Name: determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation
Uploaded: 2017-07-03T15:00:00.000+00:00
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1.通过生物素化测定星形胶质细胞中的相对细胞表面蛋白表达注意:在这里，我们说明了这种生物素化技术在细胞外基质分子层粘连蛋白对透水通道水通道蛋白-4（AQP4）的细胞表面定位的影响的研究中的应用。该测定所需的专用材料包括磺基-NHS-LC-生物素和链霉亲和素 - 琼脂糖树脂（参见材料表 ）。 <ol><li>使用Noel 等人概述的方法12 ，在测定前约2周准备皮层星形胶质细胞培养物，并在75cm 2的通气培养瓶中培养。当星形胶质细胞为80-90％融合时，使用0.05％胰蛋白酶将其从培养物表面分离出来，然后通过1:3。 </li><li>在测定前48小时，当细胞再次融合80-90％时，通过星形胶质细胞1:3（占c以培养形式变化）到60mm细胞培养皿上，使得在实验发生的当天&gt; 70％汇合。确保细胞均匀分布在盘间。 </li><li>在测定前16小时，将层管蛋白移液到培养基中至最终浓度为24nM，并在37℃下孵育。 </li><li>在测定前，准备好以下物质，然后置于冰或冷藏中:将CM-PBS（100mg / L MgCl 2· 6H 2 O和100mg / L CaCl 2在1X PBS中，pH7.4），生物素缓冲液（0.5 （CM-PBS中的mg / mL磺基-NHS-LC-生物素），猝灭缓冲液（CM-PBS中的50mM NH 4 Cl），裂解缓冲液（25mM Tris，pH 7.4,25mM甘氨酸，150mM NaCl和5mM EDTA，1％triton X-100,1X蛋白酶抑制剂混合物），3X加载缓冲液（150mM Tris，pH 6.8,6％SDS，30％甘油，300mM DTT和0.01％溴酚蓝）和洗涤缓冲液Tris（pH 7.4），1.5mM EDTA，150mM NaCl，1％Triton X-100,1X蛋白酶抑制剂混合物）。 </li><li>雷姆从培养箱中拿着星形胶质细胞培养皿，然后弃去培养基。 </li><li>用4 mL冷冻的CM-PBS将细胞洗涤3次，并将其放在碎冰上。 </li><li>将2 mL生物素缓冲液吸入每个孔中，并轻轻地将菜肴往前倾几次，以确保完全覆盖。在冰上休息30分钟。 </li><li>用吸气器取出生物素缓冲液，并用4 mL淬灭缓冲液代替。在冰上离开10分钟。 </li><li>吸入淬火缓冲液，并用相同体积的相同体积代替。再次，在冰上离开10分钟。 </li><li>弃去淬灭缓冲液，用4 mL冷冻的CM-PBS洗涤细胞三次。 </li><li>使用细胞提升器将细胞切成1mL冷冻的CM-PBS，并将悬浮液转移到微量离心管中。 </li><li>通过在100×g离心3分钟来制备粒细胞。弃去上清液，并将细胞重新悬浮于500μL裂解缓冲液中。 </li><li>将样品放在冰上30分钟，每5分钟振荡一次在4°C的端端旋转器上。 </li><li>在4℃下以14,000xg离心裂解物10分钟，以沉淀任何不溶于洗涤剂的物质。将上清液转移到新的微量离心管中。 <ol><li>保存50μL此裂解液，加入缓冲液。然后在95℃下在干浴中加热变性;这是"输入"分数，含有生物素化的细胞表面蛋白质，以及非生物素化的胞质蛋白质。 </li></ol></li><li>使用一把锋利的剪刀从其末端切掉大约0.5厘米的材料，从而扩大移液器吸头的开口。使用该吸管头，将75μL链霉抗生物素蛋白 - 琼脂糖珠（通常在4°C下储存）转移到裂解物中，并在4℃下在振荡器/摇杆上孵育3小时。 <ol><li>作为链霉抗生物素蛋白 - 琼脂糖经常作为含有50体积％的珠粒的浆料出售，悬浮在抗微生物溶液中，研磨该浆料以确保珠粒均匀悬浮，然后将150μL悬浮液移液到每个样品中。 </li></ol></li><li>颗粒链霉亲和素 - 琼脂糖珠子，在4℃下以1500xg离心30秒。 <ol><li>保存50μL上清液（加入缓冲液，并在95℃在水浴或加热块中变性）;这代表"细胞内"级分，并且主要由非生物素化的胞质蛋白组成。 </li></ol></li><li>将沉淀的珠子重悬于1mL洗涤缓冲液中，并在4℃下摇匀3分钟。颗粒珠（如步骤1.16），并弃去上清液。重复此过程4x以最小化非生物素化胞质蛋白的非特异性结合。 </li><li>通过离心（1500xg，4℃，30s）离心沉淀珠粒，并丢弃上覆的洗涤缓冲液。加入50μL的1X加载缓冲液（用裂解缓冲液稀释）。通过在95℃下将其变性从珠中释放生物素和链霉亲和素;这个分数只能含有生物素化的细胞表面蛋白质（"细胞表面"部分）。 <ol><li>通过SDS-PAGE 13分离输入，细胞表面和细胞内级分，并通过蛋白质印迹分析14 。 注意:虽然我们在我们的实验中使用了4 - 20％的预制梯度凝胶，但是在本研究中，12-14％的分离凝胶与4％堆叠层（每个含有0.1％SDS）都足以满足感兴趣的蛋白质。还应使用适当尺寸范围的分子量标准。注意，有时可以观察到生物素化蛋白质的表观分子量升高。 </li></ol></li></ol> 2.通过生物素化测定星形胶质细胞中细胞表面蛋白的内在化率注意:在下文中，我们描述了在这种情况下用于跟踪星形胶质细胞中AQP4的内吞作用的典型脉冲追踪生物素化实验。这个方法基于马德里等人使用的方法15 。所需的专门材料包括磺基-NHS-SS生物素，链霉抗生物素蛋白 - 琼脂糖树脂，还原型谷胱甘肽和碘乙酰胺（参见材料表 ）。 <ol><li>使用上一节中概述的方法，在60 mm的培养皿中制备小鼠皮层星形胶质细胞的培养物。确保细胞在测定当天约70％汇合，并且每个培养皿含有相当数量的细胞。 </li><li>在测定之前，准备好以下物质，并置于冰或冷藏中:将CM-PBS（100mg / L MgCl 2· 6H 2 O和100mg / L CaCl 2在1X PBS，pH7.4中），生物素缓冲液（0.5在CM-PBS中的mg / mL磺基-NHS-SS生物素），还原缓冲液（50mM还原型谷胱甘肽，75mM NaCl和75mM NaOH），淬灭缓冲液（50mM碘乙酰胺，1％BSA，在CM-PBS中）裂解缓冲液（25mM Tris，pH 7.4,25mM甘氨酸，150mM NaCl和5mM EDTA，1％triton X-100,1X蛋白酶抑制剂混合物），3X加载缓冲液（150mM Tris，pH 6.8,6％SDS，30％甘油，300mM DTT和0.01％溴酚蓝）和洗涤缓冲液（10mM Tris，pH 7.4,1.5mM EDTA， 150mM NaCl，1％triton X-100,1X蛋白酶抑制剂混合物）。 </li><li>准备新鲜细胞培养基（补充有10％胎牛血清（FBS），1％青霉素/链霉素和1％L-谷氨酰胺的DMEM），并将其置于37℃水浴中。 </li><li>从培养箱中取出星形胶质细胞培养物，并用吸液器抽吸培养基。 </li><li>用4 mL冷冻的CM-PBS将细胞洗涤3次，并将其放在碎冰上。 </li><li>将3 mL生物素缓冲液吸入每个培养皿中，往前后摆动几次，以确保缓冲液分布均匀，并将其放在冰上30分钟。 </li><li>吸出生物素缓冲液，并用5 mL温培养基代替。在37℃培养培养皿15分钟，并在同一温度下培养30分钟。在4点再留个菜6; C为0分钟样品。 </li><li>在孵育期结束时，弃去培养基，用4mL冷冻的CM-PBS洗涤细胞三次。在细胞上吸取6 mL还原缓冲液，并在冰上放置15分钟。 </li><li>取出还原缓冲液，然后用6 mL新鲜还原缓冲液取代。在冰上再放置15分钟。 </li><li>除去还原溶液，并用6 mL淬灭缓冲液代替。在冰上离开15分钟。 </li><li>再次重复淬火步骤。 </li><li>丢弃淬灭缓冲液，用4 mL冷冻PBS洗涤细胞三次。 </li><li>使用细胞提升器将细胞切成1mL冷冻的PBS，并将悬浮液转移到微量离心管中。 </li><li>通过在100×g离心3分钟来制备粒细胞。弃去上清液，并将细胞重悬于500μL裂解缓冲液中。 </li><li>将其放在冰上30分钟，每5分钟旋涡一次。或者，将样品放置在4°C的终端旋转器上。 </li><li>离心机在4℃下以14,000xg的速度放置10分钟以沉淀洗涤剂不溶物质，然后将上清液转移到新的微量离心管中。保存50μL此裂解液，加入缓冲液，并在95℃在干燥浴中变性;这是"输入"分数，含有生物素化的内吞蛋白质，以及非生物素化的蛋白质。 </li><li>使用切割的吸头，将150μL的链亲和素 - 琼脂糖浆液加入到裂解物中，并在4℃下在振荡器/摇臂上孵育3小时。有关此步骤的更多详细信息，请参阅1.15。 </li><li>颗粒链亲和素 - 琼脂糖珠子，在4℃下以1500×g离心30秒。 </li><li>将珠重悬于1mL洗涤缓冲液中，并在4℃下摇动3分钟。颗粒珠（按照步骤2.18），弃去上清液。重复此过程4x以最小化非生物素化胞质蛋白的非特异性结合。 </li><li>通过在4℃下以1,500×g离心30秒，并弃去颗粒珠洗涤缓冲液。加入50μL1X加载缓冲液（用裂解缓冲液稀释）。通过在95℃变性从珠粒释放生物素和链霉亲和素;这个部分应该仅含有内在的细胞表面蛋白质（"胞吞"部分）。 </li><li>通过SDS-PAGE 13分离输入，细胞表面和未结合部分，并通过蛋白质印迹分析14 。 注意:虽然我们在我们的实验中使用了4 - 20％的预制梯度凝胶，但是在本研究中，12-14％的分离凝胶与4％堆叠层（每个含有0.1％SDS）都足以满足感兴趣的蛋白质。还应使用适当尺寸范围的分子量标准。注意，有时可以观察到生物素化蛋白质的表观分子量升高。 </li></ol>

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K., Joshi, B., Moukhles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquaporin-4+Cell-Surface+Expression+and+Turnover+Are+Regulated+by+Dystroglycan,+Dynamin,+and+the+Extracellular+Matrix+in+Astrocytes.">Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes.</a> PLoS One. 11 (10), 0165439 (2016).</li></ol>

作者宣称没有利益冲突。

修改: 由于这些方法设计用于贴壁细胞，我们已经规定了使用含有100mg / L MgCl 2· 6H 2 O的PBS和 100mg / L的CaCl 2 （CM-PBS）洗涤步骤，并作为某些缓冲液的碱基，以确保细胞保持附着于培养物表面，并且细胞 - 细胞结不被破坏。然而，如果细胞在程序的每个步骤之间沉淀，则该协议也可以应用于非贴壁细胞类型。在这些情况下，可以用PBS代替CM-PBS。 此外，重要的是要注意，这里提到的每个条件都是本协议的特定内容，如果方法用于其他应用程序，则只应将其视为粗略的指导原则。特别地，应该独立地验证洗涤剂提取程序是否适用于此r细胞类型，并且离心机设置适用于细胞类型，并且适用于使用的链霉亲和素 - 琼脂糖珠粒。 最后，如果有关于非特异性结合和/或过度背景的担忧，可以用市场上目前可获得的各种其他抗生物素蛋白形式代替链霉亲和素。例如，去糖基化的抗生物素蛋白对凝集素和对于带负电荷的分子如DNA具有显着较低的亲和力。 故障排除和控制: 虽然本报告中审查的程序相当简单，但应注意一些可能影响实验结果的关键问题。首先，尽管两种测定都严重依赖于硫酸化生物素化试剂的膜不渗透性质，但某些条件可导致细胞的质膜破裂，从而导致某些细胞内的蛋白被生物素化。为了控制这种可能性，建议对已知不在质膜上表达的靶标（如β-肌动蛋白）进行探测生物素化的级分。 应注意遵守生物素化试剂的适当储存条件（通常为4或-20°C，具有某种形式的干燥），否则可能由于其高度敏感性而失败。然而，良好的做法是用链霉抗生物素蛋白-HRP检测输入，生物素化和非生物素化的级分，以确定生物素化确实发生，并且生物素化的蛋白质已被有效沉淀（这将从在非生物素化级分）。这样做应该可以消除有缺陷的试剂的可能性。 二硫键还原是内吞生物素化过程中的关键步骤，因为它可以确保在生物素化部分中仅分离内在蛋白质。纳入其中省略了还原剂的对照（ 图 1C ）允许人们获得治疗效果的印象。

细胞表面的蛋白质起着维持细胞功能的重要作用。在许多情况下，它们的活性依赖于或者通过内吞过程调节，这些内吞过程暂时将其封闭在胞内位点，或者将它们引导到降解途径1,2,3,4,5 。在这里，我们强调了2种基于生物素化的方法，旨在允许用户专门标记和分离在质膜上表达的蛋白质，以及新内化的蛋白质。通过这些方法，可以量化任何目的蛋白质的细胞表面表达和内吞速率，从而允许更清楚地评估其调节。 通过生物素化测定相对细胞表面蛋白的表达 生物素或维生素B7（以前称为维生素H 6 ）是一种小水溶性分子，可用于化学修饰生物分子的活性胺，巯基和羧基。目前的细胞表面生物素化试剂主要由生物素或其衍生物的膜 - 不渗水磺化N-羟基琥珀酰亚胺（磺基-NHS）酯组成，其与设在蛋白质赖氨酸残基侧链上的胺反应，当细胞表面在碱性条件下脱质子时，后者与生物素部分7形成酰胺键。因此，可以通过使用抗生物素蛋白（一种对生物素具有极大亲和力的66-69kDa四聚体蛋白）来分离细胞表面蛋白，以约10-15的解离常数结合后者，将其标记为最强非共价相互作用已知8 </sup&gt; ， 9 。 在以前的研究中已经使用了许多量化蛋白质在细胞表面的方法。使用针对目的蛋白质特异性的荧光标记抗体进行标记，然后通过荧光显微镜观察是常用的方法，但是严重依赖于可结合细胞外表位的抗体的可用性。最近，已经成功地使用涉及使用与暴露于酸性介质反应的具有pH敏感性荧光团的嵌合蛋白质的方法。然而，这样的测定通常涉及这些构建体在其中未被天然存在的目的蛋白质的细胞系中的外源表达。这些方法仍然能够提供关于亚细胞定位和胞外步行的有价值的信息因此如果这些工具可用，则应与本文所述的基于生物素化的方法结合使用。 在典型的生物素化测定中，首先在4℃PBS中彻底洗涤细胞。这样可以消除由培养基引入的任何痕量的血清蛋白，从而确保在下一步中这些不会消耗过量的生物素。更重要的是，降低温度会导致内吞作用显着减速。然后加入生物素化试剂。接下来，再次洗涤细胞，然后与含有甘氨酸或NH 4 Cl的淬灭缓冲液一起温育，其目的是灭活所有剩余痕量的未反应的生物素。然后将细胞裂解，随后加入琼脂糖固定的链霉抗生物素蛋白以沉淀生物素化的蛋白质。分析通常通过western印迹进行，允许各种pr的相对细胞表面表达oteins要量化。 由于该测定的基础，它仅适用于具有暴露于细胞外环境的部分的蛋白质。可能在其环区内可能具有许多反应性赖氨酸的多巴跨膜蛋白是该方法中最容易的，而单程蛋白往往不太容易被生物素化。即使在这些情况下，仍然存在构象变化或分子间相互作用可能堵塞某些活性位点的可能性，导致低于预期的生物素化产率。 通过生物素化测定细胞表面蛋白的内在化率 该测定的原理与细胞表面生物素化的原理大致相同，除了一些例外，其中最重要的是使用可逆生物素化试剂。生物素组（其中的）具有disu在其结构内的易受还原剂影响的粘合剂;这被开发以确保仅在测定期间摄入细胞内部位的细胞表面蛋白质将被遗传生物素化。测定通常以以下方式进行。首先用冷试剂洗涤细胞并生物素化，然后重新引入37℃的细胞培养基，将细胞返回培养箱;这导致标记的细胞表面蛋白质经历内吞。然后加入不能穿透膜的还原剂谷胱甘肽，以破坏与残留在细胞表面上的蛋白质连接的生物素部分的二硫键。最后，破坏的二硫键与碘乙酰胺反应，消耗不稳定的硫醇基并防止键重组。如前所述，然后将细胞裂解，并使用链霉抗生物素蛋白 - 琼脂糖沉淀标记的蛋白质。 限制光盘由于在方法之间共享的相似之处，上一节中所使用的内容也适用于此。此外，值得铭记的是，该测定中涉及的温度变化阻止了对于每次增量的时间内含量多少蛋白质的确切测定，特别是在迅速内化或快速回收蛋白质的情况下。因此，该测定仅提供了内吞率的半定量估计。全内反射荧光显微镜可用于跟踪每个加载的囊泡的摄取，并提供更精确的内吞动力学测量。因此，假设目标蛋白的荧光标记嵌合构建体可用，因此可以提供非常有用的补体。

<table><tbody><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Fisher Scientific</td><td>A661-500</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647-50</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bio-Rad</td><td>#1610404</td><td></td></tr><tr><td>cOmplete protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>11697498001</td><td></td></tr><tr><td>Disodium Ethylenediaminetetraacetate dihydrate (EDTA)</td><td>Bio-Rad</td><td>#1610729</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Bio-Rad</td><td>#1610611</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco/Thermo Fisher Scientific</td><td>11960-044</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>#21335</td><td></td></tr><tr><td>EZ-Link Sulfo-NHS-SS-Biotin</td><td>Thermo Fisher Scientific</td><td>#21331</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco/Thermo Fisher Scientific</td><td>16000-044</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G8898&amp;nbsp;</td><td></td></tr><tr><td>Iodoacetamide</td><td>Bio-Rad</td><td>#163-2109</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma-Aldrich</td><td>L2020</td><td>Thaw on ice.</td></tr><tr><td>L-glutamine</td><td>Gibco/Thermo Fisher Scientific</td><td>25030-081</td><td></td></tr><tr><td>Mouse monoclonal anti-&amp;beta;-actin antibody (AC-15)</td><td>Sigma-Aldrich</td><td>A5441</td><td></td></tr><tr><td>Mouse monoclonal anti-&amp;beta;-dystroglycan antibody (43DAG1/8D5)</td><td>Leica Biosystems</td><td>B-DG-CE</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco/Thermo Fisher Scientific</td><td>15140-122</td><td></td></tr><tr><td>Peroxidase AffiniPure Donkey anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>715-035-150</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoResearch Laboratories</td><td>111-035-045</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Gibco/Thermo Fisher Scientific</td><td>10010-023</td><td></td></tr><tr><td>Reduced glutathione</td><td>Sigma-Aldrich</td><td>G6529</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-500</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Sigma-Aldrich</td><td>862010&amp;nbsp;</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318-100</td><td></td></tr><tr><td>Streptavidin agarose resin</td><td>Thermo Fisher Scientific</td><td>#20347</td><td></td></tr><tr><td>Rabbit polyclonal anti-AQP4 antibody</td><td>Alomone</td><td>AQP-004</td><td></td></tr><tr><td>Tris base (Trizma base)</td><td>Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher Scientific</td><td>BP153-1</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr></tbody></table>

determining cell-surface expression and endocytic rate of proteins in primary astrocyte cultures using biotinylation

细胞表面蛋白介导各种功能。在许多情况下，它们的活性受调节其在质膜上的水平的内吞过程的调节。在这里，我们提供了有助于研究这些过程的两种方法的详细方案，这两种方法均基于细胞表面蛋白的生物素化原理。第一个被设计为允许半定量测定细胞表面上特定蛋白质的相对水平。其中，细胞的质膜蛋白的赖氨酸残基首先用生物素部分标记。一旦细胞被裂解，然后可以通过利用琼脂糖固定的链霉亲和素通过利用后者对于生物素的天然亲和力来特异性沉淀这些蛋白质。然后可以通过标准的Western印迹方法分析以这种方式分离的蛋白质。第二种方法提供了确定特定细胞吞噬率的方法ar细胞表面目标在一段时间内。首先用含有可切割二硫键的生物素衍生物修饰细胞表面蛋白。然后将细胞转移回正常培养条件，这导致一部分生物素化蛋白质的内吞摄取。接下来，使用膜不可渗透的还原剂谷胱甘肽使非内化生物素基团的二硫键减少。通过这种方法，内吞的蛋白质因此可以以高度的特异性被分离和定量。

使用细胞表面生物素化来评估星形胶质细胞中AQP4的质膜表达 使用所述方法将层粘连蛋白处理的星形胶质细胞培养物和未处理的对照细胞进行细胞表面生物素化。用琼脂糖缀合的链霉亲和素沉淀生物素化的蛋白质，然后通过SDS-PAGE分离。探测细胞表面级分作为细胞表面加载对照的AQP4和β-dystroglycan（β-DG），同时将输入和细胞内级分吸入AQP4和β-肌动蛋白（ 图 1A ）作为加载对照。通过光密度测定法对细胞表面级分的带强度进行定量，并且首先针对β-DG值对每组细胞的AQP4水平进行归一化，然后将这些比值与对照细胞的那些标准化。直方图（ 图1 </strong&gt; B ）代表三次独立实验的平均值±SEM。平均来说，层粘连蛋白治疗导致在细胞表面表达的AQP4增加近2倍。 使用生物素化研究AQP4的吞噬率 在30分钟内追踪星形胶质细胞中的AQP4内吞作用。简言之，首先用可切割的生物素类似物标记3个星形胶质细胞的表面蛋白质。将细胞分别转移到37℃，分别为0,15和30分钟，在此期间标记的蛋白质被内化。然后使用还原剂除去表面上残留的标记，并用琼脂糖缀合的链霉抗生物素蛋白沉淀内吞的蛋白质。在通过SDS-PAGE分离后，然后将它们探针用于AQP4（ 图 2A）。使用光密度测定法对AQP4水平进行定量，15分钟和30分钟的时间点标准化为0分钟样品。直方图（ 图 2B ）表示四个这样的实验的平均值±SEM。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55974/55974fig1.jpg" /> 图1 。 AQP4在层粘连蛋白存在下在细胞表面的表达。 （A）用AQP4和β-DG检测来自未处理的星形胶质细胞（-LN）和用24nM层粘连蛋白-111（+ LN）处理的星形胶质细胞的细胞表面，细胞内和总（输入）级分。 （B）直方图，说明AQP4未处理和层粘连蛋白处理的星形胶质细胞的细胞表面水平差异，与β-DG水平相对应。值表示归一化的平均像素强度±SEM，相对于对照细胞的值表示。星号表示统计数字通过双尾学生t检验（n = 3，* p = 0.033）确定，AQP4的显着增加。这个数字已经从Tham 等人修改16 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55974/55974fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55974/55974fig2.jpg" /> 图2 。星形胶质细胞中的AQP4内化（A）在0,15和30分钟由星形胶质细胞培养物内化的蛋白质被探测用于AQP4。 （B）直方图总结4个独立实验的AQP4的内在化率，对0分钟时间点的值进行归一化。星号表示通过双尾确定的15和30分钟内吞的AQP4的量的统计学显着增加学生的t检验（n = 3，* p = 0.017）。 （C）当使用谷胱甘肽破坏可切割生物素类似物中的二硫键时，细胞表面AQP4对生物素化部分的贡献急剧降低，导致0和15分钟样品之间有明显差异（C，左上） 。当省略此步骤时，源自细胞表面池的信号可以使两个时间点之间的变化不可检测（C，右上） 。这个数字已经从Tham 等人修改16 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55974/55974fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation at JoVE.com

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

本报告提供了两种基于生物素化的方法，设计用于测定在质膜上表达的蛋白质的细胞表面表达和内吞率。

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

determining-cell-surface-expression-endocytic-rate-proteins-primary

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.5K 次观看.  University of British Columbia. 本报告提供了两种基于生物素化的方法，设计用于测定在质膜上表达的蛋白质的细胞表面表达和内吞率。 

视频: Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

神经科学

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

重组离子通道的相对细胞表面表达的总用流式细胞仪测定

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Cell-surface Biotinylation Assay

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or &#8220;recycled&#8221; back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.

细胞表面生物素化测定

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively ...

教育

JoVE Science Education

高级生物学

细胞生物学

Assessing the Internalization of  Target Surface Proteins Using a Biotin Derivative

Source: Tham, D. K. L., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55974">Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.</a> J. Vis. Exp. (2017).
This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

使用生物素衍生物评估靶表面蛋白的内化

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Determining Internalization ...