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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<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>

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

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.

淀粉样前体蛋白胞内结构域的分离纯化和聚集

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

科研

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.

线粒体相关的ER膜（MAMS）和鞘糖脂丰富的微区（GEMS）：分离小鼠脑

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

神经科学

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.

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

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.

人工脂质纳米管网作为内质神经的底部模型的自发形成与重排

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

生物工程

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.

从兔肌肉中纯化肌内质网 Ca2+-ATP 酶

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.

在哺乳动物细胞中的内质网定位的mRNA的可视化

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.

内质网子域的培养细胞中的可视化

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).

蛋白质降解的放线菌酮大通分析<em&gt;酿酒酵母</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.

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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 ...

教育

JoVE Core

细胞生物学

Unit 1

Transmembrane Transport in Endoplasmic Reticulum and Peroxisomes

关键术语与概念

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 检索途径

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