Name: purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles
Uploaded: 2020-11-05T15:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

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。

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作者没有什么可透露的。

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?...

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。

<table><tbody><tr><td>1&amp;nbsp;M MgCl2&amp;nbsp;</td><td>Sigma</td><td>#M1028</td><td></td></tr><tr><td>1-L cell culture vessels</td><td>Techne F7610&amp;nbsp;</td><td></td><td>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&amp;rsquo;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.</td></tr><tr><td>1.5- and 2-ml tubes</td><td></td><td></td><td></td></tr><tr><td>14-ml round-bottom tubes</td><td></td><td></td><td></td></tr><tr><td>15-cm-diameter sterile culture dishes</td><td></td><td></td><td></td></tr><tr><td>15-ml screw-cap tubes</td><td></td><td></td><td></td></tr><tr><td>2-mercaptoethanol&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#M3148</td><td>2-mercaptoethanol is toxic and should be used under the hood.</td></tr><tr><td>4-(2-aminoethyl)-benzenesulfonyl fluoride&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#A8456</td><td></td></tr><tr><td>40% Acrylamide&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>#161-0140</td><td></td></tr><tr><td>5-, 10- 20-ml syringes</td><td></td><td></td><td></td></tr><tr><td>5-ml, 10-ml, 25-ml sterile pipettes</td><td></td><td></td><td></td></tr><tr><td>50-ml screw-cap tubes</td><td></td><td></td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Sigma</td><td>#A3678</td><td></td></tr><tr><td>Anti-alpha-tubulin antibody, 12G10&amp;nbsp;</td><td>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</td><td></td><td>dilution: 1/500</td></tr><tr><td>Anti-glutamylated tubulin antibody, GT335&amp;nbsp;</td><td>AdipoGen&amp;nbsp;</td><td>#AG-20B-0020</td><td>dilution: 1/20,000</td></tr><tr><td>Aprotinin&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#A1153</td><td></td></tr><tr><td>Balance (0.1 &amp;ndash; 10 g)</td><td></td><td></td><td></td></tr><tr><td>Beckman 1-l polypropylene bottles&amp;nbsp;</td><td></td><td></td><td>For collecting spinner cultures</td></tr><tr><td>Beckman Avanti J-26 XP centrifuge</td><td></td><td></td><td>For collecting spinner cultures</td></tr><tr><td>Biological stirrer&amp;nbsp;</td><td>Techne MCS-104L&amp;nbsp;</td><td></td><td>Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells</td></tr><tr><td>Bis N,N&amp;rsquo;-Methylene-Bis-Acrylamide&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>#161-0201</td><td></td></tr><tr><td>Blender IKA Ultra-Turrax&amp;reg;&amp;nbsp;</td><td></td><td></td><td>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.</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>#A7906</td><td></td></tr><tr><td>Bromophenol blue&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#1.08122</td><td></td></tr><tr><td>Carboxypeptidase A (CPA)</td><td>Sigma</td><td>#C9268</td><td>Concentration: 1.7&amp;nbsp;U/&amp;micro;l</td></tr><tr><td>Cell culture hood</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator set at 37&amp;deg;C, 5% CO2</td><td></td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>&amp;nbsp;Sigma&amp;nbsp;</td><td>#D8418</td><td>DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection.</td></tr><tr><td>DMEM medium&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#41965062</td><td></td></tr><tr><td>DTT, DL-Dithiothreitol&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#D9779</td><td></td></tr><tr><td>EDTA</td><td>Euromedex</td><td>#EU0007-C</td><td></td></tr><tr><td>EGTA</td><td>Sigma&amp;nbsp;</td><td>#E3889</td><td></td></tr><tr><td>Ethanol absolute&amp;nbsp;</td><td>Fisher Chemical&amp;nbsp;</td><td>#E/0650DF/15</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma&amp;nbsp;</td><td>#F7524</td><td></td></tr><tr><td>French pressure cell press&amp;nbsp;</td><td>Thermo electron corporation</td><td>&amp;nbsp;#FA-078A</td><td>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).</td></tr><tr><td>Glycerol</td><td>&amp;nbsp;VWR Chemicals&amp;nbsp;</td><td>#24388.295</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>#G8898</td><td></td></tr><tr><td>GTP&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#G8877</td><td></td></tr><tr><td>Heating block&amp;nbsp;</td><td>Stuart&amp;nbsp;</td><td>#SBH130D</td><td></td></tr><tr><td>Hela cells&amp;nbsp;</td><td></td><td>ATCC&amp;reg; CCL-2&amp;trade;</td><td></td></tr><tr><td>Hela S3 cells&amp;nbsp;</td><td>ATCC</td><td>ATCC&amp;reg; CCL-2.2&amp;trade;</td><td></td></tr><tr><td>Hydrochloric acid (HCl )</td><td>VWR</td><td>#20252.290</td><td></td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td></td><td></td><td>With fluorescence if cell transfection is to be verified</td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>#20842.298</td><td></td></tr><tr><td>jetPEI</td><td>Polyplus&amp;nbsp;</td><td>#101</td><td></td></tr><tr><td>JLA-8.1000 rotor&amp;nbsp;</td><td></td><td></td><td>For collecting spinner cultures</td></tr><tr><td>KOH&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#P1767</td><td>KOH is corrosive and causes burns; use eye and skin protection.</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#25030123</td><td></td></tr><tr><td>Laboratory centrifuge for 50-ml tubes</td><td>Sigma</td><td>4-16 K&amp;nbsp;</td><td></td></tr><tr><td>Leupeptin&amp;nbsp;</td><td>Sigma</td><td>#L2884</td><td></td></tr><tr><td>Liquid nitrogen&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips</td><td></td><td></td><td></td></tr><tr><td>Micropestles</td><td>Eppendorf</td><td>#0030 120.973</td><td></td></tr><tr><td>Mouse brain tissue&amp;nbsp;</td><td></td><td></td><td>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.</td></tr><tr><td>Needles 18G X 1 &amp;frac12;&amp;rdquo; (1.2 X 38 mm</td><td>Terumo</td><td>#18G</td><td></td></tr><tr><td>Needles 20G X 1 &amp;frac12;&amp;rdquo; (0.9 X 38 mm</td><td>Terumo</td><td>#20G</td><td></td></tr><tr><td>Needles 21G X 4 &amp;frac34;&amp;rdquo; (0.8 X 120 mm</td><td>B.Braun</td><td>#466 5643</td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#14190169</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>#15140130</td><td></td></tr><tr><td>pH-meter</td><td></td><td></td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>Sigma&amp;nbsp;</td><td>#P7626</td><td>PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions.</td></tr><tr><td>PIPES&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#P6757</td><td></td></tr><tr><td>Pipette-boy</td><td></td><td></td><td></td></tr><tr><td>Rotors</td><td>Beckman 70.1 Ti; TLA-100.3; and TLA 55</td><td></td><td></td></tr><tr><td>SDS-PAGE electrophoresis equipment&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>#1658001FC</td><td></td></tr><tr><td>SDS, Sodium dodecyl sulphate&amp;nbsp;</td><td>VWR&amp;nbsp;</td><td>#442444H</td><td>For preparing Laemmeli buffer&amp;nbsp;</td></tr><tr><td>SDS, Sodium dodecyl sulphate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#L5750</td><td>For preparing &#039;TUB&#039; SDS-PAGE gels</td></tr><tr><td>Sonicator&amp;nbsp;</td><td>Branson</td><td>#101-148-070</td><td>Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at &amp;ldquo;Output control&amp;rdquo; 1, &amp;ldquo;Duty cycle&amp;rdquo; 10% and time depending on the cell type used.</td></tr><tr><td>Tabletop centrifuge for 1.5 ml tubes</td><td>Eppendorf</td><td>5417R&amp;nbsp;</td><td></td></tr><tr><td>TEMED, N, N, N&amp;prime;, N&amp;prime;-Tetramethylethylenediamine&amp;nbsp;</td><td>Sigma</td><td>#9281</td><td></td></tr><tr><td>Trichostatin A (TSA)</td><td>Sigma</td><td>#T8552</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>#T9284</td><td></td></tr><tr><td>Trizma base (Tris)</td><td>Sigma&amp;nbsp;</td><td>#T1503</td><td></td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Life Technologies</td><td>#15090046</td><td></td></tr><tr><td>Ultracentrifuge rotors&amp;nbsp;</td><td></td><td>TLA-55, TLA-100.3 and 70.1 Ti rotors</td><td>Set at 4&amp;deg;C or 30&amp;deg;C based on the need of the experiment&amp;nbsp;</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>#357448&amp;nbsp;</td><td>for using with TLA-55 rotor</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>#349622</td><td>for using with TLA-100.3 rotor</td></tr><tr><td>Ultracentrifuge tubes&amp;nbsp;</td><td>Beckman</td><td>#355631&amp;nbsp;</td><td>for using with 70.1 Ti rotor</td></tr><tr><td>Ultracentrifuges</td><td>Beckman</td><td>Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent)</td><td>Set at 4&amp;deg;C or 30&amp;deg;C based on the need of the experiment&amp;nbsp;</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Water bath equipped with floaters or tube holders</td><td></td><td></td><td>Set at 30&amp;deg;C&amp;nbsp;</td></tr></tbody></table>

purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles

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。

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?...

Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

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。

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

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

purification-tubulin-with-controlled-posttranslational-modifications

Research

JoVE Journal

Biochemistry

5.0K 次观看.  Institut Curie. 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。

视频: Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

科研

生物化学

Controlling Flow Speeds of Microtubule-Based 3D Active Fluids Using Temperature

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method allows for tuning the speeds in situ without the need to manufacture new samples to reach different desired speeds. Moreover, this method enables the dynamic control of speed. Cycling the temperature leads the fluids to flow fast and slow, periodically. This controllability is based on the Arrhenius characteristic of the kinesin-microtubule reaction, demonstrating a controlled mean flow speed range of 4&#8211;8 &#181;m/s. The presented method will open the door to the design of microfluidic devices where the flow rates in the channel are locally tunable without the need for a valve.

The goal of this protocol is to use temperature to control the flow speeds of three-dimensional active fluids. The advantage of this method not only allows for regulating flow speeds in situ but also enables dynamic control, such as periodically tuning flow speeds up and down.

使用温度控制基于微管的3D活性流体的流速

We present a method for using temperature to tune the flow speeds of kinesin-driven, microtubule-based three-dimensional (3D) active fluids. This method ...

工程学

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

微管触觉的自组装

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Extracting Modified Microtubules from Mammalian Cells to Study Microtubule-Protein Complexes by Cryo-Electron Microscopy

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the posttranslational modifications, microtubules can form complexes with various interacting proteins. These microtubule-protein complexes are often implicated in human diseases. Understanding the structure of such complexes is useful for elucidating their mechanisms of action and can be studied by cryo-electron microscopy (cryo-EM). To obtain such complexes for structural studies, it is important to extract microtubules containing or lacking specific posttranslational modifications. Here, we describe a simplified protocol to extract endogenous tubulin from genetically modified mammalian cells, involving microtubule polymerization, followed by sedimentation using ultracentrifugation. The extracted tubulin can then be used to prepare cryo-electron microscope grids with microtubules that are bound to a purified microtubule-binding protein of interest. As an example, we demonstrate the extraction of fully tyrosinated microtubules from cell lines engineered to lack the three known tubulin-detyrosinating enzymes. These microtubules are then used to make a protein complex with enzymatically inactive microtubule-associated tubulin detyrosinase on cryo-EM grids.

Here, we describe a protocol to extract endogenous tubulin from mammalian cells, which can lack or contain specific microtubule-modifying enzymes, to obtain microtubules enriched for a specific modification. We then describe how the extracted microtubules can be decorated with purified microtubule-binding proteins to prepare grids for cryo-electron microscopy.

从哺乳动物细胞中提取修饰的微管以通过冷冻电子显微镜研究微管 - 蛋白质复合物

Microtubules are an important part of the cytoskeleton and are involved in intracellular organization, cell division, and migration. Depending on the ...

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects.
Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues.
The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 &#215; g) to remove debris. The first ultracentrifugation step (100,000 &#215; g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 &#215; g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Microtubules, which are tubulin polymers, play a crucial role as a cytoskeleton component in eukaryotic cells and are known for their dynamic instability. This study developed a method for fractionating microtubules to separate them into stable microtubules, labile microtubules, and free tubulin to evaluate the stability of microtubules in various mouse tissues.

定量微管分离技术分离小鼠组织中稳定的微管、不稳定的微管和游离微管蛋白

Microtubules, composed of &#945;/&#946;-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit ...

<meta charset="utf8"></head><body>从神经组织中高效分离微管蛋白

Optimizing Tubulin Yield from Porcine Brain Tissue

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we present a procedure for extracting tubulin that can be employed, with minor modifications, for neural tissue from multiple sources. In the presented protocol, a new clarification step of the crude lysate has been introduced, which led to a significant reduction in the initial amount of insoluble debris before the first polymerization step occurred. This additional step allowed the processing of additional tissue while using the same instrumentation and, thus, increasing the relative volume of the processed homogenate. The newly introduced step has no significant effect on the quality of purified tubulin, as confirmed by in vitro activity assays and transmission electron microscopy. The described procedure contains all crucial steps, including tissue collection, transport, tissue homogenization, tubulin isolation cycles, and final polishing by ion exchange chromatography using FPLC and subsequent activity measurement assays. The homogeneity of purified tubulin was more than 97%, as confirmed by MS/MS analysis utilizing electrospray ionization and MALDI-TOF.

<meta charset="utf8"></head><body>作者聚焦：纯化高质量微管蛋白以研究蛋白质动力学和治疗应用

This protocol describes a technique for the high-yield isolation of tubulin from the porcine brain optimized for small-scale instrumentation. The isolation procedures are complemented by procedures for determining tubulin polymerization activity in vitro using co-sedimentation assays and transmission electron microscopy.

优化猪脑组织的微管蛋白产量

Neural tissue from any source is an excellent material for tubulin isolation, as the dendrites and axons of neurons are rich in microtubules. Here, we ...

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

摘要

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此视频中的章节

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

使用温度控制基于微管的3D活性流体的流速

通过TIRF显微镜在体外同时可视化交联和单微管的动力学

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

微管触觉的自组装

从哺乳动物细胞中提取修饰的微管以通过冷冻电子显微镜研究微管 - 蛋白质复合物

定量微管分离技术分离小鼠组织中稳定的微管、不稳定的微管和游离微管蛋白

优化猪脑组织的微管蛋白产量

优化猪脑组织的微管蛋白产量

优化猪脑组织的微管蛋白产量

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

摘要

探索更多视频

此视频中的章节

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

使用温度控制基于微管的3D活性流体的流速

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Sf9纯化超加工驱动蛋白-3系列电机的单分子分析

微管触觉的自组装

从哺乳动物细胞中提取修饰的微管以通过冷冻电子显微镜研究微管 - 蛋白质复合物

定量微管分离技术分离小鼠组织中稳定的微管、不稳定的微管和游离微管蛋白

优化猪脑组织的微管蛋白产量

优化猪脑组织的微管蛋白产量

优化猪脑组织的微管蛋白产量

通过TIRF显微镜在体外同时可视化交联和单微管的动力学