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Mar 2020

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Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues
存在不良成核核苷酸类似物的微管种晶组装   

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Abstract

Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy.

Keywords: Tubulin (微管蛋白), Microtubules (微管), GTP analogues (GTP类似物), Microtubule nucleation (微管形成), Seeded microtubule assembly (微管种晶组装), Spectrophotometry (分光光度法), Cryo-electron microscopy (冷冻电子显微技术)

Background

Nucleotide analogues are commonly used to investigate the conformational changes that the α-β tubulin heterodimer undergoes during microtubule assembly and hydrolysis of the GTP bound to its exchangeable (E) site. However, apart from GMPCPP, most analogues are poor microtubule nucleators. To overcome this difficulty, GMPCPP-stabilized seeds are classically used to elongate microtubules in the presence of other analogues (Maurer et al., 2011 and 2014; Zhang et al., 2015), resulting in the assembly of mixed nucleotide-bound lattices. Alternatively, glycerol at relatively high concentration (e.g., 25%) can be added to the reaction mix to stimulate tubulin self-assembly. However, high glycerol concentrations might not be desirable, for instance to analyze microtubule structure by cryo-electron microscopy, since this compound matches the protein density and reduces the image contrast.

To avoid the use of GMPCPP-seeds or high glycerol concentrations in the final reaction mix, we have developed a protocol that uses seeds assembled in the presence of the same nucleotides that will be used to assemble the microtubules. As a first step, glycerol is used to polymerize microtubules in the presence of the nucleotide analogue. These microtubules are then fragmented by sonication to produce seeds, which are further diluted in the tubulin mix in the absence of glycerol. Microtubules elongated onto these seeds can be analyzed by cryo-electron microscopy without the risk to mistake microtubules assembled in the presence of GMPCPP with those assembled in the presence of the nucleotide analogue of interest.

Our protocol describes the step-by-step preparation of the seeds and the assembly of microtubules, which is monitored by turbidimetry at 350 nm. It includes a cooling step at the end of the assembly process to assess the presence of depolymerizing microtubules. These conditions were used to assemble GDP-BeF3--microtubules, whose lattices were further characterized by cryo-electron microscopy (Estévez-Gallego et al., 2020). We have successfully applied this protocol to microtubules assembled in the presence of other analogues such as GTPγS, GDP-AlF3, GMPPCP and GMPCP, all of them being poor microtubule nucleators.

Materials and Reagents

  1. Pipette tips
  2. Open-top thinwall polypropylene centrifuge tubes, 175 μl, 5 x 20 mm (Beckman Coulter, catalog number: 342630 )
  3. Beryllium sulfate (BeSO4·4H2O) (Sigma-Aldrich, catalog number: 14270 )
  4. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  5. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  6. Guanosine 5’-triphosphate sodium salt hydrate (GTP) (Sigma-Aldrich, catalog number: G8877 )
  7. Magnesium chloride solution (MgCl2) (Sigma-Aldrich, catalog number: 63069 )
  8. Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757 )
  9. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 30603 )
  10. Sodium Fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
  11. BRB80 buffer solution 5x (see Recipes)
  12. GDP solution (see Recipes)
  13. Tubulin stocks (see Recipes)
  14. NaF solution (see Recipes)
  15. BeSO4 solution (see Recipes)
  16. G-BRB80-BeF3- (see Recipes)
  17. BRB80-BeF3- solution (see Recipes)

Equipment

  1. Pipetman Classic P100, P20, P10 (Gilson, catalog numbers: F123615 , F123600 , F144802 )
  2. Airfuge Air-Driven (Beckmann Coulter, catalog number: 340401 )
  3. Fixed-angle rotor A-110 (Beckman Coulter, catalog number: 347596 )
  4. Refrigerator MEDIKA 100 ECT-F (fiocchetti)
  5. Compressed air cooler BEKOBLITZZ LC12 (Beko Technologies, catalog number: 4020103 )
  6. Compressed air filter Clearpoint, 1 μm (Beko Technologies, catalog number: S040SWT )
  7. Membrane dryer Drypoint M plus (Beko Technologies, catalog number: 4007626 )
  8. Condensate drain ECD model ED12
  9. B2000 Ultrasonic Cleaner (Branson)
  10. Dri-Block heater DB-2D (Techne)
  11. Spectrophotometer Uvikon XS (Secomam, Bio-Tek Instruments)
  12. Quartz cuvette Ultra-Micro cells (Hellma, catalog number: 105-201-15-40 )
  13. Julabo F30-C (Julabo) with Thermal HY fluid, -60 °C to +65 °C, (Julabo, catalog number: 8940104 )
  14. Julabo MP-5 (Julabo) with Thermal HY fluid, -60 °C to +65 °C, (Julabo, catalog number: 8940104 )
  15. Three-way valves, PFTE (Bürkle, catalog number: 8610-0006 )

Software

  1. LabPower v3.50 (Secomam, Bio-Tek Instruments)
  2. Excel (Microsoft)
  3. Affinity Designer (Serif Ltd)

Procedure

  1. Spectrophotomer preparation
    1. Switch on the spectrophotometer and make sure that it uses the UV lamp at 350 nm.
      Note: The default value to switch from the UV (D2) to the halogen lamp is 340 nm on the Uvikon XS. This value must be changed to, e.g., 360 nm, since the UV lamp must be used with the Ultra-Micro-cell quartz cuvettes at 350 nm.
    2. Select the autorate mode at 350 nm.
    3. Switch on the warm water bath, set the temperature to 35 °C and let equilibrate for 15 min.
      Note: The temperatures of the warm and cold circulating baths must be adjusted using a small thermocouple inside the quartz cuvette filled with water. The goal is to reach 35 °C and 4 °C for polymerization and depolymerization conditions, respectively. Variations in ambient temperature ( the cold water bath, set the temperature to 2 °C and let equilibrate for 30 min.e.g., between summer and winter) may require periodic control and adjustment of these settings. Do not exceed 35 °C during polymerization, since tubulin denaturates easily above this temperature (Weis et al., 2010).
    4. Switch on the cold water bath, set the temperature to 2 °C and let equilibrate for 30 min.
    5. Circulate the 35 °C fluid inside the spectrophotometer thermostated cell holder (Figure 1).


      Figure 1. Temperature control of the spectrophotometer. A. Two water baths regulated at 35 °C and 2 °C are connected to the thermostated 6+6 cell changer of the spectrophotometer. Three-ways valves allow rapid switch of the temperature from 35 °C (bottom) to 4 °C (top). The temperature of the cold water bath must be set below 4 °C due to warming up of the fluid in the tubing system. The actual warm and cold temperatures must be checked regularly inside the cell holder using a small thermocouple. B. Warm (left) and cold (right) bather baths. C. Three-way valves. These must be switched simultaneously.

  2. Microtubule seeds preparation (for 30 µl)
    1. Thaw rapidly between fingers a 10 μl aliquot of tubulin (initial concentration 22 mg/ml) and keep at 4 °C until use.
      Note: Tubulin was purified from porcine brains by two cycles of assembly in a high-molarity buffer (Castoldi and Popov, 2003), followed by a cycling step to obtain tubulin liganded with GDP at the E-site (Ashford et al., 1998). Cycled tubulin is commercially available, e.g., from PurSolution (https://puresoluble.com/cycled-tubulin/).Tubulin at a final concentration of 22 mg/ml was aliquoted in 100 µl and 10 µl aliquots, snap frozen in liquid nitrogen, and stored at -80 °C until use. Once tubulin has been defrost, try to minimize all steps until polymerization since it will rapidly denaturates if kept in its unassembled form, even at 4 °C.
    2. Add 5.5 µl of the tubulin solution to 24.5 μl of G-BRB80-BeF3- in a polypropylene centrifuge tube.
    3. Centrifuge the tube in the Airfuge at 141,000 x g (A-110 rotor, 22 psig), 5 min, 4 °C (Figure 2).
      Note: The airfuge is not regulated in temperature. It must be placed in a cold room, or alternatively in a freezer such as in Figure 2. In both cases, the incoming air flow must also be cooled, otherwise the rotor will warm up (see manufacturer’s instructions if installed in a cold room).
    4. Incubate the supernatant in the dry block heater, 1 h, 35 °C.
    5. Sonicate the microtubules in the ultrasonic cleaner, 1 min.
    6. Keep microtubule seeds at 35 °C until use in the following step.


      Figure 2. Temperature control of the Airfuge. The Airfuge is maintained at 4 °C inside a refrigerator. The incoming compressed air must also be cooled before reaching the centrifuge to avoid warming-up of the rotor during centrifugation.

  3. Microtubule seeded assembly followed by spectrophotometry at 350 nm (for 100 µl)
    1. Thaw rapidly a 20 µl aliquot of tubulin (initial concentration 22 mg/ml).
    2. Add 18 µl of this tubulin solution to 82 μl of BRB80-BeF3- in a polypropylene centrifuge tube.
    3. Centrifuge the tube in the Airfuge at 141,000 x g (A-110 rotor, 22 psig), 5 min, 4 °C.
    4. Incubate the supernatant in the dry block heater, 5 min, 35 °C.
    5. Pipet 90 µl of the pre-warmed tubulin solution and add 10 µl of pre-warmed seeds.
    6. Transfer the mix to the quartz cuvette and place it inside the spectrophotometer.
    7. Press autozero and start recording at 2 points/min.
    8. After about 1 h of polymerization, switch the fluid to the 2 °C thermostated bath to induce microtubule depolymerization (Figure 1).
    9. Stop recording when the OD baseline trace remains stable.
    10. Save data in Excel format (.xls) and plot the variation in optical density at 350 nm (∆OD 350 nm, a.u.) versus time (min) in Excel (Figure 3).


      Figure 3. Assembly of GDP-BeF3--microtubules in the presence and absence of seeds. The OD decreases below zero after cold depolymerization of the microtubules assembled in the presence of seeds (plain line), since these latter contributed to the initial turbidity of the specimen. In the absence of seeds (dotted line), the OD remains almost stable after switching to 4 °C. This indicates that the small increase in OD was essentially due to the formation of tubulin aggregates.

Data analysis

The LabPower software was used to control the Uvikon XS spectrophotometer and acquire data in autorate mode, which were further saved in Excel (.xls) format. Data were imported into Excel for visualizing the variations in optical density versus time plots. Figures were prepared in Affinity Designer.

Recipes

  1. BRB80 buffer solution 5x
    0.4 M PIPES, adjusted to pH 6.8 with KOH
    5 mM MgCl2
    5 mM EGTA
    Store at 4 °C for up to one month
  2. GDP solution
    20 mM in BRB80 1x
    Split into 20 µl aliquots
    Store at -20 °C for up to one year
  3. Tubulin stocks
    22 mg·ml-1 purified porcine brain tubulin
    Store at -80 °C for up to one year
  4. NaF solution
    200 mM in BRB80 1x
    Store at 4 °C for up to one month
  5. BeSO4 solution
    20 mM in BRB80 1x
    Store at 4 °C for up to one month
  6. G-BRB80-BeF3-
    1x BRB80
    20 mM NaF
    5 mM BeSO4
    1 mM GDP
    25% Glycerol
    Keep at 4 °C until use.
  7. BRB80-BeF3- solution
    1x BRB80
    20 mM NaF
    5 mM BeSO4
    1 mM GDP
    Keep at 4 °C until use.
    Note: Prepare fresh BRB80-BeF3- solution since BeF3- salts are not stable.

Acknowledgments

This work was funded by the Agence Nationale de la Recherche (ANR-16-C11-0017-01). This protocol was derived from Estévez-Gallego et al., 2020.

Competing interests

The authors declare no competing interests.

References

  1. Ashford, A. J., Anderson, S. S. L. and Hyman, A. A. (1998). Cell Biology: A Laboratory Handbook. 2nd edition. Vol 2. In: Celis, J. E. (Ed.). Academic Press, 205-212.
  2. Castoldi, M. and Popov, A. V. (2003). Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr Purif 32(1): 83-88.
  3. Estévez-Gallego, J., Josa-Prado, F., Ku, S., Buey, R. M., Balaguer, F. A., Prota, A. E., Lucena-Agell, D., Kamma-Lorger, C., Yagi, T., Iwamoto, H., Duchesne, L., Barasoain, I., Steinmetz, M. O., Chrétien, D., Kamimura, S., Díaz, J. F. and Oliva, M. A. (2020). Structural model for differential cap maturation at growing microtubule ends. Elife 9: e50155.
  4. Maurer, S. P., Bieling, P., Cope, J., Hoenger, A. and Surrey, T. (2011). GTPγS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs). Proc Natl Acad Sci U S A 108(10): 3988-3993.
  5. Maurer, S. P., Fourniol, F. J., Hoenger, A. and Surrey, T. (2014). Seeded microtubule growth for cryoelectron microscopy of end-binding proteins. Methods Mol Biol 1136: 247-260.
  6. Weis, F., Moullintraffort, L., Heichette, C., Chrétien, D. and Garnier, C. (2010). The 90-kDa heat shock protein Hsp90 protects tubulin against thermal denaturation. J Biol Chem 285(13): 9525-9534.
  7. Zhang, R., Alushin, G. M., Brown, A. and Nogales, E. (2015). Mechanistic origin of microtubule dynamic instability and its modulation by EB proteins. Cell 162(4): 849-859.

简介

[摘要 ] 微管动态不稳定性是由与α - β 微管蛋白异二聚体的β- 亚基结合的GTP水解驱动的。核苷酸类似物通常用于模拟微管蛋白GTPase循环的不同步骤,但其中大多数是不良的微管成核剂。通常,微管组装是通过胍基-(α ,β )-亚甲基-二膦酸酯(GMPCPP)或甘油来接种的,它们可能是限制其他核苷酸类似物对其聚合作用的监测因素。 在这里,我们描述了一种协议,该协议允许在核苷酸类似物存在的情况下组装微管,而无需异质种子,并且最终甘油浓度低。首先在感兴趣的类似物和甘油的存在下组装微管以促进组装。然后对这些微管进行超声处理,以产生种子,这些种子将在不存在甘油的情况下用于组装微管。这种策略产生了均质的核苷酸结合的微管,可以通过生化或结构方法(如冷冻电子显微镜)进一步分析。

[背景 ] 核苷酸类似物通常用于研究的构象变化,该α - β 微管组装和水解绑定到其可交换的(E)的网站上的GTP的期间微管蛋白异源二聚体经历。但是,除GMPCPP以外,大多数类似物都是微管成核剂。为克服这一困难,GMPCPP稳定化的种子通常用于存在其他类似物的情况下延长微管(Maurer 等人,2011和2014; Zhang 等人,2015),从而导致混合核苷酸结合的晶格的组装。或者,可以将相对较高浓度(例如25%)的甘油加入反应混合物中以刺激微管蛋白自组装。然而,高甘油浓度可能是不希望的,例如通过冷冻电子显微镜分析微管结构,因为该化合物与蛋白质密度匹配并降低了图像对比度。

为了避免在最终反应混合物中使用GMPCPP种子或高甘油浓度,我们开发了一种协议,该协议使用在相同核苷酸存在下组装的种子,这些核苷酸将用于组装微管。第一步,在核苷酸类似物的存在下,甘油被用于聚合微管。然后通过超声处理将这些微管破碎以产生种子,将其在不存在甘油的微管蛋白混合物中进一步稀释。可以通过冷冻电子显微镜分析伸长到这些种子上的微管,而没有在GMPCPP存在下组装的微管与在感兴趣的核苷酸类似物存在下组装的微管出错的风险。

我们的协议描述了种子的逐步制备和微管的组装,可通过比浊法在350 nm处进行监测。它在组装过程结束时包括一个冷却步骤,以评估解聚微管的存在。这些条件用于组装GDP-BEF 3 - -microtubules,其晶格由低温电子显微镜进行进一步表征(埃斯特韦斯-Gallego 等人,2020)。我们已经成功地应用这个协议在其他类似物如存在组装的微管GTP γ 小号,GDP-的AlF 3 ,GMPPCP和GMPCP,它们都是微管差成核剂。

关键字:微管蛋白, 微管, GTP类似物, 微管形成, 微管种晶组装, 分光光度法, 冷冻电子显微技术

材料和试剂


 


P ipette提示
开顶薄壁聚丙烯离心管,175 微升,5×20毫米(Beckman Coulter公司,目录号:342630)
硫酸铍(BeSO 4· 4H 2 O)(Sigma-Aldrich,目录号:14270)
乙二醇双(2-氨基乙基醚)-N,N,N',N'- 四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
甘油(Sigma-Aldrich,目录号:G5516)
鸟苷5'-三磷酸钠水合物(GTP)(Sigma-Aldrich,目录号:G8877)
氯化镁溶液(MgCl 2 )(Sigma-Aldrich,目录号:63069)
哌嗪-N,N'-双(2-乙磺酸)(PIPES)(Sigma-Aldrich,目录号:P6757)
氢氧化钾(KOH)(Sigma-Aldrich,目录号:30603)
氟化钠(NaF )(Sigma-Aldrich,目录号:S7920)
BRB80缓冲溶液5 x (请参阅配方)
GDP解决方案(请参见食谱)
微管蛋白库存(请参见食谱)
NaF 解决方案(请参阅食谱)
BeSO 4 解决方案(请参阅食谱)
G-BRB80-BEF 3 - (见配方)
BRB80-BEF 3 - 溶液(见食谱)
 


设备


 


Pipetman Classic P100,P20,P10(Gilson,目录号:F123615,F123600,F144802)
Airfuge 空气驱动(贝克曼库尔特,目录号:340401)
固定角转子的A-110(Beckman Coulter公司,CATA 日志号码:347596)
冰箱MEDIKA 100 ECT-F(抽屉)
压缩空气冷却器BEKOBLITZZ LC12(Beko Technologies,目录号:4020103)
压缩空气过滤器CLEARPOINT ,1 μ M(贝科技术,目录号码:S040SWT)
膜式干燥机Drypoint M plus(Beko Technologies,目录号:4007626)
冷凝水排放ECD型号ED12
B2000超声波清洗器(布兰森)
DRI -块加热器DB-2D(Techne公司)
Uvikon XS 分光光度计(Secomam ,Bio-Tek Instruments)
石英比色皿超微池(Hellma,目录号105-201-15-40)
具有导热HY流体的Julabo F30-C(Julabo ),-60°C至+65°C,(Julabo ,目录号:8940104)
具有热HY流体的Julabo MP-5(Julabo ),-60°C至+65°C,(Julabo ,目录号:8940104)
三通阀,PFTE (Bürkle ,目录号:8610-0006)
 


软件


 


LabPower v3.50(Secomam ,Bio-Tek仪器)
Excel(微软)
亲和力设计师(Serif Ltd)
 


程序


 


分光光度剂的制备
开启分光光度计,并确保其使用350 nm的紫外灯。
注意:在Uvikon XS 上,从UV(D2)切换到卤素灯的默认值为340 nm 。该值必须更改为例如360 nm,因为必须将UV灯与350 nm的超微池石英比色皿一起使用。


选择350 nm 的自动速率模式。
打开温水浴,将温度设置为35°C,使其平衡15分钟。
注意:必须使用装满水的石英比色杯内的小型热电偶来调节冷热循环浴的温度。目标是在聚合和解聚条件下分别达到35°C和4°C。环境温度的变化(例如,夏季和冬季之间)可能需要定期控制和调整这些设置。聚合过程中不要超过35°C,因为微管蛋白在高于该温度时很容易变性(Weis et al。,2010)。


交换机上的冷水浴,将温度设定为2℃并让平衡30分钟。
使分光光度计恒温池支架内的35°C流体循环(图1)。
 


D:\重新格式化\ 2020-7-1 \ 2003101--1487DenisChrétien861629 \无花果jpg \图1.jpg


图1.分光光度计的温度控制。A.将两个在35°C和2°C下调节的水浴连接到分光光度计的恒温6 + 6电池更换器。三通阀允许将温度从35°C(底部)快速切换到4°C(顶部)。所述的温度冷水浴必须低于4℃被设定,由于在管道系统中的流体的预热。必须使用小型热电偶定期检查电池座内部的实际高温和低温温度。B.温暖(左)和冷(右)沐浴池。C.三通阀。这些必须同时切换。


 


微管种子准备(30(升)
在手指之间快速解冻10μl 等分试样的微管蛋白(初始浓度22 mg / ml),并保持在4°C直至使用。
注意:通过在高分子量缓冲液中组装两个周期从猪脑中纯化微管蛋白(Casolddi 和Popov,2003年),然后进行循环步骤以在E位点获得与GDP配伍的微管蛋白(Ashford等人,1998年)。 )。循环微管蛋白是可商购的,例如购自PurSolution (https://puresolve.com/cycled-tubulin/)。Ť 在22的终浓度ubulin毫克/毫升在100μl和10μl等份等分,急速冷冻在液氮中,并储存在-80℃直至使用。微管蛋白解冻后,请尽量减少所有步骤,直到聚合为止,因为如果以未组装的形式保存,即使在4°C时,它也会迅速变性。


添加5.5微升的微管蛋白溶液中,以24.5 微升G-BRB80-BEF的3 - 在聚丙烯离心管中。
将离心管在Airfuge中以141,000 x g (A-110转子,22 psig )离心5分钟,4°C(图2)。
注意:呼吸机的温度不受调节。它必须在冰箱中在图2中被放置在冷室中,或可替代地,如在这两种情况下,所述进入空气流也必须被冷却,否则转子将预热(见manufactu RER 的指令,如果安装在冷室)。


将上清液在干式加热器中孵育1小时,温度为35°C。
在超声清洁器中超声微管1分钟。
将微管种子保持在35°C,直到用于下一步。
 


D:\重新格式化\ 2020-7-1 \ 2003101--1487DenisChrétien861629 \图jpg \图2.jpg


图2. 气浮器的温度控制。将Airfuge 保持在冰箱内4°C的温度下。进入的压缩空气在到达离心机之前也必须进行冷却,以避免离心过程中转子预热。


 


微管接种的组件,然后在350 nm处进行分光光度法(对于100(l)
快速解冻20μl微管蛋白的等分试样(初始浓度22 mg / ml)。
添加18个OFL 此微管蛋白溶液中,以82 微升BRB80-BEF的3 - 在聚丙烯离心管中。
将离心管在Airfuge中以141,000 xg (A-110转子,22 psig )离心5分钟,4°C。
将上清液在干式加热器中孵育5分钟,温度为35°C。
移取90毫升预热的微管蛋白溶液,并加入10微升预热的种子。
将混合物转移到石英比色杯中,并将其放入分光光度计内。
按自动归零,以2点/分钟的速度开始录制。
聚合大约1小时后,将流体切换至2°C 恒温槽以引发微管解聚(图1)。
当OD基线跟踪保持稳定时,停止记录。
将数据保存为Excel格式(.xls ),并绘制350 nm(∆ OD 350 nm,au 。)处的光密度变化与时间(min)的关系(图3)。
 


D:\重新格式化\ 2020-7-1 \ 2003101--1487DenisChrétien861629 \无花果jpg \ Fig3-word.jpg


图3. GDP-BEF的组装3 - -microtubules在种子的存在和不存在。在种子存在下组装的微管冷解聚后,OD降低至零以下(纯系),因为这些微管有助于样品的初始浊度。在没有种子的情况下(虚线),OD切换至4°C后几乎保持稳定。这表明OD的少量增加主要是由于微管蛋白聚集体的形成。


 


数据分析


 


所述LabPower 使用软件来控制UVIKON 在XS分光光度计和获取数据的自动定速率模式,其进一步保存在Excel(.xls的)格式。将数据导入Excel以可视化光密度与时间图的变化。数字是在Affinity Designer中准备的。


 


菜谱


 


BRB80缓冲溶液5 x
0.4 M PIPES,用KOH调节至pH 6.8
5 mM MgCl 2
5 mM EGT A
在4°C下保存长达一个月
GDP解决方案
在BRB80中为20 mM 1 x
分成20份等分试样,
在-20° C下储存长达一年
微管蛋白储备液
22 mg · ml -1 纯化的猪脑微管蛋白
在-80°C下储存长达一年
在BRB80中的200 mM NaF 溶液1 x 在4°C下储存长达一个月


BeSO 4 溶液
在BRB80中为20 mM 1 x
在4°C的温度下保存长达一个月
G-BRB80-BEF 3 -
1 X BRB80
20mM的氟化钠
的5mM BESO 4
1mM的GDP
25 %甘油
ķ 在4℃下EEP直至使用。
BRB80-BEF 3 - 解决方案
1 X BRB80
20mM的氟化钠
的5mM BESO 4
1mM的GDP
ķ 在4℃EEP直至使用。
不E:准备新鲜BRB80-BEF 3 - 解决方案SI NCE BEF 3 - 盐并不稳定。


 


致谢


 


这项工作由法国国家侦察局(ANR-16-C11-0017-01)资助。该协议源自Estévez-Gallego 等人,2020。


 


利益争夺


 


作者宣称没有利益冲突。


 


参考文献


 


Ashford,AJ,Anderson,SSL和Hyman,AA(1998)。细胞生物学:实验室手册。第2版ITIO ñ。第2卷。在:塞利斯,JE (主编)。学术出版社,205-212。
Castoldi ,M.和Popov,AV(2003)。通过在高分子量缓冲液中进行两个聚合-解聚循环来纯化脑微管蛋白。Protein Expr Purif 32(1):83-88。              
Estévez- Gallego,J.,Josa -Prado,F.,Ku,S.,Buey ,RM,Balaguer,FA,Prota ,AE,Lucena-Agell ,D.,Kamma-Lorger ,C.,Yagi,T., Iwamoto,H.,Duchesne,L.,Barasoain ,I.,Steinmetz,MO,Chrétien,D.,Kamimura ,S.,Díaz,JF和Oliva,MA(2020)。微管末端生长时差异帽成熟的结构模型。eLife 9:e50155。              
Maurer,SP,Bieling ,P.,Cope,J.,Hoenger ,A。和Surrey,T。(2011)。GTP γ 小号微管模拟由端结合蛋白(EBS)认识到越来越微管末端结构。PROC国家科科学院科学USA 108(10):3988-3993。              
毛勒(SP),富尼奥尔(Fourniol),FJ,亨格(A. 种子微管生长,用于末端结合蛋白的冷冻电子显微镜检查。方法分子生物学1136:247-260。
韦斯,F.,Moullintraffort ,L.,Heichette ,C.,CHR é 田,D。和卡尼尔,C.(2010)。90 kDa的热激蛋白Hsp90保护微管蛋白免受热变性。生物化学杂志285(13):9525-9534。              
Zhang,R.,Alushin ,GM,Brown,A.和Nogales,E.(2015年)。微管动态不稳定性的机制起源及其受EB蛋白的调节。细胞162(4):849-859。
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Copyright Ku et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ku, S., Heichette, C., Duchesne, L. and Chrétien, D. (2020). Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues. Bio-protocol 10(16): e3714. DOI: 10.21769/BioProtoc.3714.
  2. Estévez-Gallego, J., Josa-Prado, F., Ku, S., Buey, R. M., Balaguer, F. A., Prota, A. E., Lucena-Agell, D., Kamma-Lorger, C., Yagi, T., Iwamoto, H., Duchesne, L., Barasoain, I., Steinmetz, M. O., Chrétien, D., Kamimura, S., Díaz, J. F. and Oliva, M. A. (2020). Structural model for differential cap maturation at growing microtubule ends. Elife 9: e50155.
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