密度梯度超速离心研究哺乳动物细胞内吞循环
Summary
本文旨在提出一种利用蔗糖密度梯度超速离心制备哺乳动物细胞内体回收的方案。
Abstract
内体运输是调节各种生物事件的重要细胞过程。蛋白质从质膜内化，然后运输到早期内体。内化的蛋白质可以转运到溶酶体进行降解或再循环回质膜。需要一个强大的内吞循环途径来平衡膜材料从内吞作用中的去除。据报道，各种蛋白质可以调节该途径，包括ADP-核糖基化因子6（ARF6）。密度梯度超速离心是细胞分级分离的经典方法。离心后，将细胞器沉淀在其等光表面。这些馏分被收集并用于其他下游应用。这里描述的是使用密度梯度超速离心从转染的哺乳动物细胞中获取回收的含内体部分的方案。对分离的馏分进行标准的蛋白质印迹以分析其蛋白质含量。通过采用该方法，我们发现吞噬和细胞动力1（ELMO1）的质膜靶向，一种与Ras相关的C3肉毒杆菌毒素底物1（Rac1）鸟嘌呤核苷酸交换因子，是通过ARF6介导的内吞循环。
Introduction
内体运输是一个基本的生理过程，涉及各种生物事件1，例如，信号受体，离子通道和粘附分子的运输。定位于质膜上的蛋白质通过内吞作用2内化。然后由早期内体3对内化蛋白进行分类。一些蛋白质被靶向溶酶体进行降解4。然而，大量的蛋白质通过快速回收和缓慢的回收过程被回收回细胞表面。在快速循环中，蛋白质离开早期内体并直接返回质膜。相反，在缓慢循环中，蛋白质首先被分选到内吞细胞回收室，然后被输送回质膜。各种货物蛋白，例如克拉菊酯、逆转录蛋白复合物、寻回物复合物和Wiskott-Aldrich综合征蛋白，以及SCAR同系物（WASH）复合物，参与这种膜循环过程4、5、6、7、8、9。内吞作用和再循环事件的平衡对细胞存活至关重要，并有助于各种细胞事件10，例如，细胞粘附，细胞迁移，细胞极性和信号转导。
ARF6是一种小型GTP酶，是内吞细胞运输7，11，12的调节因子。有趣的是，各种研究小组已经说明了ARF6在内吞细胞回收中的重要性13，14，15，16，17。该研究旨在研究ARF6介导的神经突生长与内吞细胞循环之间的关系。之前的报告表明，ARF6的激活是通过作用于细胞分裂1（DOCK180）复合物18的ELMO1-奉献物而处于Rac1活性的上游。然而，ARF6如何触发ELMO1-DOCK180介导的Rac1信号仍不清楚。采用密度梯度超速离心研究了ARF6介导的内吞细胞循环在该过程中的作用。通过使用该，从细胞裂解物19获得含有内体的回收部分。对该馏分进行蛋白质印迹以进行蛋白质含量分析。免疫印迹结果表明，在富含脑的接合蛋白FE65存在下，活性ARF6显著提高了ELMO1在含有内体的循环部分中的水平。以下方案包括（1）转染哺乳动物细胞的程序;（2）制备样品和密度梯度柱;（3）获得回收的含内体部分。
Protocol
1. 哺乳动物细胞培养和转染 在100mm培养皿中平板2×106 个细胞。每次转染使用四个培养皿。注意：不同的细胞系所需的细胞数量可能会有所不同。在继续隔离步骤之前，可能需要进行优化。 第二天，根据制造商的说明，用利泊酚胺转染细胞。 2. 细胞采集 转染后48小时弃置培养基。 用冰冷的PBS（10mM磷酸钠，2.68mM氯化钾，140mM氯化钠?…
Representative Results
通过密度梯度超速离心对未转染的HEK293细胞进行分馏后，从梯度顶部开始收集12个组分。收获的馏分用稀释缓冲液以1：1的比例稀释并进行第二轮离心。然后将样品进行蛋白质印迹以分析其蛋白质含量。 如图1所示，回收内体标记物Rab11在馏分720中被检测到。还探测了其他亚细胞标志物，包括β-COP，COX IV，GAPDH，EEA1，Rab7和Lamp1。在分数7中也检测到正EEA1信号。…
Discussion
上述方案概述了通过超速离心从培养细胞中分离回收内体的程序。这种方法的可靠性已经由最新的出版物22证明，证明回收内体成功地从其他细胞器中分离出来（图1），如高尔基体和线粒体。为了获得良好的分离结果，需要注意一些关键步骤。在制备蔗糖溶液时，建议使用折光率仪验证溶液的折射率。62%、35%和25%蔗糖溶液在室温下的折射率分别为1.44、1.39?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了香港研究资助局、香港中文大学直接资助计划、联合学院捐赠基金和TUYF慈善信托基金的资助。这项工作中的数字改编自我们之前的出版物，”ARF6-Rac1信号介导的神经突起生长通过编排ARF6和ELMO1由神经元适配器FE65增强”于2020年10月发表在FASEB杂志上。
Materials
| 1 mL, Open-Top Thickwall Polypropylene Tube, 11 x 34 mm | Beckman Coulter | 347287 | |
| 100 mm tissue culture dish | SPL | 20100 | |
| 13.2 mL, Certified Free Open-Top Thinwall Ultra-Clear Tube, 14 x 89 mm | Beckman Coulter | C14277 | |
| 5x Sample Buffer | GenScript | MB01015 | |
| cOmplete, EDTA-free Protease Inhibitor Cocktail | Roche | 11873580001 | |
| COX IV (3E11) Rabbit mAb | Cell Signaling Technology | 4850S | Rabbit monoclonal antibody for detecting COX IV. |
| Cycloheximide | Sigma-Aldrich | C1988 | |
| Dounce Tissue Grinder, 7 mL | DWK Life Sciences | 357542 | |
| Dulbecco's Modified Eagle Medium (DMEM) with low glucose | HyClone | SH30021.01 | |
| ELMO1 antibody (B-7) | Santa Cruz Biotechnology | SC-271519 | Mouse monoclonal antibody for detecting ELMO1. |
| EndoFree Plasmid Maxi Kit | QIAGEN | 12362 | |
| FE65 antibody (E-20) | Santa Cruz Biotechnology | SC-19751 | Goat polyclonal antibody for detecting FE65. |
| Fetal Bovine Serum, Research Grade | HyClone | SV30160.03 | |
| GAPDH Monoclonal Antibody (6C5) | Ambion | AM4300 | Mouse monoclonal antibody for detecting GAPDH. |
| ImageLab Software | Bio-Rad | Measurement of band intensity | |
| Imidazole | Sigma-Aldrich | I2399 | |
| Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668019 | |
| Monoclonal Anti-β-COP antibody | Sigma | G6160 | Mouse monoclonal antibody for detecting β-COP. |
| Myc-tag (9B11) mouse mAb | Cell Signaling Technology | 2276S | Mouse monoclonal antibody for detecting myc tagged proteins. |
| OmniPur EDTA, Disodium Salt, Dihydrate | Calbiochem | 4010-OP | |
| Optima L-100 XP | Beckman Coulter | 392050 | |
| Optima MAX-TL | Beckman Coulter | A95761 | |
| Opti-MEM I Reduced Serum Media | Gibco | 31985070 | |
| PBS Tablets | Gibco | 18912014 | |
| PhosSTOP | Roche | 4906845001 | |
| RAB11A-Specific Polyclonal antibody | Proteintech | 20229-1-AP | Rabbit polyclonal antibody for detecting Rab11. |
| Sucrose | Affymetrix | AAJ21931A4 | |
| SW 41 Ti Swinging-Bucket Rotor | Beckman Coulter | 331362 | |
| TLA-120.2 Fixed-Angle Rotor | Beckman Coulter | 362046 | |
| Trypsin-EDTA (0.05%), phenol red | Gibco | 25300062 |
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Cite This Article
Chan, W. W. R., Zhai, Y. Q., Lau, K. Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells. J. Vis. Exp. (172), e62621, doi:10.3791/62621 (2021).