), 923-930 (1996).</li><li>Kurts, C., Kosaka, H., Carbone, F. R., Miller, J. F., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+I-restricted+cross-presentation+of+exogenous+self-antigens+leads+to+deletion+of+autoreactive+CD8(+)+T+cells.">Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.</a> J Exp Med. 186 (2), 239-245 (1997).</li><li>Bonifaz, L., 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=Efficient+targeting+of+protein+antigen+to+the+dendritic+cell+receptor+DEC-205+in+the+steady+state+leads+to+antigen+presentation+on+major+histocompatibility+complex+class+I+products+and+peripheral+CD8++T+cell+tolerance.">Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.</a> J Exp Med. 196 (12), 1627-1638 (2002).</li><li>Heath, W. R., Carbone, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-presentation+in+viral+immunity+and+self-tolerance.">Cross-presentation in viral immunity and self-tolerance.</a> Nat Rev Immunol. 1 (2), 126-134 (2001).</li><li>Imai, J., Hasegawa, H., Maruya, M., Koyasu, S., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+antigens+are+processed+through+the+endoplasmic+reticulum-associated+degradation+(ERAD)+in+cross-presentation+by+dendritic+cells.">Exogenous antigens are processed through the endoplasmic reticulum-associated degradation (ERAD) in cross-presentation by dendritic cells.</a> Int Immunol. 17 (1), 45-53 (2005).</li><li>Houde, 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=Phagosomes+are+competent+organelles+for+antigen+cross-presentation.">Phagosomes are competent organelles for antigen cross-presentation.</a> Nature. 425 (6956), 402-406 (2003).</li><li>Guermonprez, P., 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=ER-phagosome+fusion+defines+an+MHC+class+I+cross-presentation+compartment+in+dendritic+cells.">ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells.</a> Nature. 425 (6956), 397-402 (2003).</li><li>Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+mechanistic+separation+of+cross-presentation+and+endogenous+antigen+presentation.">Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.</a> Nat Immunol. 9 (5), 558-566 (2008).</li><li>Liz&#233;e, G., 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=Control+of+dendritic+cell+cross-presentation+by+the+major+histocompatibility+complex+class+I+cytoplasmic+domain.">Control of dendritic cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain.</a> Nat Immunol. 4 (11), 1065-1073 (2003).</li><li>Basha, G., 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=A+CD74-dependent+MHC+class+I+endolysosomal+cross-presentation+pathway.">A CD74-dependent MHC class I endolysosomal cross-presentation pathway.</a> Nat Immunol. 13 (3), 237-245 (2012).</li><li>Saveanu, L., Carroll, O., Hassainya, Y., van Endert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity,+contradictions,+and+conundrums:+studying+post-proteasomal+proteolysis+in+HLA+class+I+antigen+presentation.">Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation.</a> Immunol Rev. 207 (1), 42-59 (2005).</li><li>Wiertz, E. 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=Sec61-mediated+transfer+of+a+membrane+protein+from+the+endoplasmic+reticulum+to+the+proteasome+for+destruction.">Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction.</a> Nature. 384 (6608), 432-438 (1996).</li><li>Hampton, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER-associated+degradation+in+protein+quality+control+and+cellular+regulation.">ER-associated degradation in protein quality control and cellular regulation.</a> Curr Opin Cell Biol. 14 (4), 476-482 (2002).</li><li>Tsai, B., Ye, Y., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-translocation+of+proteins+from+the+endoplasmic+reticulum+into+the+cytosol.">Retro-translocation of proteins from the endoplasmic reticulum into the cytosol.</a> Nat Rev Mol Cell Biol. 3 (4), 246-255 (2002).</li><li>Nyathi, Y., Wilkinson, B. M., Pool, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-translational+targeting+and+translocation+of+proteins+to+the+endoplasmic+reticulum.">Co-translational targeting and translocation of proteins to the endoplasmic reticulum.</a> Biochim Biophys Acta. 1833 (11), 2392-2402 (2013).</li><li>Varabyova, A., Stojanovski, D., Chacinska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+homeostasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloned+dendritic+cells+can+present+exogenous+antigens+on+both+MHC+class+I+and+class+II+molecules.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+subcellular+compartment+in+which+exogenous+antigens+undergo+endoplasmic+reticulum-associated+degradation+from+dendritic+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phagosome-to-cytosol+pathway+for+exogenous+antigens+presented+on+MHC+class+I+molecules.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+endoplasmic+reticulum+protein+retro+translocation+machinery+during+cross+presentation+by+dendritic+cells.">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., Tamp&#233;, R., Cresswell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phagosomes+in+dendritic+cells+form+a+cellular+compartment+sufficient+for+cross+presentation+of+exogenous+antigens.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IRAP+identifies+an+endosomal+compartment+required+for+MHC+class+I+cross-presentation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+translocon+protein+Sec61+mediates+antigen+transport+from+endosomes+in+the+cytosol+for+cross-presentation+to+CD8(+)+T+cells.">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

Universidad de Cartagena UDEC

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

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

網膜色素上皮細胞に貪食アッセイのためにブタまたはウシの感光体外側セグメントの大規模精製

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

免疫・感染学

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

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Echinococcus granulosusからの小さな細胞外小胞の単離と樹状細胞の取り込み

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Intralumenal Vesicles and Multivesicular Bodies

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing numerous ILVs is called a multivesicular body (MVB). ILVs contain internalized molecules such as antigens, nucleic acids, proteins, and metabolites. Some of these molecules are released from the MVBs inside exosomes and are transported to other cells. Other MVBs contain molecules that are retained in the ILVs and are later degraded within the cell.
MVBs were first observed in neuronal cells in 1955 using electron microscopy. MVBs have a diameter of 400-500 nm, are spherical, and were initially thought to be a form of the late endosome; however, recent studies have shown that MVBs are an intermediate form of these organelles between the early and the late endosomes.
ILV formation takes place when a ubiquitin-tagged protein enters the cell in an endocytic vesicle and fuses with the early endosome. As the early endosome matures, the endosomal membrane bends inwards to form ILVs. An ESCRT protein complex mediates ILV formation. Phosphatidylinositol 3-phosphate (PtdIns3P) is also required for ILV formation. PtdIns3P acts as a docking site for ESCRTs for the bending of the endosomal membrane.
The ESCRT complex and the ATPase, Vps4, required for ILV formation can be hijacked by enveloped retroviruses that infect host cells. Normally, ILVs are formed inside maturing endosomes with the help of ESCRTs within the cell, and the ubiquitin-tagged proteins are recognized and sorted into ILVs. In contrast, retroviruses use the ESCRTs to be released outside the cell. For example, the human immunodeficiency virus (HIV) codes for proteins, such as Gag, NC-p2, and Tat, that require ubiquitination for export. This ubiquitination is essential because ESCRTs and Vps4 recognize and sort only ubiquitin-tagged proteins into viral buds. Once the viral particles bud off from an infected cell, they are released into the extracellular space to infect other host cells.

内腔小胞と多胞体

Intraluminal vesicles (ILVs) are small vesicles 50-80 nm in diameter formed during the maturation of early endosomes. A specialized endosome containing ...

Education

JoVE Core

細胞生物学

ユニット １

エンドサイトーシスとエキソサイトーシス

KEY TERMS AND CONCEPTS

Receptor Downregulation in MVBs

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal growth factor (EGF) and its receptor (EGFR) form a complex that can be internalized through endocytosis, sorted into an MVB, and later degraded.
The EGFR can initiate signaling pathways that&#160; lead to cell proliferation, migration, and differentiation. Overexpression of EGFR&#160; stimulates cells to proliferate. Excessive&#160; EGFR activation may result in cancer; therefore, EGFR expression must be tightly regulated. The degradation of the EGF-EGFR complex through the endocytic pathway is a means of downregulating EGFR and preventing its overexpression.
EGF binding to EGFR induces EGFR dimerization. EGFR is ubiquitinated by a ubiquitin ligase. Ubiquitination triggers the endocytosis of the dimerized EGF-EGFR complex. Once inside the cell, the complex is first sorted into the early endosome. As the early endosome matures, ESCRT proteins, or endosomal sorting complexes required for transport, translocate&#160; the EGFR-EGF complex into intraluminal vesicles (ILVs). Several ILVs are formed inside the maturing endosome, eventually becoming multivesicular bodies (MVBs). MVBs containing the EGF-EGFR complex then fuses with a lysosome for degradation of the EGFR-EGF complex.
Similar to EGFR, cytokine receptors are also internalized and downregulated by MVBs. Ubiquitinated receptor-cytokine complexes are endocytosed and later delivered to MVBs for degradation. The degradation weakens the cytokine signaling. In the case of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF&#593;) or interleukin-1 (IL-1), any defect in ubiquitination, endocytosis, or degradation of the receptor-cytokine complex through MVB can cause enhanced or sustained signaling. This may lead to inflammatory diseases like Crohn&#39;s disease or rheumatoid arthritis.

MVBにおける受容体のダウンレギュレーション

Multivesicular bodies (MVBs) are mature endosomes that sort ubiquitinated proteins and then fuse with lysosomes to degrade the sorted proteins. Epidermal ...

Export of Misfolded Proteins out of the ER

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for degradation.
Different modes of ERAD pathways
Hrd1 is a ubiquitin ligase and a retro-translocation channel for misfolded polypeptides. It complexes with another membrane protein, Hrd3, to form the core ERAD machinery. The other players associated with these core proteins depend upon the topology of the misfolded protein. The retrotranslocation complex depends on the luminal lectin chaperones to identify misfolded proteins. Studies in the yeast system suggest the presence of ERAD-C, ERAD-L, and ERAD-M pathways to export misfolded proteins from various parts of the ER. The target proteins of the ERAD-L and ERAD-M pathways use the Hrd1 channel to exit the ER lumen and membrane, respectively, while the ERAD-C pathway utilizes the Doa10p ubiquitin ligase complex to degrade the cytoplasmic domains of the ER membrane proteins. Sometimes, these pathways can overlap as some misfolded proteins need translocation machinery from two different ERAD pathways to exit the ER. The mammalian ERAD machinery is more complex than yeast and has multiple proteins that export and mark proteins for proteasomal degradation. Eventually, all ERAD pathways converge due to their dependence on a cytoplasmic AAA-ATPase to provide the mechanical force to extract proteins through the membrane.
Degradation of non-glycosylated proteins
The ER has another lectin-independent system that monitors and clears the non-glycosylated proteins from ER. It consists of the resident ER chaperone, BiP protein, and the ER-localized DnaJ family members (ERdjs). ER stress induces the production of HERP, a ubiquitin-like membrane protein, which assembles with other ERAD proteins like the Hrd1 and AAA-ATPase, forming a complex. BiP uses some ERdj proteins as cofactors to recruit non-glycosylated misfolded proteins to the HERP complex for retrotranslocation and ubiquitination of the misfolded protein. HERP is associated with the proteasome on the cytosolic side, where it delivers the misfolded proteins for degradation.

ERからのミスフォールドタンパク質の輸出

After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for ...

小胞体とペルオキシソームにおける膜貫通輸送

Antigen Presenting Cells

The immune system is a complex network of cells and molecules that protects the body from foreign invaders. T cells, a type of white blood cell, play a crucial role in this process. They recognize and attack foreign substances, such as pathogens, that enter the body.
T cells require the help of antigen-presenting cells (APCs), which process foreign antigens into smaller fragments that can be recognized by T cells. These APCs are highly specialized cells that efficiently internalize antigens through phagocytosis or receptor-mediated endocytosis.
Dendritic cells, a type of professional APC, are particularly efficient at antigen processing and presentation. They are highly mobile and can be found in tissues first exposed to foreign antigens, such as the skin, mucosal surfaces, and lymphoid organs.
Macrophages may also function as APCs, and are found in all tissues. They can engulf and process foreign antigens and release cytokines, which activate T cells and other immune cells, thereby coordinating the immune response.
B cells are another type of APC that can present antigens to T cells. When an antigen is presented to a B cell, it activates the B cell to produce antibodies against the antigen. This activation process involves the binding of the antigen to the B cell receptor, followed by a series of molecular signaling events that produce antibodies.
When an APC engulfs an antigen, it breaks into smaller fragments and combines them with major histocompatibility complex (MHC) molecules. The antigen-MHC complex is then transported to the APC&#39;s plasma membrane, where it is presented to a T cell.
The appropriate T cell receptor recognizes the processed antigen complex, triggering an immune response against the foreign invader. This process is crucial for protecting the body from infection and disease.

抗原提示細胞

Antigen-presenting cells, also known as APCs, process foreign antigens, such as fragments from pathogens, for recognition by T cells.
When an APC engulfs ...

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

この動画の章

網膜色素上皮細胞に貪食アッセイのためにブタまたはウシの感光体外側セグメントの大規模精製

完全精製成分を用いたMsp1抽出活性の再構成

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

サルモネラの分離-大食細胞のファゴゾームを含む

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

Echinococcus granulosusからの小さな細胞外小胞の単離と樹状細胞の取り込み

内腔小胞と多胞体

MVBにおける受容体のダウンレギュレーション

ERからのミスフォールドタンパク質の輸出

抗原提示細胞

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

この動画の章

網膜色素上皮細胞に貪食アッセイのためにブタまたはウシの感光体外側セグメントの大規模精製

完全精製成分を用いたMsp1抽出活性の再構成

マウスの樹状細胞サブセットの単離のための効率的かつ高収率方法

サルモネラの分離-大食細胞のファゴゾームを含む

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

Echinococcus granulosusからの小さな細胞外小胞の単離と樹状細胞の取り込み

内腔小胞と多胞体

MVBにおける受容体のダウンレギュレーション

ERからのミスフォールドタンパク質の輸出

抗原提示細胞

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