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Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics
采用滑动分析法分析基于微管的运动蛋白动力学   

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Abstract

The purpose of this protocol is to provide an updated method of performing microtubule gliding assays and visualizing it using fluorescence microscopy.

Keywords: Gliding assay(滑动分析法), Mitotic motor(有丝分裂马达蛋白), in vitro motility(体外运动), Polarity-marked microtubules(标记极性的微管), Glass chamber(玻璃室)

Background

Mitotic spindles are protein machinery that dominate mitosis. The mitotic spindle utilizes microtubule-based motor proteins to organize itself, and exert forces to drive cell division. Microtubule-based motor proteins produce mechanical work using energy derived from ATP hydrolysis (Coppin et al., 1997). Motor proteins translocate microtubules in a unidirectional manner. The behavior of motility can be observed by in vitro gliding assay (Tao and Scholey, 2010), in which the motors are affixed onto a glass surface and supplied with microtubules and ATP. The motility of the motor proteins can then be studied using fluorescence microscopy and the details of their dynamic behavior can be observed in real time. This updated protocol will allow analysis of microtubule-based motor protein function with the use of in vitro microtubule gliding assays (Tao et al., 2006 and 2016).

Materials and Reagents

  1. Pipette tips (Corning, catalog number: 4860 )
  2. Centrifuge tubes (Corning, catalog number: 430290 )
  3. Thickwall polycarbonate tubes (Beckman Coulter, catalog number: 343775 )
  4. Coverslips (Sigma-Aldrich, catalog number: Z692263 )
  5. Microscope slides (Fisher Scientific, catalog number: S17466A )
  6. Coverslips (Fisher Scientific, catalog number: S175211A )
  7. Guanosine-5’-[(α,β)-methyleno]triphosphate (GMPCPP) (10 mM) (Jena Bioscience, catalog number: NU-405S )
  8. Tubulin (CYTOSKELETON, catalog number: TL238A )
  9. Rhodamine tubulin (CYTOSKELETON, catalog number: TL590M )
  10. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
  11. N-ethylmaleimide (Sigma-Aldrich, catalog number: E3876 )
  12. Guanosine 5’-triphosphate (GTP) (Sigma-Aldrich, catalog number: G8877 )
  13. Hydrogen chloride (HCl) (Sigma-Aldrich, catalog number: 295426 )
  14. Casein (Sigma-Aldrich, catalog number: C7078 )
  15. ATP (Sigma-Aldrich, catalog number: A6419 )
  16. Paclitaxel (Taxol) (Sigma-Aldrich, catalog number: T7402 )
  17. Piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) (Sigma-Aldrich, catalog number: P6757 )
  18. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
  19. EGTA (Sigma-Aldrich, catalog number: E3889 )
  20. Tris base (Sigma-Aldrich, catalog number: T1503 )
  21. Potassium chloride (KCl) (Sigma-Aldrich: P5405 )
  22. 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 )
    Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
    Leupeptin (Sigma-Aldrich, catalog number: L2884 )
    Soybean trypsin inhibitor (SBTI) (Sigma-Aldrich, catalog number: T6522 )
    Tert-Amyl methyl ether (TAME) (Sigma-Aldrich, catalog number: 283096 )
  23. Catalase (Sigma-Aldrich, catalog number: C1345 )
  24. Glucose oxidase (Sigma-Aldrich, catalog number: C6766 or G7141 )
    Note: The product “ C6766 ” has been discontinued.
  25. Glucose (Sigma-Aldrich, catalog number: D9434 )
  26. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P5368 )
  27. BRB80 buffer solution (see Recipes)
  28. Buffer L (see Recipes)
  29. Anti-fade solution (see Recipes)

Equipment

  1. Micropipette  
    2-20 μl (Sigma-Aldrich, catalog number: Z717304 )
    10-100 μl (Sigma-Aldrich, catalog number: Z717312 )
  2. Ultra-centrifuge (Beckman Coulter, model: TLA-100 )
  3. NanoDrop (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
  4. Fluorescence microscope (Nikon Instruments, model: Eclipse E600 )
  5. Water bath (Thermo Fisher Scientific, model: PrecisionTM General Purpose Baths , catalog number: TSGP02)

Software

  1. ImageJ (Version 1.51J, https://imagej.nih.gov/ij/)

Procedure

  1. Polarity marked microtubules
    1. Make bright GMPCPP seed, which serves as a ‘primer’ for microtubule polymerization as well as an indicator of the microtubule minus-end.
      1. Make a 10 mg/ml solution of tubulin by dissolving 250 µg of tubulin into 25 µl of BRB80.
      2. Make a 10 mg/ml solution of rhodamine tubulin by dissolving 20 µg into 2 µl of BRB80.
      3. Mix 4 µl of the 10 mg/ml tubulin solution, 2 µl of the10 mg/ml rhodamine tubulin solution, 18 µl of BRB80, 3 µl of 10 mM DTT, and 3 µl 10 mM GMPCPP. Store mix on ice.
      4. Centrifuge at 410,000 x g for 7 min at 4 °C.
      5. Take out 3 µl of bright seed mix supernatant and incubate in water bath at 37 °C for 15 min.
    2. Make N-ethylmaleimide tubulin
      1. Make a 10 mg/ml tubulin solution on ice (see step A1a).
      2. Make a fresh 1 mM N-ethylmaleimide (NEM) solution.
        Add 0.5 μl 50 mM NEM to 25 μl 10 mg/ml tubulin. Let the solution sit at room temperature for 5 min.
      3. Add 0.5 µl of 1 M DTT to 25 µl NEM-tubulin solution. Let the solution sit on ice for 30 min.
      4. Centrifuge NEM-tubulin solution at 350,000 x g for 15 min at 4 °C. Collect the supernatant.
      5. Measure the concentration of supernatant with NanoDrop at 280 nm absorbance.
    3. Make dim elongation mix
      1. To make 10 μl elongation mix:
        1.34 μl unlabeled tubulin (10 mg/ml)
        0.17 μl rhodamine tubulin (10 mg/ml)
        3 μl NEM-tubulin (4 mg/ml)
        1 μl 10 mM DTT (diluted 1 M DTT into 10 mM DTT first)
        0.2 μl 50 mM GTP
        4.29 μl 1x BRB80
      2. Incubate the mix in water bath at 37 °C for 1 min.
    4. Make polarity marked microtubules.
      1. Add 2 µl of bright seed mix to 10 µl dim elongation mix and gently mix.
      2. Incubate the solution in water bath at 37 °C for 30 min, and add taxol stepwise to 10 µM final solution. Store the microtubules at room temperature and protect from light.

  2. Gliding assay
    1. Make glass chamber
      1. Acid wash coverslips (22 x 22 mm) with HCl for 24 h and rinse with ddH2O for 10 min each, 3 times. Air dry the coverslips for 24 h.
      2. Attach the coverslip to a glass slide (75 x 25 mm) using two pieces of double-sided tape on the top and bottom. There should be open slits on both sides (Figure 1).


        Figure 1. Illustration of glass slide chamber using double-sided tape

      3. Flow in 25 µl of 1 mg/ml casein into the chamber and let it sit for 5 min. Casein serves as a cushion to anchor motor proteins.
      4. Wash out the unbinding casein with 3 x 20 µl buffer L.
      5. Flow in 10-100 nM purified motor protein (in buffer L), and let it sit for 5 min. Full-length kinesin motor proteins are expressed and purified from baculoviral expression system.
      6. Wash out unbinding proteins with 3 x 20 µl buffer L.
      7. Flow in 25 µl polarity marked microtubules at a 1/250 dilution into the chamber.
        For 100 µl volume, mix 95.4 µl buffer L, 1 µl 100 mM ATP, 1 µl 1 mM taxol, 2 µl anti-fade, 0.2 µl 1 M DTT, and 0.4 µl MT’s.
    2. Observe microtubule motility under fluorescence microscope (Nikon E600, excitation wavelength, 547 nm; emission wavelength, 576 nm. Also, see following Video 1).

      Video 1. Example of microtubule gliding assay

Data analysis

  1. Figure 2 illustrates the principle of a gliding assay. The motor proteins are fixed onto the coverslip and then an aliquot of fluorescent microtubules is introduced into the chamber and caught by the motor. Individual fluorescent microtubules move continuously in a unidirectional manner. If the motor is plus-end directed, the microtubule itself will translocate in the minus-end leading direction, and vice versa for a minus-end directed motor.


    Figure 2. Illustration of a gliding assay. Full-length motor protein is fixed onto coverslip, then polarity-marked microtubules are introduced to the chamber. Microtubule motility is observed under fluorescence microscope.

  2. Figure 3 shows a plus-end directed motor moving microtubules. The microtubule is in the minus-end leading direction. ImageJ was used to measure the distance of the microtubule movement, and the velocity was subsequently calculated.


    Figure 3. Polarity-marked microtubule translocation in a gliding assay. MT gliding driven by purified kinesin-5 protein. MT is minus-end leading, which suggests that kinesin-5 is a plus-end-directed motor. Scale bar = 5 µm (Tao et al., 2006).

Notes

  1. In the gliding assay, the motor proteins must be freshly purified to ensure maximum activity.
  2. While making polarity marked microtubules, it is recommended to prepare the bright GMPCPP seed and the dim elongation mix simultaneously. Bright seed mix needs to be added to dim elongation mix right after the seed is made.

Recipes

  1. BRB80 buffer solution
    1.80 mM PIPES
    1 mM MgCl2
    1 mM EGTA, pH 6.8
  2. Buffer L
    1.20 mM Tris
    75 mM KCl
    2 Mm MgCl2
    1 mM DTT
    0.1 mM ATP
    Adjust pH to 8.0
    Protease inhibitors (Final concentrations: Aprotinin, 2 μg/ml; Benzamidine, 20 μg/ml; Leupeptin, 1 μg/ml; Pepstatin A, 1 μg/ml; PMSF, 0.1 mM; SBTI, 100 μg/ml; TAME, 40 μg/ml), added just before use
  3. Anti-fade solution
    1. Mix 10 mg of catalase, 3 mg of glucose oxidase, 180.2 mg glucose, 100 µl 1 M DTT, 1.902 mg EGTA, and 100 µl 10x PBS
    2. Fill with ddH2O up to 1 ml
    3. Aliquot into 20 µl, and store at -80 °C

Acknowledgments

This method is adopted from Tao et al. (2016) and Tao et al. (2010). The above work was funded by NIH grant GM046409 to W.S. and NIH grant GM 55507 to J.M.S.

References

  1. Coppin, C. M., Pierce, D. W., Hsu, L. and Vale, R. D. (1997). The load dependence of kinesin's mechanical cycle. Proc Natl Acad Sci U S A 94(16): 8539-8544.
  2. 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.
  3. 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.
  4. Tao, L. and Scholey, J. M. (2010). Purification and assay of mitotic motors. Methods 51(2): 233-241.

简介

该协议的目的是提供一种更新的微管滑翔测定方法,并使用荧光显微镜对其进行可视化。

有丝分裂主轴是主导有丝分裂的蛋白质机械。有丝分裂纺锤体利用基于微管的运动蛋白来组织自身,并施加力量驱动细胞分裂。基于微管的运动蛋白使用源自ATP水解的能量产生机械工作(Coppin等人,1997)。运动蛋白以单向方式转移微管。运动的行为可以通过体外滑行测定(Tao和Scholey,2010)来观察,其中将电机固定在玻璃表面上并提供微管和ATP。然后可以使用荧光显微镜研究运动蛋白的运动性,并且可以实时观察其动态行为的细节。该更新的方案将允许使用体外微管滑动测定法(Tao等人,2006和2016)分析基于微管的运动蛋白功能。

关键字:滑动分析法, 有丝分裂马达蛋白, 体外运动, 标记极性的微管, 玻璃室

材料和试剂

  1. 移液器提示(康宁,目录号:4860)
  2. 离心管(Corning,目录号:430290)
  3. 厚壁聚碳酸酯管(Beckman Coulter,目录号:343775)
  4. 盖片(Sigma-Aldrich,目录号:Z692263)
  5. 显微镜幻灯片(Fisher Scientific,目录号:S17466A)
  6. 盖玻片(Fisher Scientific,目录号:S175211A)
  7. 鸟苷-5' - [(α,β) - 甲基]三磷酸(GMPCPP)(10mM)(Jena Bioscience,目录号:NU-405S)
  8. 微管蛋白(CYTOSKELETON,目录号:TL238A)
  9. 罗丹明微管蛋白(CYTOSKELETON,目录号:TL590M)
  10. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  11. N-乙基马来酰亚胺(Sigma-Aldrich,目录号:E3876)
  12. 鸟苷5'-三磷酸(GTP)(Sigma-Aldrich,目录号:G8877)
  13. 氯化氢(HCl)(Sigma-Aldrich,目录号:295426)
  14. 酪蛋白(Sigma-Aldrich,目录号:C7078)
  15. ATP(Sigma-Aldrich,目录号:A6419)
  16. 紫杉醇(紫杉醇)(Sigma-Aldrich,目录号:T7402)
  17. 哌嗪-N,N'-双(2-乙磺酸)(PIPES)(Sigma-Aldrich,目录号:P6757)
  18. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M4880)
  19. EGTA(Sigma-Aldrich,目录号:E3889)
  20. Tris碱(Sigma-Aldrich,目录号:T1503)
  21. 氯化钾(KCl)(Sigma-Aldrich:P5405)
  22. 蛋白酶抑制剂:
    抑肽酶(Sigma-Aldrich,目录号:A3428)
    苄脒(Sigma-Aldrich,目录号:B6505或12072)
    注意:产品"B6505"已经停产。
    消化抑素A(Sigma-Aldrich,目录号:P5318)
    苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
    亮肽素(Sigma-Aldrich,目录号:L2884)
    大豆胰蛋白酶抑制剂(SBTI)(Sigma-Aldrich,目录号:T6522)
    叔戊基甲基醚(TAME)(Sigma-Aldrich,目录号:283096)
  23. 过氧化氢酶(Sigma-Aldrich,目录号:C1345)
  24. 葡萄糖氧化酶(Sigma-Aldrich,目录号:C6766或G7141)
    注意:产品"C6766"已经停产。
  25. 葡萄糖(Sigma-Aldrich,目录号:D9434)
  26. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P5368)
  27. BRB80缓冲溶液(参见食谱)
  28. 缓冲液L(见配方)
  29. 防褪色解决方案(见配方)

设备

  1. 微量移液器
    2-20μl(Sigma-Aldrich,目录号:Z717304)
    10-100μl(Sigma-Aldrich,目录号:Z717312)
  2. 超离心机(Beckman Coulter,型号:TLA-100)
  3. NanoDrop(Thermo Fisher Scientific,Thermo Scientific TM ,型号:NanoDrop TM 2000)
  4. 荧光显微镜(Nikon Instruments,型号:Eclipse E600)
  5. 水浴(Thermo Fisher Scientific,型号:Precision TM 通用浴,目录号:TSGP02)

软件

  1. ImageJ(版本1.51J, https://imagej.nih.gov/ij/

程序

  1. 极性标记微管
    1. 使明亮的GMPCPP种子,作为微管聚合的"底漆"以及微管负端的指示剂。
      1. 通过将250微克微管蛋白溶解在25微升的BRB80中,制成10毫克/毫升的微管蛋白溶液
      2. 通过将20μg溶解在2μlBRB80中制成10mg/ml的罗丹明微管蛋白溶液。
      3. 混合4微升10毫克/毫升微管蛋白溶液,2微升10毫克/毫升罗丹明微管蛋白溶液,18微克BRB80,3微升10毫米DTT和3微升10毫摩尔GMPCPP。冰上的商店搭配。
      4. 在4℃下以410,000 x g离心7分钟。
      5. 取出3μl亮的种子混合上清液,并在37℃的水浴中孵育15分钟
    2. 制备N-乙基马来酰亚胺微管蛋白
      1. 在冰上制成10毫克/毫升的微管蛋白溶液(参见步骤A1a)。
      2. 制备新鲜的1mM N-乙基马来酰亚胺(NEM)溶液。
        加入0.5μl50 mM NEM至25μl10 mg/ml微管蛋白。让溶液在室温下放置5分钟
      3. 向25μlNEM微管蛋白溶液中加入0.5μl1 M DTT。让溶液坐在冰上30分钟。
      4. 在4℃下以350,000×g离心NEM-微管蛋白溶液15分钟。收集上清液。
      5. 用280nm波长下的纳米脱落物测量上清液的吸光度
    3. 使暗淡的伸长混合物
      1. 使10μl伸长率混合:
        1.34μl未标记的微管蛋白(10mg/ml)
        0.17μl若丹明微管蛋白(10mg/ml)
        3微升NEM微管蛋白(4毫克/毫升)
        1μl10mM DTT(首先将1M DTT稀释到10mM DTT中) 0.2μl50 mM GTP
        4.29μl1x BRB80
      2. 将混合物在37℃的水浴中孵育1分钟
    4. 使极性标记为微管。
      1. 加入2μl明亮的种子混合物至10μl暗淡伸长率混合物,轻轻混合
      2. 将溶液在37℃的水浴中孵育30分钟,并逐渐加入紫杉醇至10μM终浓度。将微管储存在室温下,防止光照。

  2. 滑翔测定
    1. 做玻璃室
      1. 用HCl洗涤盖玻片(22×22mm),用HCl洗涤24小时,并用ddH 2 O洗涤10分钟,每次3次。空气干燥盖玻片24小时。
      2. 使用顶部和底部的两片双面胶带将盖玻片连接到玻璃滑块(75 x 25 mm)上。两侧应有开放狭缝(图1)。


        图1.使用双面胶带的玻璃滑动室的图示

      3. 将25μl1mg/ml酪蛋白流入室中,放置5分钟。酪蛋白作为运动蛋白锚定垫。
      4. 用3 x 20μl的缓冲液L冲洗未结合的酪蛋白
      5. 在10-100nM纯化的运动蛋白(缓冲液L)中流动,并使其静置5分钟。从杆状病毒表达系统表达和纯化全长驱动蛋白运动蛋白
      6. 用3×20μl缓冲液L冲洗脱粘蛋白
      7. 流出25μl极性标记的微管,以1/250稀释进入腔室。
        对于100μl体积,混合95.4μl缓冲液L,1μl100mM ATP,1μl1mM紫杉醇,2μl抗褪色剂,0.2μl1M DTT和0.4μlMT。
    2. 观察荧光显微镜下的微管运动(Nikon E600,激发波长,547nm;发射波长,576nm,另见视频1)。

      Video 1. Example of microtubule gliding assay

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


数据分析

  1. 图2说明滑翔测定的原理。将运动蛋白固定在盖玻片上,然后将等分的荧光微管引入室中并被马达捕获。单个荧光微管以单向方式连续移动。如果电动机是正端导向的,微管本身将以负端导向方向移位,反之亦然。

    图2.滑翔测定图。将全长运动蛋白固定在盖玻片上,然后将极性标记的微管引入腔室。在荧光显微镜下观察微管运动
  2. 图3显示了移动微管的正端导向马达。微管处于负端引导方向。 ImageJ用于测量微管运动的距离,随后计算速度

    图3.滑翔测定中极性标记的微管易位。由纯化的驱动蛋白-5蛋白驱动的MT滑移。 MT是负端领先,这表明驱动蛋白-5是一个正端导向的马达。比例尺=5μm(Tao等人,2006)。

笔记

  1. 在滑翔测定中,运动蛋白必须新鲜纯化,以确保最大程度的活动
  2. 在极性标记微管的同时,建议同时制备光亮的GMPCPP种子和昏暗的伸长率混合物。在种子制作之后,需要加入明亮的种子混合物到暗淡的伸长率混合物。

食谱

  1. BRB80缓冲溶液
    1.80 mM PIPES
    1mM MgCl 2
    1mM EGTA,pH6.8
  2. 缓冲区L
    1.20 mM Tris
    75 mM KCl
    2 Mm MgCl 2
    1 mM DTT
    0.1 mM ATP
    将pH调节至8.0
    蛋白酶抑制剂(终浓度:抑肽酶2μg/ml;苯脒20μg/ml;亮肽素1μg/ml;抑胃酶A,1μg/ml; PMSF,0.1mM; SBTI,100μg/ml; TAME, μg/ml),在使用前加入
  3. 防褪色解决方案
    1. 混合10 mg过氧化氢酶,3 mg葡萄糖氧化酶,180.2 mg葡萄糖,100μl1 M DTT,1.902 mg EGTA和100μl10x PBS
    2. 填充ddH 2 O至1 ml
    3. 等分成20μl,并储存在-80℃

致谢

这种方法是从陶等人采用的。 (2016)和陶等人。 (2010)。上述工作由NIH授予GM046409资助给W.S. NIH将GM 55507授予J.M.S.

参考文献

  1. Coppin,CM,Pierce,DW,Hsu,L.and Vale,RD(1997)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9238012"target ="_ blank">驱动蛋白机械循环的负载依赖性。 Proc Natl Acad Sci USA 94(16):8539-8544。
  2. 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.
  3. Tao,L.,Mogilner,A.,Civelekoglu-Scholey,G.,Wollman,R.,Evans,J.,Stahlberg,H.and Scholey,JM(2006)。< a class ="ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/17141610"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。
  4. Tao,L.and Scholey,JM(2010)。  有丝分裂马达的纯化和测定。 51(2):233-241。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shim, A., Tezuka, G., Kupcha, L. and Tao, L. (2017). Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics. Bio-protocol 7(7): e2210. DOI: 10.21769/BioProtoc.2210.
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