通过聚合-去聚合周期，通过有限来源的受控后翻译修改和异种型对图布林进行净化
Summary
本协议使用聚合和去聚合周期描述从中小型来源（如培养细胞或单个小鼠大脑）提纯的图布林。纯化的图布林富含特定的同位素或具有特定的翻译后修饰，可用于体外重组测定，以研究微管动力学和相互作用。
Abstract
微管细胞学研究的一个重要方面是在体外重组实验中对微管行为的研究。它们允许分析微管的内在特性，如动力学，以及它们与微管相关蛋白质（MAPs）的相互作用。”图布林代码”是一个新兴的概念，指向不同的图布林同位素同位素和各种翻译后修改 （PTM） 作为微管属性和功能的调节器。要探索图布林密码的分子机制，必须使用具有特定同位素和PTM的纯化图布林进行体外重组实验。
迄今为止，这在技术上是具有挑战性的，因为脑图布林，这是广泛应用于体外实验，窝藏许多PTM，并有一个定义的异构型组成。因此，我们开发了这种协议，利用聚合和去聚合周期的经典方法，从不同来源、不同同型成分和受控PTM中纯化图布林。与现有基于亲和力纯化的方法相比，该方法产生纯聚合能力强的图布林，因为在连续的净化步骤中，对聚合或去聚合具有抗性的图布林被丢弃。
我们描述从细胞系中提炼的图布林，无论是在悬浮中生长还是作为粘附培养物生长，以及从单一小鼠大脑中生长。该方法首先描述了在悬架和附定设置（裂解步骤）中细胞质量的生成，然后是通过聚合-去聚合周期进行图布林纯化的连续阶段。我们的方法产生图布林，可用于解决图布林代码对微管的内在特性和微管与相关蛋白质相互作用的影响的实验。
Introduction
微管在许多细胞过程中起着关键作用。它们为细胞提供形状，为染色体分离构建髓和线粒体主轴，并作为细胞内运输的轨道。为了执行这些不同的功能，微管以不同的方式组织自己。该领域的一个耐人寻味的问题是了解分子机制，使结构和进化保存的微管能够适应这种过多的组织和功能。一个潜在的机制是微管的多样化，这是由被称为”图布林代码“1，2，3的概念定义。图布林密码包括两个主要组成部分：将α和β图布林基因产物（图布林异构型）的微分结合到微管中，以及图布林翻译后修饰（PTM）。
自20世纪70年代以来，体外重组实验结合不断发展的光显微镜技术，为有关微管特性的重要发现铺平了道路：动态不稳定4和跑步5，以及它们的其他机制和功能6、7、8、9、10、11、12、13、14、15。到目前为止，几乎所有的体外实验都是基于使用重复的聚合和去聚合周期从脑组织中纯化的图布林16，17。虽然从脑组织中提纯赋予了大量（通常克量）获得高质量图布林的优势，但一个重要的缺点是异质性，因为从脑组织中纯化的图布林是不同图布林异种型的混合物，并富含许多图布林PTM。这种异质性使得无法描述特定的图布林PTM或同位素在微管特性和功能控制中的作用。因此，生产具有受控图布林PTM和同质异型成分的组装能力图布林对于解决图布林代码的分子机制至关重要。
最近，一种利用酵母Stu2p的微管结合托格（肿瘤过度表达基因）域的亲和色谱净化图布林的方法已经开发出来。在这种方法中，细胞或组织粗裂解的图布林通过一个柱子，它与矩阵固定的托格域结合，从而能够分析给定甚至非常小的样品的整个图布林池。近年来，人们还描述了一种期待已久的净化重组管蛋白的方法。它基于巴库洛病毒系统，其中含有α和β-图布林基因的双柠檬载体在昆虫细胞19中表达。然而，这种方法是非常繁琐和耗时的，因此主要用于研究图布林突变的影响20和图布林等型21，22，23体外。
在目前的协议中，我们描述了一种方法，它使用成熟和广泛使用的聚合去聚化方法作为蓝图，从细胞系或小鼠脑组织24产生不同程度的修饰图布林。在此过程中，图布林在可溶性（4 °C的图布林二毛钱）和聚合形式（在瓜诺辛 5′-三磷酸 [GTP] 存在的情况下在 30 °C 处的微管）之间循环。每种形式都通过连续的离心步骤分离：在冷（4 °C）旋转后，图布林调光器将留在超自然体中，而微管将在30°C下喷出。 此外，在高管氨酸-N，N+-bis（2-乙醇离子酸）（PIPES）浓度下进行一个聚合步骤，允许从微管中去除微管相关蛋白质，从而从最终纯化的图布林中去除。从 HeLa S3 细胞中纯化为悬浮或粘附培养物的 Tubulin 几乎没有任何图布林 PTM，并已用于最近的体外重组实验25，26，27，28.我们进一步调整了从单只小鼠大脑中净化图布林的方法，可用于大量小鼠模型，这些模型具有图布林异种类型和PTM的变化。
在协议中，我们首先描述了源材料（细胞质量或脑组织）的生成，其裂解（图1A），然后是图布林聚合和去聚合的连续步骤，以净化图布林（图1B）。我们进一步描述了评估纯化吐布林的纯度（图2A，B）和数量（图3A，B）的过程。该方法可以通过在图布林纯化（图4B）之前在细胞中过度表达修改酶来生产富含选PTM的图布林。或者，在净化过程中，可以将调子蛋白修改酶添加到图布林中。最后，我们可以从缺乏相应图布林修饰酶（图4B）29的小鼠大脑中提炼出缺乏特定同型或PTM的图蛋白。
我们在这里描述的方法有两个主要优点：（一） 它允许在相对较短的时间内生产足够大量的图布林， （ii） 它产生高品质， 纯图布林， 与特定的图布林异型组成或PTM.在本手稿的相关视频中，我们重点介绍了此程序中涉及的一些关键步骤。
Protocol
根据欧洲共同体的建议（2010/63/UE），对这项研究进行动物护理和使用。实验程序是居里CEEA-IC#118（国家管理局授权号04395.03）根据国际准则特别批准的。 1. 为图布林净化准备试剂 注：所有用于图布林净化的缓冲剂应含有钾盐，而不是钠盐30。 准备 1 L 的完整介质：杜尔贝科的改良鹰介质 （DMEM）， 10% 胎儿牛血清 （FBS， 100 mL）， …
Representative Results
该方法的主要目标是生产出质量高、装配能力强的图布林，其数量足以对纯化成分进行反复的体外实验。从这种管蛋白组装的微管可用于重组测定，基于全内部反射荧光（TIRF）显微镜技术与动态或稳定的微管，在实验测试微管动力学，与MAP或分子电机的相互作用，并强制发电的电机25。它们还可用于微管-MAP共聚检测和固态NMR光谱28。 在整?…
Discussion
这里描述的方法提供了一个平台，从细胞线和单个鼠标大脑中快速产生高质量、具有组装能力的中量图布林。它是基于黄金标准协议的图布林纯化牛脑用于该领域多年16，17。该方法的一个特别优点是使用 HeLa S3 细胞的悬浮培养物，一旦建立，就会产生大量的细胞，同时几乎不需要动手时间。这使得该协议在任何细胞生物学实验室中都相对容易执行，而?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了ANR-10-IDEX-0001-02、LabEx单元’n’Scale ANR-11-LBX-0038和收敛Q-life ANR-17-CONV-0005研究所的支持。CJ由居里研究所支持，法国国家研究机构（ANR）授予ANR-12-BSV2-0007和ANR-17-CE13-0021， 国家癌症学院（INCA）赠款2014-PL BIO-11-ICR-1，以及雷彻切医学基金会（FRM）赠款DEQ20170336756。MMM由Vaincre老年痴呆症基金会赠款FR-16055p支持，并得到法国阿尔茨海默氏症赠款AAP SM 2019 n+2023的支持。JAS 得到了欧盟”地平线 2020″研究和创新计划的支持，该计划由玛丽·斯考多夫斯卡-库里赠款协议第 675737 号和 FRM 赠款 FDT201904008210 提供。SB 得到了 FRM 赠款 FDT201805005465 的支持。
我们感谢Janke实验室的所有成员，特别是J.苏普龙，以及G.拉基西奇（米卡利斯研究所、农业巴黎技术研究所）和A.高特劳（理工学院）在制定议定书期间的帮助。我们要感谢居里动物设施在老鼠繁殖和护理方面所提供帮助。
抗体12G10由J.弗兰克尔和M.纳尔逊开发，来自由NICCHD赞助、爱荷华大学维护的发展研究杂交瘤银行。
Materials
| 1 M MgCl2 | Sigma | #M1028 | |
| 1-L cell culture vessels | Techne F7610 | Used for spinner cultures. Never stir the empty spinner bottles. When spinner bottles are in the cell culture incubator, always keep the lateral valves of spinner bottles slightly open to facilitate the equilibration of media with incubator’s atmosphere. After use, fill the spinner bottles immediately with tap water to avoid drying of remaining cells on the bottle walls. Wash the bottles with deionised water, add app 200 ml of deionised water and autoclave. Under a sterile cell culture hood remove the water and allow the bottles to dry completely, still under the hood, for several hours. Never use detergents for cleaning the spinner bottles because any trace amounts of the detergent can be deleterious to the cells. | |
| 1.5- and 2-ml tubes | |||
| 14-ml round-bottom tubes | |||
| 15-cm-diameter sterile culture dishes | |||
| 15-ml screw-cap tubes | |||
| 2-mercaptoethanol | Sigma | #M3148 | 2-mercaptoethanol is toxic and should be used under the hood. |
| 4-(2-aminoethyl)-benzenesulfonyl fluoride | Sigma | #A8456 | |
| 40% Acrylamide | Bio-Rad | #161-0140 | |
| 5-, 10- 20-ml syringes | |||
| 5-ml, 10-ml, 25-ml sterile pipettes | |||
| 50-ml screw-cap tubes | |||
| Ammonium persulfate (APS) | Sigma | #A3678 | |
| Anti-alpha-tubulin antibody, 12G10 | Developed by J. Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa | dilution: 1/500 | |
| Anti-glutamylated tubulin antibody, GT335 | AdipoGen | #AG-20B-0020 | dilution: 1/20,000 |
| Aprotinin | Sigma | #A1153 | |
| Balance (0.1 – 10 g) | |||
| Beckman 1-l polypropylene bottles | For collecting spinner cultures | ||
| Beckman Avanti J-26 XP centrifuge | For collecting spinner cultures | ||
| Biological stirrer | Techne MCS-104L | Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells | |
| Bis N,N’-Methylene-Bis-Acrylamide | Bio-Rad | #161-0201 | |
| Blender IKA Ultra-Turrax® | For lysing brain tissue, use 5-mm probe, with the machine set at power 6 or 7. Blend the brain tissue 2-3 times for 15 s on ice. | ||
| Bovine serum albumin (BSA) | Sigma | #A7906 | |
| Bromophenol blue | Sigma | #1.08122 | |
| Carboxypeptidase A (CPA) | Sigma | #C9268 | Concentration: 1.7 U/µl |
| Cell culture hood | |||
| Cell culture incubator set at 37°C, 5% CO2 | |||
| Dimethyl sulfoxide (DMSO) | Sigma | #D8418 | DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection. |
| DMEM medium | Life Technologies | #41965062 | |
| DTT, DL-Dithiothreitol | Sigma | #D9779 | |
| EDTA | Euromedex | #EU0007-C | |
| EGTA | Sigma | #E3889 | |
| Ethanol absolute | Fisher Chemical | #E/0650DF/15 | |
| Fetal bovine serum (FBS) | Sigma | #F7524 | |
| French pressure cell press | Thermo electron corporation | #FA-078A | with a #FA-032 cell; for lysing big amounts of cells. Set at medium ratio, and the gauge pressure of 1,000 psi (corresponds to 3,000 psi inside the disruption chamber). |
| Glycerol | VWR Chemicals | #24388.295 | |
| Glycine | Sigma | #G8898 | |
| GTP | Sigma | #G8877 | |
| Heating block | Stuart | #SBH130D | |
| Hela cells | ATCC® CCL-2™ | ||
| Hela S3 cells | ATCC | ATCC® CCL-2.2™ | |
| Hydrochloric acid (HCl ) | VWR | #20252.290 | |
| Inverted microscope | With fluorescence if cell transfection is to be verified | ||
| Isopropanol | VWR | #20842.298 | |
| jetPEI | Polyplus | #101 | |
| JLA-8.1000 rotor | For collecting spinner cultures | ||
| KOH | Sigma | #P1767 | KOH is corrosive and causes burns; use eye and skin protection. |
| L-Glutamine | Life Technologies | #25030123 | |
| Laboratory centrifuge for 50-ml tubes | Sigma | 4-16 K | |
| Leupeptin | Sigma | #L2884 | |
| Liquid nitrogen | |||
| Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips | |||
| Micropestles | Eppendorf | #0030 120.973 | |
| Mouse brain tissue | Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines. | ||
| Needles 18G X 1 ½” (1.2 X 38 mm | Terumo | #18G | |
| Needles 20G X 1 ½” (0.9 X 38 mm | Terumo | #20G | |
| Needles 21G X 4 ¾” (0.8 X 120 mm | B.Braun | #466 5643 | |
| Parafilm | |||
| PBS | Life Technologies | #14190169 | |
| Penicillin-Streptomycin | Life Technologies | #15140130 | |
| pH-meter | |||
| Phenylmethanesulfonyl fluoride (PMSF) | Sigma | #P7626 | PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions. |
| PIPES | Sigma | #P6757 | |
| Pipette-boy | |||
| Rotors | Beckman 70.1 Ti; TLA-100.3; and TLA 55 | ||
| SDS-PAGE electrophoresis equipment | Bio-Rad | #1658001FC | |
| SDS, Sodium dodecyl sulphate | VWR | #442444H | For preparing Laemmeli buffer |
| SDS, Sodium dodecyl sulphate | Sigma | #L5750 | For preparing 'TUB' SDS-PAGE gels |
| Sonicator | Branson | #101-148-070 | Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at “Output control” 1, “Duty cycle” 10% and time depending on the cell type used. |
| Tabletop centrifuge for 1.5 ml tubes | Eppendorf | 5417R | |
| TEMED, N, N, N′, N′-Tetramethylethylenediamine | Sigma | #9281 | |
| Trichostatin A (TSA) | Sigma | #T8552 | |
| Triton X-100 | Sigma | #T9284 | |
| Trizma base (Tris) | Sigma | #T1503 | |
| Trypsin | Life Technologies | #15090046 | |
| Ultracentrifuge rotors | TLA-55, TLA-100.3 and 70.1 Ti rotors | Set at 4°C or 30°C based on the need of the experiment | |
| Ultracentrifuge tubes | Beckman | #357448 | for using with TLA-55 rotor |
| Ultracentrifuge tubes | Beckman | #349622 | for using with TLA-100.3 rotor |
| Ultracentrifuge tubes | Beckman | #355631 | for using with 70.1 Ti rotor |
| Ultracentrifuges | Beckman | Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent) | Set at 4°C or 30°C based on the need of the experiment |
| Vortex mixer | |||
| Water bath equipped with floaters or tube holders | Set at 30°C |
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Diesen Artikel zitieren
Bodakuntla, S., Jijumon, A., Janke, C., Magiera, M. M. Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles. J. Vis. Exp. (165), e61826, doi:10.3791/61826 (2020).