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

这项工作被支持由高崎大学健康和福利。

1.生长细胞与外源性抗原另外 <ol><li>准备宝物使用生物素蛋白标记试剂盒随着制造商 &#39; s 协议。 
	注意: 通常，宝物平均包含 2 M 生物素每 1 M OVA。</li>
	<li>成长 DC2.4 细胞在 RPMI 1640 辅以 2 毫米 L-谷氨酰胺、 丙酮酸钠 1 毫米、 0.1 毫米非必需氨基酸，100 U/mL 青霉素-链霉素、 2-巯基乙醇 55 毫米，10 毫米复 (pH 7.5)，10%胎牛血清 (以下简称 RPMI) 在 37 ° C 在 5%CO 2 在湿的孵化器 （以下简称未提及，细胞孵育在这种情况）。它也是可以使用辅以 3.7 g/L 碳酸氢钠 3 (以下简称 DMEM) 代替 RPMI 10%胎牛血清的 DMEM。</li>
	<li>提纯前, 一天，细胞分成 RPMI 细胞培养板 1 × 10 5 细胞/毫升。避免将存放在汇合状态细胞。DC2.4 细胞可以高度纳入外源性抗原直到半汇合的国家，但迅速达到过度汇合状态后失去能力。</li>
	<li>前 DC2.4 细胞半汇合，添加 250 微克/毫升的宝物为 1 x 10 6 细胞/毫升，孵育 2 4 h. 
	注: 下游在试验期间，所有缓冲区及试剂均都保持 4 ° C 除非另有说明。</li>
</ol> 2。微粒的制备 <ol><li>收获 DC2.4 细胞吹打温柔从组织板成一种新型的 50 毫升锥形管。</li>
	<li>在 1,000 x g 离心 5 分钟，4 ° C 和小心取出宝物的介质由志向。</li>
	<li>洗 DC2.4 细胞两次 40 毫升的 PBS 缓冲 (1.37 mM NaCl，8.1 毫米 Na 2 HPO 4，2.68 毫米 KCl，1.47 毫米 KH 2 PO 4) 在 1 5 分钟，4 ° C，x 000g 离心，弃上清每次吸入。</li>
	<li>重悬颗粒在步骤 2.3 在 1-2 毫升 （1/20-1/10 体积的培养基） 的均质介质 （0.25 M 蔗糖，1 毫米乙二胺四乙酸，10 毫米复-氢氧化钠 [pH 7.4]） 与 1/1,000 容量的蛋白酶抑制剂的鸡尾酒和转让再悬浮 DC2.4 细胞移植冰冷的 Dounce 均质机。</li>
	<li>破坏悬浮的 DC2.4 细胞轻轻由 10-20 笔画用冰冷的 Dounce 均质机。 
	注意: 在中断步骤中，Dounce 均质机在冰上冷却</li>
	<li>转移到新的 15 毫升锥形管中细胞破坏悬浮。</li>
	<li>8 9 毫升同质化培养基添加 10 毫升总和离心 10 分钟 x g 2000 及 4 的锥形管 ° C.</li>
	<li>将上清液转移到新的 15 毫升锥形管，若要删除连续的细胞和细胞核，和离心机的锥形管再 10 分钟 x g 2000 及 4 ° C.</li>
	<li>上清液转移到新的离心管和离心机 45 分钟在 100000 x g 及 4 ° C.</li>
	<li>吸出上清液和悬小球仔细在 1-2 毫升的均质介质与 1/1,000 容量的蛋白酶抑制剂的鸡尾酒吹打数次，使下游实验的微粒分数解决方案。</li>
	<li>将微粒体部分转移到新 5 毫升圆底管。 
	注: 此步骤完成后，有可能通过努力丰富客观微粒碘密度梯度离心法根据制造商 &#39; s 协议，删除非特定微粒体。对外源性抗原的峰值分数是收集，然后遭受净化 （步骤 3） 的下一步。</li>
</ol> 3。宝物的微粒净化术 ERAD <ol><li>添加 1/100 卷的新鲜 SA 磁珠步 2.11 在 5 毫升的微粒分数圆底管。 
	注意: 在此步骤中之前, 有可能预先通过控制磁性微球，以减少非特定微粒污染物清除微粒分数。</li>
	<li>轻轻拌匀，旋转圆底管慢慢为 30 分钟，在 4 ° C.</li>
	<li>2-3 毫升的均质介质 4 毫升总成圆底管加上轻轻拌匀。</li>
	<li>将圆底管放在磁性表座和孵育 10 分钟在 4 ° c。由于绑定到囊泡的珠子都被磁铁吸引，棕色微珠便会逐渐积聚到管壁靠近磁铁。</li>
	<li>与管剩余的磁性的立场，认真上清液通过放弃愿望要删除未绑定的囊泡。</li>
	<li>洗磁珠绑定到两次用 5 毫升的均质介质囊泡的磁站 10 分钟在 4 ° C，弃上清，每次通过仔细的愿望。 
	注: 无磁性的支架，通过在 2,000 x g 10 分钟洗涤净化 4 ° C 也是可用。</li>
	<li>重悬磁珠绑定到 100 µ L 均质介质中仔细囊泡吹打数次解决方案。</li>
	<li>中，再悬浮小泡将转入新的微管作为纯化微粒体下游实验。 
	注意: 通常情况下，包含 5-20 微克蛋白质从 1 × 10 7 细胞的 50 µ L 微粒分数可以是孤立。</li>
</ol> 4。分析纯化微粒 <ol><li>重磁悬珠绑定到囊泡从加强开展自评、 缓冲区的 100-200 微升 3.8 (20 毫米三 HCl pH 7.4，150 毫米氯化钠，0.5 M EDTA 1%Nonidet P-40) 与 1/1,000 容量的蛋白酶抑制剂的鸡尾酒而不是同质化介质。</li>
	<li>转移裂解步骤 4.1 到新 1.5 毫升捩。</li>
	<li>通过使用 BCA 试剂按制造商确定蛋白质浓度的步骤 4.2 &#39; s 协议。 
	注意: 通常情况下，5-10 µ L 裂解液从步 4.2 就足以确定蛋白质浓度。</li>
	<li>转入裂解步骤 4.2 （的银染蛋白 2 微克和 10 微克的蛋白质印迹） 从新微细管。</li>
	<li>微细管放在 95 ° C 和煮蛋白 1 x SDS 凝胶加载缓冲区中的加热块 (100 毫米三 HCl pH 6.8，200 毫米醇，4%的 SDS，0.2%溴酚蓝，20%的甘油) 5 分钟</li>
	<li>离心 10 分钟 x g 21,500 和 4 微细管 ° C.收集上清液到新微细管来删除不溶性小数。</li>
	<li>分析步骤 4.6 （2 微克） 的标准 SD 页的解决的蛋白质。</li>
	<li>可视化蛋白条带的银染采用银染技术工具包之后制造商 &#39; s 协议。</li>
	<li>分析步骤 4.6 （10 微克） 的标准 SD 页和蛋白印迹的解决的蛋白质。按制造商使用试剂 (SA-HRP) &#39; s 协议和透视的照相机通过可视化。</li>
</ol> 5。在体外重构的 ERAD 泛素化的宝物使用纯化微粒体 <ol><li>转让纯化微粒 （5-10 微克的蛋白） 从 50%体积的网织红细胞裂解液 (RL)，在 x 反应缓冲区 (50 毫米三 pH 7.4，3 毫米 ATP，0.5 毫米氯化镁 1 步 3.82)，和 12:02 下午旗子标记泛素新的微管。这个实验的最终数量是 20-40 微升。</li>
	<li>孵育 2 h 37 微 ° C. 
	注意: 如果步骤 5.12 中泛素化宝物量太小，免疫印迹法检测，添加 MG132 10 μ M 或随着由蛋白酶体抑制的泛素化宝物处理 2 μ M。</li>
	<li>阻止通过放置在冰上的微反应</li>
	<li>解决微粒体加入 500 µ L 开展自评、 缓冲区 1/1,000 量的蛋白酶抑制剂的鸡尾酒。</li>
	<li>微管离心机以离心 10 分钟 21,50x g 和 4 0 ° C.收集上清液到新的微管，要删除的不溶性的分数，包含在 DC2.4 前测定 体外 泛素化的泛素化宝物。</li>
	<li>将上清液转移到新的微管和添加新 SA-磁珠 （通常为 500 µ L 的上清液 5 μ） 的 1/100 卷。</li>
	<li>轻轻拌匀，慢慢为 30 分钟微圆管内旋转在 4 ° C.</li>
	<li>离心 10 分钟 x g 21,500 和 4 微 ° C.Discard 的渴望恢复的宝物和泛素化宝物上清绑定与 SA 磁珠。</li>
	<li>洗两次收集的 SA-磁珠与 1ml 上层缓冲液离心 10 分钟 x g 21,500 和 4 ° C.丢弃每次吸上清液。</li>
	<li>煮 1 x SDS 凝胶缓冲液 5 分钟在 95 ° C 加热块以 SA 磁珠解决纯化的蛋白对 SA-磁珠。</li>
	<li>离心 10 分钟 x g 21,500 和 4 微 ° C.收集上清液到新的微管，要删除的不溶性的分数。</li>
	<li>分析绑定到蛋白质的标准 SD 页和蛋白印迹 SA 磁珠。使用抗体 （抗旗、 反-多-Ub 和抗鼠 IgG HRP） 和试剂 （SA-辣根过氧化物酶） 是按制造商 &#39; s 协议和可视化的透视的照相机。</li>
</ol>

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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>

作者没有透露。

在以往研究中的 CP，注册外源抗原在禁区的晚体膜或 ER 通过免疫荧光显微镜16，30，，3132积累。据估计在这些专业领域的 ER 或晚体膜进行 ERAD 样运输和加工的外源性抗原细胞室是由蔗糖或碘密度梯度离心法分离使用标识泛素化作为一种 ERAD 样处理标记的宝物。后刺激他们先天免疫，CP 效率显著提高，...

MHC 我用内源性抗原，由泛素-蛋白酶体系统在胞浆1处理短抗原肽表达上所有的有核细胞表面的分子。经处理后，抗原肽被送入内质网 (ER) 腔通过肽转运蛋白水龙头。在 ER 腔，一系列特定的分子伴侣的协助肽加载和正确折叠的 MHC I 复合。这一系列的分子称为肽加载复杂 (PLC)，指示 ER 车厢中部为肽加载在 MHC2。后加载，多肽 MHC 我分子被运送到细胞表面，在发挥关键作用自适应免疫系统自我标记，以及使 CD8+细胞毒性 T 淋巴细胞 (Ctl) 来检测癌细胞或传染性病原体的抗原从非自我蛋白质3肽。
在抗原提呈细胞 (APC)，从外源抗原的抗原肽也出示 MHC 我4，，56，7，8 通过CP，其中主要进行由 DCs9，，1011。CP 是必不可少的两个为激活天真 CD8+ T 细胞和 CD8 记忆+ T 细胞抗感染和抗肿瘤 Ctl12，13，和在维持免疫耐受的失活自署理幼稚 T 细胞14，15。CP 许多重要作用中自适应的免疫系统，然而 CP 的分子机制尚未予以详细说明。以前对 CP 的研究揭示了外源性抗原被本地化在急诊室和内体和通过 ERAD，暗示外源抗原从内体运到 ERAD 样加工和肽加载16 ER 进行处理.然而，越来越多的证据表明 CP 的肽加载进行不在急诊室，而在于非经典的内吞车厢，也有特色的 ER (图 1)17，18 ，19，，2021。通过来避免退化的抗原肽前体肽氨基肽酶活性较高22胞液、 加工和多肽在 CP 中加载发生在近端地区的这些非经典的内吞车厢 (图 1)。虽然这些细胞内吞的车厢的刻画是有争议的没有现有的特定分子，除了这个包间的外源性抗原。
ERAD 是细胞通路，专门从急诊室中移除错误折叠的蛋白质。ERAD 通路中错误折叠的蛋白是逆行通过对细胞质 ER 膜运输和处理由泛素-蛋白酶体系统23，，2425。大分子，比如蛋白质，运输时通过脂质双分子层，这些分子通过分子的装置称为解，如 Sec61 复杂和德林在 ER26，和汤姆复杂复杂和蒂姆在复杂线粒体27。当外部添加抗原是通过内质网膜，他们必须穿透脂质双分子层中复杂的 translocons，例如 Sec61 复杂。这里介绍的方法通过利用这些膜穿透的分子标志物在细胞内吞的隔间作为纯化有针对性的囊泡。
这里介绍的方法是一种新的使用作为外源性抗原的树突状细胞线 DC2.428和生物素化的卵清蛋白 （宝物） 的囊泡净化协议。内吞的车厢进行纯化的链霉亲和 (SA)-磁珠作为制造商使用膜穿透宝物。在这纯净的微粒，一些外部添加宝物仍保持膜组分中但被运到外面的微粒体，然后泛素化和处理体外29。这纯净的微粒体载不仅吞隔间特异性蛋白二村居民蛋白质 ERAD 和肽加载复杂;建议细胞隔间 CP29前瞻性吞车厢。本议定书依赖外源性抗原，这种并不是也适用于其他 DC 亚群和其他细胞类型，如巨噬细胞、 B 细胞和内皮细胞，以澄清精通 CP DCs 的精确分子机制。

<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

树突状细胞 (Dc) 是高度能够处理和后主要组织相容性类 (MHC) 的内化外源抗原呈递我分子也被称为跨演示文稿 (CP)。CP 扮演重要的角色，不仅在天真 CD8 的刺激+ T 细胞和 CD8 记忆+ T 细胞的传染性，而且在自立式灭活肿瘤免疫 t 细胞的 T 细胞无能或 T 细胞删除。虽然 CP 的关键分子机制仍有待澄清，越来越多的证据表明外源抗原进行处理通过内质网关联降解 (ERAD) 后从非古典出口内吞隔间。直到最近，刻画这些细胞内吞的车厢都有限，因为有没有除了外源性抗原的特异分子标记。这里介绍的方法是一种新的囊泡隔离协议，允许纯化这些细胞内吞的车厢。我们使用此纯化的微粒，重组 ERAD 像运输、 泛素化和处理的外源性抗原体外，暗示泛素-蛋白酶体系统后从这出口加工外源性抗原细胞的隔间。该协议可以进一步应用于其他细胞类型以澄清 CP 的分子机制。

为了阐明 CP 的分子机制，是有必要确定细胞的隔间，外源抗原在哪里接受 ERAD 样运输和加工。虽然通过免疫荧光显微镜或电子显微镜的观察确定外源抗原在那里积累16，17，，1819，细胞室 30，，31<s...

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

这里介绍的方法是一种新的囊泡隔离协议，允许纯化细胞的车厢外源性抗原处理由内质网关联退化跨演示文稿中的地方。

纯化膜隔间跨演示文稿中的外源抗原内质网关联降解

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 次观看.  Takasaki University of Health and Welfare. 这里介绍的方法是一种新的囊泡隔离协议，允许纯化细胞的车厢外源性抗原处理由内质网关联退化跨演示文稿中的地方。

视频: 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.

猪或牛光感受器外段的细胞吞噬分析对视网膜色素上皮细胞大规模纯化

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

科研

免疫与感染

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.

用完全纯化的组分重构Msp1提取活性

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

生物化学

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.

小鼠树突状细胞亚群的分离的高效和高产的方法

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.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

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.

研究免疫突触形成过程中B细胞中的细胞动力学

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.

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

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.

腔内囊泡和多泡体

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

教育

JoVE Core

细胞生物学

Unit 1

Endocytosis and Exocytosis

关键术语与概念

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 中的受体下调

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.

从 ER 中导出错误折叠的蛋白质

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

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

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.

抗原呈递细胞

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