In vitro Microtubule Bundling Assay under Physiological Conditions

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Nature Communications
Apr 2016



Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions.

Keywords: in vitro bundling assay (体外成束测定), Fluorescence microtubules (荧光微管), Kinesin motors (动力蛋白马达), DEAE-Dextran coated glass chamber (DEAE-葡聚糖涂层玻璃室), Physiological [ATP] (生理[ATP])


Microtubule-based motor proteins are important as they use the chemical energy from ATP to generate force to translocate microtubules (MTs) vectorially. Of these MT based motor proteins, the superfamily known as kinesins, are responsible for directional transport and movement along microtubules. Some kinesins have MT-binding sites at both ends of the holoenzyme, so they can crosslink MTs into bundles under physiological ATP conditions (Tao et al., 2006). Due to this bundling activity, they have important roles in organizing and maintaining the mitotic spindle, whose action depends upon the polarity patterns of its microtubules (van den Wildenberg et al., 2008). Previous bundling assays didn’t include ATP, or used a non-hydrolysable ATP analog, which could generate artificial results. Here we developed a method using physiological ATP conditions. By purifying these full-length motor proteins, it has allowed us to determine their crosslinking activity under physiological conditions.

Materials and Reagents

  1. Microcentrifuge tubes (1.5 ml) (Sigma-Aldrich, catalog number: Z336769 )
  2. Aluminum foil
  3. Double sided tape
  4. Coverslip, No. 1.5-22 x 22 mm (Sigma-Aldrich, catalog number: Z692263 )
  5. Microscope slides (AmScope, catalog number: BS-50P )
  6. Kimwipe (Sigma-Aldrich, catalog number: Z188956 )
  7. Pipette tips (Corning, catalog number: 4860 )
  8. Tubulin (CYTOSKELETON, catalog number: MT001-A )
  9. Glycerol (Sigma-Aldrich, catalog number: G2025 )
  10. Rhodamine tubulin (CYTOSKELETON, catalog number: TL590M-A )
  11. GTP (Sigma-Aldrich, catalog number: G8877 )
  12. Hydrochloric acid (HCl [37%, v/v]) (Sigma-Aldrich, catalog number: 320331 )
  13. Full-length kinesin motor proteins are expressed and purified from baculoviral expression system
  14. ATP (Sigma-Aldrich, catalog number: A6419 )
  15. Piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757 )
  16. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
  17. EGTA (Sigma-Aldrich, catalog number: 03777 )
  18. Potassium hydroxide (KOH)
  19. Paclitaxel (Sigma-Aldrich, catalog number: T7402 )
  20. DMSO (Sigma-Aldrich, catalog number: D2650 )
  21. DEAE-dextran (Sigma-Aldrich, catalog number: 30461 )
  22. Tris base (Sigma-Aldrich, catalog number: T1503 )
  23. Potassium chloride (KCl) (Sigma-Aldrich: P5405 )
  24. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  25. Protease inhibitors
    Aprotinin (Sigma-Aldrich, catalog number: A3428 )
    Benzamidine (Sigma-Aldrich, catalog number: B6505 or 12072 )
    Note: The product “ B6505 ” has been discontinued.
    Pepstatin A (Sigma-Aldrich, catalog number: P5318 )
    Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
    Leupeptin (Sigma-Aldrich, catalog number: L2884 )
    Soy Bean Trypsin inhibitor (SBTI) (Sigma-Aldrich, catalog number: T6522 )
    tert-Amyl methyl ether (TAME) (Sigma-Aldrich, catalog number: 283096 )
  26. Catalase (Sigma-Aldrich, catalog number: C1345 )
  27. Glucose oxidase (Sigma-Aldrich, catalog number: C6766 or G7141 )
    Note: The product “ C6766 ” has been discontinued.
  28. Glucose (Sigma-Aldrich, catalog number: G7021 or D9434 )
  29. 10x phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5493 )
  30. BRB80 (see Recipes)
  31. 1 µM, 10 µM, and 100 µM taxol (see Recipes)
  32. DEAE-dextran (see Recipes)
  33. Buffer T (see Recipes)
  34. Antifade (see Recipes)


  1. Nichipet EX-Plus II pipette 2-20 μl (Nichiryo, catalog number: 00-NPLO2-20 )
  2. Nichipet EX-Plus II pipette 10-100 μl (Nichiryo, catalog number: 00-NPLO2-100 )
  3. Water bath, from ambient to 100 °C (Thermo Fisher Scientific, Thermo ScientificTM, model: PrecisionTM General Purpose Baths , catalog number: TSGP02)
  4. Flow chamber (see Procedure)
  5. Inverted fluorescence microscope (12-volt 100-watt tungsten-halide lamp, excitation wavelength, 547 nm; emission wavelength, 576 nm, in conjunction with a 570 nm dichroic mirror, objective: CFI Plan Fluor 100x oil) (Nikon Instruments, model: Eclipse E600 )


  1. Polymerization of fluorescent MTs
    1. In a 1.5 ml microcentrifuge tube, dissolve 0.25 mg tubulin in 60 µl of BRB80 solution (containing 10% glycerol). Store on ice.
    2. Add 20 µg of Rhodamine tubulin into the 60 µl solution. Mix gently for 30 sec and store tube half submerged in an ice bucket.
    3. Add 1/10 the total volume of the 60 µl of tubulin mix, worth of 10 mM GTP, to the 60 µl tubulin mix, with a final concentration of 1 mM GTP. Mix gently by inverting.
    4. Add 1/10 the total volume of mixture step 1c, worth of 1 µM taxol, to the mix, with a final taxol concentration of 0.1 µM. Incubate (in water bath) at 37 °C for 10 min.
    5. Add 1/10 the total volume of mixture step 1d, worth of 10 µM taxol, to the mix, with a final taxol concentration of 1 µM. Incubate (in water bath) at 37 °C for 10 min.
    6. Add 1/10 the total volume of mixture step 1e, worth of 100 µM taxol, to the mix, with a final taxol concentration of 10 µM. Incubate (in water bath) at 37 °C for 10 min.
    7. Store the polymerized MTs at room temperature, with the tube covered in aluminum foil and stored in a closet to protect the mixture from light. The MTs are stable for use for up to two weeks.
  2. Prepare chamber

    Figure 1. Infusion of chamber. Using two pieces of double sided tape, a coverslip is attached to a microscope slide, completing the preparation of the flow chamber. Using a pipette with appropriate volumes, infuse chamber by pipetting solution into the opening of the flow chamber. A Kimwipe is used to facilitate diffusion of the solution across the chamber and absorb excess liquid at the end.

    1. Acid wash coverslip with HCl (37%, v/v) for 24 h, rinse with de-ionized water and air dry for 24 h.
    2. After cleaning, put a drop of 0.2 mg/ml DEAE-dextran on coverslip and let sit for 5 min.
    3. Absorb remaining liquid with Kimwipe and air dry coverslip.
    4. Place two strips of double sided tape (L x 22 mm) parallel to each other on microscope slide at a distance of 22 mm from each other. Place the coverslip down, matching the alignment of the tape, with the DEAE-dextran coated side facing towards the microscope slide. Refer to Figure 1.
  3. Prepare bundling mixture.
    1. Prepare 25 µl mix in buffer T, with the following reagents to a final concentration of; 2.5 mM MTs, 0.2 mM motor proteins, 10 µM taxol, and 1 mM ATP.
      Note: For motor proteins, we use hydrodynamic assays (Tao et al., 2016) to determine the MW. For MT, we use MW of tubulin dimer (α tubulin + β tubulin) to calculate the concentration of MTs.
    2. Shake the mixture at 50 rpm at room temperature for 30 min.
  4. Infuse mix from step 3 into flow chamber:
    1. Infuse 25 µl into the flow chamber(s), let sit for 3 min. Refer to Figure 1.
    2. Wash out any unstuck proteins with a continuous flow of 25 µl buffer T solution (including reagents with a final concentration of 1 mM ATP, 10 mM taxol, and Antifade), repeated two additional times. Use a similar technique as infusion. Refer to Figure 1.
    3. Observe and record MT bundling using inverted fluorescence microscope (12-volt 100-watt tungsten-halide lamp, excitation wavelength, 547 nm; emission wavelength, 576 nm in conjunction with a 570 nm dichroic mirror, objective: CFI Plan Fluor 100x oil, Nikon Instruments, model: Eclipse E600).

Data analysis

The microtubule (MT) bundling assay was repeated three times, with three separate batches of reagents. 15 images were collected for each experiment. To ensure consistency, it is recommended that the assay be repeated three times, with 15 images collected for each. Figure 2 shows a kinesin holoenzyme crosslinking MTs into bundles. Figure 2A shows a robust bundle due to crosslinking activity of the kinesin motor. However, in Figure 2B the kinesin-1 head dimers do not bundle MTs, suggesting that the binding sites at opposite ends of the motor are required for crosslinking, rather than action of two heads at one end.

Figure 2. Example of bundling assay. Fluorescence microscopy of purified kinesin holoenzyme bundling MTs (A) while kinesin-1 head dimers unable to bundle MTs under same condition (B). Scale bar = 10 µm. (Tao et al., 2016)


  1. To ensure maximum bundling activity, purify the motor proteins within 30 min before use in bundling assay.
  2. For best results, fluorescent microtubules should be used within two weeks of their polymerization, with the mixture being stored in aluminum foil covered container, kept in a dark location.
  3. Full-length kinesin motor cDNA was cloned into a Gateway Baculovirous expression vector (pDEST8) with a 6xHis tag. After verification by sequencing, the recombinant expression vector was used to generate recombinant Baculoviruses. Baculovirus-infected Sf9 cells were collected and then recombinant motor protein was purified on a Ni-NTA column using standard protocol (Tao et al., 2010).


  1. BRB80*
    80 mM PIPES
    1 mM MgCl2
    1 mM EGTA
    Adjust pH to 6.8 with KOH
  2. 1 µM, 10 µM, and 100 µM taxol
    20 µl of 1 mM taxol dissolved in 180 µl DMSO = 200 µl of 100 µM taxol
    6 µl of 100 µM taxol dissolved in 54 µl DMSO = 60 µl of 10 µM taxol
    6 µl of 10µM taxol dissolved in 54 µl DMSO = 60 µl of 1 µM taxol
  3. DEAE-dextran
    0.2 mg/ml of ddH2O
  4. Buffer T*
    20 mM Tris
    150 mM KCl
    2 mM MgCl2
    1 mM DTT
    Adjust pH to 8.0 with KOH
    Protease inhibitors with final concentration of Aprotinin 2 µg/ml, Benzamidine 20 µg/ml, Pepstatin A 1 µg/ml, PMSF 0.1 mM, Leupeptin 1 µg/ml, SBTI 100 µg/ml, Tame 40 µg/ml, added just before use
  5. Antifade
    10 mg catalase
    3 mg glucose oxidase
    180 mg glucose
    100 µl 1 M DTT
    1.90 mg EGTA
    100 µl 10x PBS
    Fill to 1 ml with ddH2O

*Note: Listed concentrations are final concentrations, buffer is prepared in ddH2O.


This MT bundling assay was inspired by ‘Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor’ (Tao et al., 2016) and ‘Purification and assay of mitotic motors’ (Tao and Scholey, 2010). This work was funded by the NIH grant GM046409 to W.S. and NIH grant GM 55507 to J.M.S.


  1. Tao, L., Fasulo, B., Warecki, B. and Sullivan, W. (2016). Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor. Nat Commun 7: 11182.
  2. Tao, L., Mogilner, A., Civelekoglu-Scholey, G., Wollman, R., Evans, J., Stahlberg, H. and Scholey, J. M. (2006). A homotetrameric kinesin-5, KLP61F, bundles microtubules and antagonizes Ncd in motility assays. Curr Biol 16(23): 2293-2302.
  3. Tao, L. and Scholey, J. M. (2010). Purification and assay of mitotic motors. Methods 51(2): 233-241.
  4. van den Wildenberg, S. M., Tao, L., Kapitein, L. C., Schmidt, C. F., Scholey, J. M. and Peterman, E. J. (2008). The homotetrameric kinesin-5 KLP61F preferentially crosslinks microtubules into antiparallel orientations. Curr Biol 18(23): 1860-1864.



基于微管的运动蛋白是重要的,因为它们使用ATP的化学能产生力以便向量转移微管(MT)。在这些基于MT的运动蛋白中,称为驱动蛋白的超家族负责定向运输和沿微管的运动。一些运动蛋白在全酶的两端具有MT结合位点,因此它们可以在生理ATP条件下将MT交联成束(Tao等人,2006)。由于这种捆绑活动,它们在组织和维持有丝分裂纺锤体方面具有重要作用,其主要作用取决于其微管的极性模式(van den Wildenberg等人,2008)。以前的捆绑测定不包括ATP,或者使用可以产生人为结果的不可水解的ATP类似物。这里我们开发了一种使用生理ATP条件的方法。通过纯化这些全长运动蛋白,它使我们能够在生理条件下测定其交联活性。

关键字:体外成束测定, 荧光微管, 动力蛋白马达, DEAE-葡聚糖涂层玻璃室, 生理[ATP]


  1. 微量离心管(1.5ml)(Sigma-Aldrich,目录号:Z336769)
  2. 铝箔
  3. 双面胶带
  4. 盖片,1.5-22×22mm(Sigma-Aldrich,目录号:Z692263)
  5. 显微镜幻灯片(AmScope,目录号:BS-50P)
  6. Kimwipe(Sigma-Aldrich,目录号:Z188956)
  7. 移液器提示(康宁,目录号:4860)
  8. 微管蛋白(CYTOSKELETON,目录号:MT001-A)
  9. 甘油(Sigma-Aldrich,目录号:G2025)
  10. 罗丹明微管蛋白(CYTOSKELETON,目录号:TL590M-A)
  11. GTP(Sigma-Aldrich,目录号:G8877)
  12. 盐酸(HCl [37%,v/v])(Sigma-Aldrich,目录号:320331)
  13. 从杆状病毒表达系统表达和纯化全长驱动蛋白运动蛋白
  14. ATP(Sigma-Aldrich,目录号:A6419)
  15. 哌嗪-N,N'-双(2-乙磺酸)(PIPES)(Sigma-Aldrich,目录号:P6757)
  16. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M4880)
  17. EGTA(Sigma-Aldrich,目录号:03777)
  18. 氢氧化钾(KOH)
  19. 紫杉醇(Sigma-Aldrich,目录号:T7402)
  20. DMSO(Sigma-Aldrich,目录号:D2650)
  21. DEAE-葡聚糖(Sigma-Aldrich,目录号:30461)
  22. Tris碱(Sigma-Aldrich,目录号:T1503)
  23. 氯化钾(KCl)(Sigma-Aldrich:P5405)
  24. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  25. 蛋白酶抑制剂
  26. 过氧化氢酶(Sigma-Aldrich,目录号:C1345)
  27. 葡萄糖氧化酶(Sigma-Aldrich,目录号:C6766或G7141)
  28. 葡萄糖(Sigma-Aldrich,目录号:G7021或D9434)
  29. 10倍磷酸缓冲盐水(PBS)(Sigma-Aldrich,目录号:P5493)
  30. BRB80(见配方)
  31. 1μM,10μM和100μM紫杉醇(参见食谱)
  32. DEAE葡聚糖(参见食谱)
  33. 缓冲T(见配方)
  34. 防风(见食谱)


  1. Nichipet EX-Plus II移液管2-20μl(Nichiryo,目录号:00-NPLO2-20)
  2. Nichipet EX-Plus II移液管10-100μl(Nichiryo,目录号:00-NPLO2-100)
  3. 水浴,从环境温度到100°C(Thermo Fisher Scientific,Thermo Scientific TM,型号:Precision TM通用浴,目录号:TSGP02)
  4. 流动室(见程序)
  5. 倒置荧光显微镜(12伏100瓦卤钨灯,激发波长,547nm;发射波长,576nm,结合570nm分色镜,目标:CFI Plan Fluor 100x油)(Nikon Instruments,型号: Eclipse E600)


  1. 荧光MT的聚合
    1. 在1.5ml微量离心管中,将0.25mg微管蛋白溶于60μlBRB80溶液(含有10%甘油)中。存放在冰上。
    2. 加入20微克罗丹明微管蛋白至60微升溶液。轻轻搅拌30秒,将管子半淹没在冰桶中。
    3. 将60μl微管蛋白混合物(价值10mM GTP)的总体积的1/10加入到60μl微管蛋白混合物中,终浓度为1mM GTP。反转轻轻混合。
    4. 加入混合物的混合物步骤1c的总体积的1/10,其值为1μM紫杉醇,最终的紫杉醇浓度为0.1μM。孵育(在水浴中)在37°C 10分钟
    5. 将混合物步骤1d的总体积的1/10加入到混合物中,最终紫杉醇浓度为1μM。孵育(在水浴中)在37°C 10分钟
    6. 将混合物步骤1e(价值100μM紫杉醇)的总体积的1/10加至混合物中,最终紫杉醇浓度为10μM。孵育(在水浴中)在37℃下10分钟。
    7. 将聚合的MT存放在室温下,将管覆盖在铝箔上并储存在衣柜中以保护混合物免受光照。 MT稳定使用长达两周。
  2. 准备室

    1. 用HCl(37%,v/v)酸洗盖玻片24小时,用去离子水冲洗干燥24小时。
    2. 清洁后,将一滴0.2mg/ml DEAE-葡聚糖放在盖玻片上,放置5分钟。
    3. 用Kimwipe和空气干燥盖玻片吸收剩余液体。
    4. 在显微镜载玻片上放置两条双面胶带(长x 22毫米)彼此平行,距离彼此相距22毫米。将盖玻片向下放置,与胶带对齐,与面向显微镜载玻片的DEAE-葡聚糖涂层面对齐。参见图1。
  3. 准备捆扎混合物。
    1. 在缓冲液T中制备25μl混合物,用以下试剂至最终浓度; 2.5mM MT,0.2mM运动蛋白,10μM紫杉醇和1mM ATP。
    2. 在室温下以50rpm摇动混合物30分钟。
  4. 将步骤3的混合物浸入流动室中:
    1. 将25μl注入流动室,放置3分钟。参见图1.
    2. 用连续流动的25μl缓冲液T溶液(包括最终浓度为1mM ATP,10mM紫杉醇和Antifade的试剂)洗涤任何未松解的蛋白质,重复两次。使用类似的技术作为输液。参见图1.
    3. 使用倒置荧光显微镜(12伏100瓦钨卤化物灯,激发波长547nm;发射波长576nm,结合570nm二向色镜,目的:CFI Plan Fluor 100x油,Nikon)观察并记录MT束仪器,型号:Eclipse E600)。



图2.捆绑测定实施例当驱动蛋白-1头二聚体在相同条件下不能捆绑MT时,纯化的驱动蛋白全酶MT(A)的荧光显微镜检查(B)。刻度棒=10μm。 (Tao等人,2016)


  1. 为了确保最大的捆绑活动,在捆绑测定中使用前,在30分钟内净化运动蛋白。
  2. 为了获得最佳效果,荧光微管应在其聚合两周内使用,将混合物储存在铝箔覆盖的容器中,保存在黑暗的位置。
  3. 将全长驱动蛋白马达cDNA克隆到具有6xHis标签的Gateway Baculovetic表达载体(pDEST8)中。经测序验证后,重组表达载体用于产生重组杆状病毒。收集杆状病毒感染的Sf9细胞,然后使用标准方案在Ni-NTA柱上纯化重组运动蛋白(Tao等人,2010)。


  1. BRB80 *
    80 mM PIPES
    1mM MgCl 2
    1 mM EGTA
  2. 1μM,10μM和100μM紫杉醇
    6μl100微摩尔紫杉酚溶于54微升DMSO = 60微升10微克紫杉醇
    6μl10μM紫杉醇溶于54μlDMSO =60μl1μM紫杉醇
  3. DEAE葡聚糖
    0.2mg/ml的ddH 2 O
  4. 缓冲区T *
    20 mM Tris
    150 mM KCl
    2mM MgCl 2
    1 mM DTT
    用KOH调节pH至8.0 蛋白酶抑制剂,最终浓度为抑肽酶2μg/ml,Benzamidine20μg/ml,Pepstatin A1μg/ml,PMSF 0.1mM,亮肽素1μg/ml,SBTI100μg/ml,驯鹿40μg/ml,刚刚加入使用
  5. 防祸
    3 mg葡萄糖氧化酶
    100μl1 M DTT
    1.90mg EGTA
    100μl10x PBS
    用ddH 2 O填充至1 ml



这种MT捆绑测定的灵感来自于"Tum/RacGAP功能作为启动Pav/kinesin-6电机的开关"(陶瓷等,2016)和"有丝分裂马达的纯化和测定"(Tao和Scholey,2010年)。这项工作由NIH授予GM046409资助至W.S. NIH将GM 55507授予J.M.S.


  1. Tao,L.,Fasulo,B.,Warecki,B。和Sullivan,W。(2016)。  Tum/RacGAP用作激活Pav/kinesin-6电机的开关。 7:11182.
  2. Tao,L.,Mogilner,A.,Civelekoglu-Scholey,G.,Wollman,R.,Evans,J.,Stahlberg,H.and Scholey,JM(2006)。< a class ="ke-insertfile" href =""target ="_ blank">同源三聚体驱动蛋白-5,KLP61F,束状微管,并在动力测定中拮抗Ncd。 Curr Biol 16(23):2293-2302。
  3. Tao,L.and Scholey,JM(2010)。  有丝分裂马达的纯化和测定。 方法 51(2):233-241。
  4. van den Wildenberg,SM,Tao,L.,Kapitein,LC,Schmidt,CF,Scholey,JM和Peterman,EJ(2008)。< a class ="ke-insertfile"href ="http:"target ="_ blank">同源四聚体驱动蛋白-5 KLP61F优先将微管交联成反向平行取向。 18(23):1860 -1864。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Geisterfer, Z., Tezuka, G. and Tao, L. (2017). In vitro Microtubule Bundling Assay under Physiological Conditions. Bio-protocol 7(7): e2217. DOI: 10.21769/BioProtoc.2217.