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Deflagellation and Regeneration in Chlamydomonas
衣藻的鞭毛脱落和再生   

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Abstract

Eukaryotic cilia/flagella are one of the only cellular structures that can be removed without injuring cells, can be highly purified for biochemical analysis, and, in many cells, can be completely reassembled within 90 minutes. Following amputation, the expression of many flagellar genes is up-regulated, and many are packaged and associated with intraflagellar transport (IFT) particles for transport to flagellar bases and into growing flagella. Studies of deciliation and ciliary growth provide insight to mechanisms that regulate microtubule assembly and length, mechanisms that regulate the transport of soluble cytoplasmic proteins into the ciliary compartment and their assembly into microtubules, and mechanisms that regulate trafficking of membrane proteins and lipids to the plasma membrane or to ciliary bases and their movement into and out of the cilium. These are important for motility and for signal transduction.
Deciliation methods for many cells have been developed and most require extracellular calcium ions and activation of signaling pathways that regulate microtubule severing (Quarmby, 2009). Deciliation occurs at the distal end of the basal bodies and, as soon as axonemes are severed, the membrane reseals and basal bodies begin to regenerate cilia.
Chlamydomonas is an ideal organism with which to study ciliary regeneration. Cells are easily and inexpensively cultured, flagellar amputation and regeneration is uniform in all cells in a population and growth can be assayed by observing fixed or living cells with a phase contrast microscope equipped and a 40x objective lens. Flagellar regeneration on individual living cells can be observed using paralyzed mutants immobilized in agarose. Because deflagellation leaves cells intact, the released flagella can be purified without contamination with cellular debris. The most reliable deciliation and regeneration method is the pH shock method developed by Reference 5 (also see References 4 and 11). Other methods are reviewed by Quarmby, (2009). The pH shock method is primarily used for Chlamydomonas but can be used for deciliation and regeneration of Tetrahymena cilia (Gaertig et al., 2013).


Figure 1. Typical flagellar regeneration curve showing phase contrast images of Chlamydomonas cells photographed before deflagellation and during regeneration. The average flagellar lengths of a population of regenerating cells is shown in red and the average flagellar lengths on a population of nondeflagellated cells is in blue.

Materials and Reagents

  1. Cells
    Chlamydomonas cells can be obtained from a variety of sources and pure strains of a variety of flagellar mutants can be obtained from the Chlamydomonas Resource Center (http://chlamycollection.org/contact-us/).
  2. Media and cell culture (see Notes)
  3. 0.5 N acetic acid
  4. 0.5 M NaOH
  5. 2% glutaraldehyde in M medium or Lugols iodine (see Recipes)  
    Note: For electron microscopy, one should use fresh glutaraldehyde from sealed ampuoles. To measure flagellar lengths, the age of the glutaraldehyde is not critical.

    Optional:
  6. 2.5% low EEO agar (Thermo Fisher Scientific, FisherBiotechTM, catalog number: BP160-100 )
    Note: It is used to observe flagellar growth or maintenance on individual living cells.
  7. 5 mM (final concentration) colchicine (to inhibit microtubule assembly)
    Note: These experiments are carried out in phosphate-buffered Minimal medium. Avoid Tris-containing buffers because they may inhibit the effects of colchicine (Margulis et al., 1969).
  8. 10 µg/ml (final concentration) Cycloheximide
    Note: It is used to inhibit protein synthesis.
  9. VALAP (see Recipes)
    Note: It is used to support coverslips to examine living cells without inducing deflagellation by coverslip pressure.

Equipment

  1. pH meter calibrated for pH 4-7
    Note: Some gel-filled electrodes are not accurate across this pH range.
  2. Centrifuge and tubes
    Note: For small scales, a clinical centrifuge and conical tubes.
  3. Phase contrast microscope or brightfield microscope
    Note: If cells are fixed with Lugol’s iodine, a 40x lens is ideal for flagellar length measurements.
  4. Orbital shaker
  5. Magnetic stirrer
  6. Stir bar

Software

  1. Image J (http://imagej.nih.gov/ij/)

Procedure

  1. Check cells with a 40x lens and a phase microscope to be certain that cells are healthy, contain two flagella, and are motile (unless paralyzed mutants are studied). Dividing cells lack flagella and are surrounded by a refractile cell wall containing 4-8 cells. Cultures with dividing cells are not synchronous and are not used for regeneration experiments.
  2. Harvest cells (~5x105 cells/ml) using a clinical centrifuge, for up to 120 ml of culture, or low speed preparative centrifuge (3 min, 1,500 x g) pour off the supernatant.
  3. Suspend cells in 20-100 ml of fresh medium in a beaker by gentle swirling or gently drawing cells up and down through a large-bore pipette (I break off the tips of plastic pipettes.).
  4. Check cells with a phase microscope to insure that they are uniformly flagellated and have not been deflagellated during centrifugation and resuspension.
    Optional: Agarose to observe flagella on immobilized living cells.
    1. Dissolve 2.5% low EEO agar in culture medium and maintain at 45 °C for use.  
    2. Apply cells to a clean coverslip, draw off most of the liquid, and apply ~20 microliters of agarose at 40 °C.
    3. Rapidly invert over a clean microscope slide and seal the edges with VALAP (1:1:1 Vaseline, Lanolin, Paraffin).  
    4. Flatten the bead of VALAP around the edge of the coverslip with a warm spatula.
      Note : To capture time-lapse images of regenerating flagella use a microscope equipped with a UV filter.  Keep the illumination low and be certain that non-deflagellated cells maintain full length throughout the observation period (see Reference 1).
  5. Stir cells using a magnetic stirrer and dropwise add 0.5 N acetic acid until the pH reaches 4.5-4.0.  Add acetic acid as rapidly as possible but be certain that it is well mixed as it is added to the medium. Keep at pH 4 for no longer than ~1 minute and then raise the pH to 6.8-7.2 by dropwise adding 0.5 N KOH. For 100 ml of cells in M medium, use approximately 4.5 ml of 0.5 N acetic acid and 5.2 ml of 0.5 M KOH.  For most purposes, it is best to monitor the pH change but, for mass-deflagellations in microtitre plates, adding defined quantities of acetic acid and KOH is useful.
  6. For regeneration
    1. Pellet cells using a clinical centrifuge, and suspend in fresh M medium to ~105 cells/ml. It is not critical to suspend in fresh medium but I generally do so.
    2. Incubate cells in light with aeration using bubblers or by gentle rotation in an Erlenmeyer flask on an orbital shaker.
    3. Flagella generally will start to grow within 10-15 min and will be fully grown by 60-90 min.
    4. To measure flagella on regenerating cells, fix samples at 5-10 min intervals with glutaraldehyde or Lugol’s iodine.
      1. Fix: 2% glutaraldehyde in M medium. Dilute 100 microliters of cells + 100 microliters of 2% glutaraldehyde and let settle in microfuge tubes. Measure flagella within 1-2 days. Longer fixation will lead to clumping of cells at the bottom of the tube, which makes measurement of individual flagella very difficult.
      2. Alternatively, fix by diluting 80 microliters of cells + 20 microliters Lugol’s iodine. Measure flagella within 1-2 days to avoid clumping of flaglellated cells.
      3. Regeneration can be inhibited reversibly by the addition of colchicine, an inhibitor of microtubule assembly.  If cycloheximide is added at the time of deflagellation, flagellar regeneration will be limited to half-length due to the exhaustion of a pool of proteins critical for flagellar assembly (Rosenbaum et al., 1969).
  7. Measuring flagella: There are a variety of ways to measure flagella and one has to balance accuracy with the ease of measuring large numbers of flagella to minimize error.  Even if flagella are planar when fixed, they are curved and are viewed at various angles, depending on the orientation of the cell on the microscope slide. Very short flagella may be difficult to see if the cell is not properly oriented to reveal the flagella.  It also is difficult to identify the flagellar base because it is surrounded by the cell wall, which can obscure the first micron of a flagellum above the basal body. Thus, the observer must use their judgment when measuring flagella and be consistent with all measurements. One can use complex image analysis involving the Z-axis (see Reference 9) but one also could simply deflagellate cells at various time points, photograph isolated flagella attached to a coverslip, and measure their lengths with a variety of image programs.
    The easiest method to measure flagella is to use an ocular micrometer and record measurements of flagella on fixed cells by hand.  I prefer to use a ccd or video camera that interacts with Image J, a free software program (http://imagej.nih.gov/ij/).  Trace the flagella using a mouse, save the measurements, and determine the average lengths and distributions of length (using histograms) using Excel or similar spreadsheets.
  8. Flagella are easily purified from pH-shocked cells following methods described elsewhere (these protocols).

Notes

  1. Notes about media and cell culture
    1. Culture media recipes are available at http://www.chlamy.org/media.html.
    2. I prefer to culture cells in at room temperature in  Sager and Granick (1953) minimal (M) medium (http://www.chlamy.org/SG.html; Harris, 1989) and synchronize cells on a 12 h dark/light cycle to insure that all cells are at the same point in the cell cycle.  This is important because Chlamydomonas cells disassemble their flagella prior to division and one mutant, pf18, gradually disassembles its flagella 4-6 h after the beginning of the light cycle (Tuxhorn et al., 1998). Cell density is not critical but cells grown to stationary stage (> 106 cells/ml) often are not 100% flagellated.
    3. Autoclave media in Erlenmeyer flasks fitted with a foam plug and cotton-plugged glass Pasteur pipette for aeration with house air or an aquarium pump.  For deflagellation and regeneration, cultures of 50-100 ml of cells grown to ~ 5 x 105 cells/ml is adequate.  
    4. To isolate flagella, culture 8-16 L of cells in aerated 4 L bottles and concentrate cells to 100-200 ml for pH shock.

Recipes

  1. Lugol’s Iodine
    Mix 6% potassium iodide in distilled water and add 4% iodine crystals
    Mix by stirring overnight and store indefinitely

Acknowledgments

This protocol was adapted from Lefebvre (1995), Margulis et al. (1969) and Witman (1986).

References

  1. Dentler, W. L. and Adams, C. (1992). Flagellar microtubule dynamics in Chlamydomonas: cytochalasin D induces periods of microtubule shortening and elongation; and colchicine induces disassembly of the distal, but not proximal, half of the flagellum. J Cell Biol 117(6): 1289-1298.
  2. Gaertig, J., Wloga, D., Vasudevan, K. K., Guha, M. and Dentler, W. (2013). Discovery and functional evaluation of ciliary proteins in Tetrahymena thermophila. Methods Enzymol 525: 265-284.
  3. Harris, E. H. (1989). A comprehensive guide to biology and laboratory use. The Chlamydomonas Sourcebook. Academic Press
  4. Lefebvre, P. A. (1995). Flagellar amputation and regeneration in Chlamydomonas. Methods Cell Biol 47: 3-7.
  5. Margulis, L., Banerjee, S. and White, T. (1969). Colchicine-inhibited cilia regeneration: explanation for lack of effect in tris buffer medium. Science 164(3884): 1177-1178.
  6. Quarmby, L. M. (2009). Deflagellation. In: Witman, G. B. (ed). The Chlamydomonas Sourcebook second edition. Academic Press Vol 3:43-69.
  7. Rosenbaum, J. L., Moulder, J. E. and Ringo, D. L. (1969). Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins. J Cell Biol 41(2): 600-619.
  8. Sager, R. and Granick, S. (1953). Nutritional studies with Chlamydomonas reinhardi. Ann N Y Acad Sci 56(5): 831-838.
  9. Saggese, T., Young, A. A., Huang, C., Braeckmans, K. and McGlashan, S. R. (2012). Development of a method for the measurement of primary cilia length in 3D. Cilia 1(1): 11.
  10. Tuxhorn, J., Daise, T. and Dentler, W. L. (1998). Regulation of flagellar length in Chlamydomonas. Cell Motil Cytoskeleton 40(2): 133-146.
  11. Witman, G. B. (1986). Isolation of Chlamydomonas flagella and flagellar axonemes. Methods Enzymol 134: 280-290.

简介

真核纤毛/鞭毛是唯一的细胞结构之一,其可以被去除而不伤害细胞,可以被高度纯化用于生化分析,并且在许多细胞中,可以在90分钟内完全重新组装。截肢后,许多鞭毛基因的表达被上调,并且许多被包装和与用于运输到鞭毛基部和进入生长鞭毛的鞭毛内转运(IFT)颗粒相关联。脱落和睫毛生长的研究提供了调节微管装配和长度,调节可溶性细胞质蛋白质运输到睫状隔室和它们装配到微管中的机制以及调节膜蛋白和脂质运输到质膜的机制的机制或睫状基质及其进入和离开纤毛的运动。这些对于动力和信号转导是重要的。
已经开发了许多细胞的脱落方法,并且大多数需要细胞外钙离子和调节微管切断的信号通路的激活(Quarmby,2009)。脱屑发生在基体的远端,并且一旦轴突断裂,膜结合和基底体开始再生纤毛。
衣藻是治疗睫状体再生的理想生物体。细胞容易且廉价地培养,鞭毛截肢和再生在群体中的所有细胞中是均匀的,并且生长可以通过用装备有相差显微镜和40倍物镜的观察固定或活细胞来测定。使用固定在琼脂糖中的麻痹突变体可以观察对单个活细胞的鞭毛再生。因为絮凝使细胞完整,所以可以纯化释放的鞭毛而不污染细胞碎片。最可靠的脱胶和再生方法是由参考文献5开发的pH休克方法(也参见参考文献4和11)。其他方法由Quarmby,(2009)综述。 pH休克方法主要用于衣藻,但可用于四膜虫的纤毛脱落和再生(Gaertig等人,2013)。


图1.典型的鞭毛再生曲线显示在脱落前和再生期间拍摄的衣藻细胞的相衬图像。再生细胞群的平均鞭毛长度显示为红色和 在非鞭毛细胞群上的平均鞭毛长度是蓝色的。

材料和试剂

  1. 单元格
    衣藻细胞可以从多种来源获得,并且多种鞭毛突变体的纯株可以从衣藻资源中心获得( http://chlamycollection.org/contact-us/)。
  2. 培养基和细胞培养(见注释)
  3. 0.5 N乙酸
  4. 0.5 M NaOH
  5. 2%戊二醛的M培养基或Lugols碘(见Recipes) 
    注意:对于电子显微镜,应该使用来自密封安瓿瓶的新鲜戊二醛。 为了测量鞭毛长度,戊二醛的年龄不是关键的。

    可选 :
  6. 2.5%低EEO琼脂(Thermo Fisher Scientific,FisherBiotech< sup>,目录号:BP160-100)
    注意:它用于观察鞭毛生长或维持个体活细胞。
  7. 5mM(终浓度)秋水仙碱(以抑制微管装配)
    注意:这些实验在磷酸盐缓冲的最小培养基中进行。 避免含有Tris的缓冲液,因为它们可能会抑制秋水仙碱的作用(Margulis et al。,1969)。
  8. 10μg/ml(终浓度)环己酰亚胺
    注意:它用于抑制蛋白质合成。
  9. VALAP(参见配方)
    注意:它用于支持盖玻片以检查活细胞,而不会由于盖玻片压力而引起玷污。

设备

  1. pH计,校准pH 4-7
    注意:一些凝胶电极在这个pH范围内不准确。
  2. 离心机和试管
    注意:对于小规模,临床离心机和锥形管。
  3. 相差显微镜或明场显微镜
    注意:如果细胞用Lugol的碘固定,40x镜头是鞭毛长度测量的理想选择。
  4. 轨道振动器
  5. 磁力搅拌器
  6. 搅拌棒

软件

  1. 图片J( http://imagej.nih.gov/ij/

程序

  1. 检查单元格与一个40倍镜头和相位显微镜,以确定细胞是健康的,包含两个鞭毛,并动态(除非瘫痪突变体研究)。 分裂细胞缺乏鞭毛并被包含4-8个细胞的折射性细胞壁包围。 具有分裂细胞的培养物不是同步的,并且不用于再生实验
  2. 使用临床离心机,至多120ml培养物或低速制备型离心机(3分钟,1,500xg)灌注收获细胞(〜5×10 5个细胞/ml) 离开上清液。
  3. 将细胞悬浮在烧杯中的20-100ml新鲜培养基中,通过轻柔旋转或通过大口径移液管轻轻地抽吸细胞(我断开塑料移液管的尖端)。
  4. 用相位显微镜检查细胞,以确保它们均匀鞭毛,并且在离心和重悬期间没有萎缩。
    可选:琼脂糖在固定的活细胞上观察鞭毛。
    1. 将2.5%低EEO琼脂溶解在培养基中并保持在45℃下使用。  
    2. 将细胞应用于干净的盖玻片,抽出大部分液体,并在40℃下应用〜20微升的琼脂糖。
    3. 快速倒置在干净的显微镜载玻片上,并用VALAP(1:1:1凡士林,羊毛脂,石蜡)密封边缘。  
    4. 用温暖的抹刀将VALAP的边缘覆盖在盖玻片的边缘。
      注意  :捕获再生鞭毛的延时图像使用a 显微镜,配备UV过滤器。保持照明低和 确保非絮凝细胞在整个过程中保持全长  观察期(见参考文献1)。
  5. 使用磁力搅拌器搅拌细胞,并滴加0.5N乙酸,直到pH达到4.5-4.0。尽可能快地加入乙酸,但要确保当加入到培养基中时它充分混合。保持在pH4不超过〜1分钟,然后通过滴加0.5N KOH将pH升高到6.8-7.2。对于在M培养基中的100ml细胞,使用约4.5ml的0.5N乙酸和5.2ml的0.5M KOH。对于大多数目的,最好监测pH变化,但是对于微量滴定板中的质量减少,添加规定量的乙酸和KOH是有用的。
  6. 用于再生
    1. 使用临床离心机沉淀小细胞,并悬浮在新鲜的M培养基中 至约10 5个细胞/ml。在新鲜培养基中悬浮并不重要,但我 一般这样做。
    2. 使用鼓泡器或通过在定轨振荡器上的锥形瓶中轻轻旋转在光照下孵育细胞。
    3. 鞭毛一般将在10-15分钟内开始生长,并且将完全生长60-90分钟。
    4. 为了测量再生细胞上的鞭毛,以5-10分钟间隔用戊二醛或Lugol碘固定样品。
      1. 固定:2%戊二醛在M培养基中。 稀释100微升细胞+ 100微升的2%戊二醛并且在微量离心管中沉降。 在1-2天内测量鞭毛。 较长的固定会导致结块 的细胞在管的底部,这使得测量 个体鞭毛非常困难。
      2. 或者,修复 稀释80微升细胞+ 20微升Lugol碘。 在1-2天内测量鞭毛,以避免结块的斑点 细胞
      3. 再生可以被可逆地抑制 加入秋水仙碱,微管装配的抑制剂。如果 环己酰亚胺在絮凝时加入,鞭毛 再生将由于a的耗尽而限于半长 对鞭毛集合至关重要的蛋白质库(Rosenbaum等人, 1969)。
  7. 测量鞭毛:有多种方法来测量鞭毛,一个必须平衡精度与容易测量大量鞭毛,以最大限度地减少错误。即使鞭毛在固定时是平面的,它们是弯曲的并且以各种角度观察,这取决于显微镜载玻片上的细胞的取向。如果细胞没有正确定向以显露鞭毛,则很短的鞭毛可能很难看到。还难以鉴定鞭毛基部,因为其被细胞壁包围,这可以遮蔽基部本体上方的鞭毛的第一微米。因此,观察者在测量鞭毛时必须使用他们的判断,并且与所有测量一致。可以使用涉及Z轴的复杂图像分析(参见参考文献9),但是也可以在不同时间点简单地使细胞瘪缩,将附着于盖玻片的分离的鞭毛照相,并且用各种图像程序测量它们的长度。 > 测量鞭毛最简单的方法是使用眼测微计,并用手记录固定细胞上鞭毛的测量值。我更喜欢使用与Image J(一个免费软件程序)互动的ccd或视频相机( http://imagej。 nih.gov/ij/)。使用鼠标跟踪鞭毛,保存测量,并使用Excel确定长度的平均长度和分布(使用直方图) 或类似的电子表格
  8. 根据其他地方(这些方案)描述的方法,鞭毛容易从pH-震荡细胞中纯化。

笔记

  1. 关于媒体和细胞培养的注意事项
    1. 您可以在 http://www.chlamy.org/media.html 上查看文化媒体食谱。 br />
    2. 我更喜欢在Sager和Granick(1953)中在室温下培养细胞, 最小(M)媒体( http://www.chlamy.org/SG.html ; Harris ,1989) 在12小时黑暗/光周期上同步细胞以确保所有细胞 在细胞周期的同一点。 这是很重要的,因为 衣藻细胞在分裂前分解它们的鞭毛   突变体pf18,后4-6 h逐渐解体其鞭毛 光周期的开始(Tuxhorn等人,1998)。 细胞密度不是   但是细胞常常生长至静止期(> 10 6细胞/ml) 不是100%鞭毛。
    3. 高压灭菌器在锥形瓶中 配有泡沫塞和棉塞玻璃巴斯德吸管 室内空气或水族馆泵的曝气。 为了deflagellation和 再生,生长至〜5×10 5个细胞/ml的50-100ml细胞的培养物   是足够的。  
    4. 为了分离鞭毛,在充气的4L瓶中培养8-16L细胞,并将细胞浓缩至100-200ml用于pH休克。

食谱

  1. Lugol的碘
    将6%碘化钾在蒸馏水中混合,加入4%碘晶体 通过搅拌混合过夜并无限期储存

致谢

该方案改编自Lefebvre(1995),Margulis等人(1969)和Witman(1986)。

参考文献

  1. Dentler,W.L.and Adams,C。(1992)。 衣藻中的鞭毛微管动力学:细胞松弛素D诱导微管缩短和伸长的时期;并且秋水仙碱诱导鞭毛的远端但不是近端的一半的拆卸。          117(6):1289-1298。
  2. Gaertig,J.,Wloga,D.,Vasudevan,K.K.,Guha,M.and Dentler,W。(2013)。 热敏四膜虫中睫状蛋白的发现和功能评价。 Methods Enzymol 525:265-284。
  3. Harris,E.H。(1989)。生物学和实验室使用的综合指南。 The Chlamydomonas Sourcebook 。学术出版社
  4. Lefebvre,P.A。(1995)。 衣藻中的鞭毛截肢和再生。 方法细胞生物学 47:3-7。
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引用:Dentler, W. (2014). Deflagellation and Regeneration in Chlamydomonas. Bio-protocol 4(12): e1155. DOI: 10.21769/BioProtoc.1155.
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