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Fluorescence Recovery after Photobleaching (FRAP) Assay to Measure the Dynamics of Fluorescence Tagged Proteins in Endoplasmic Reticulum Membranes of Plant Cells
荧光漂白恢复法(FRAP)进行植物细胞内质网膜中荧光标记蛋白的动力学分析

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Abstract

In this protocol, we used fluorescence recovery after photobleaching (FRAP) to measure the influence that some mutations and drug treatment have on mobility of a green fluorescent protein (GFP)-fused viral transmembrane protein into endoplasmic reticulum membranes (Serra-Soriano et al., 2014). The proteins of interest were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermic cells by agro-infiltration. To minimize transient overexpression artifacts, fluorescence intensity values were gathered at 36 hpi using an inverted Zeiss LSM 780 confocal microscope. Only epidermic cells showing moderated expression levels and homogenous distribution through the ER of the GFP-tagged proteins were used for further experiments. To examine the role of actin polymerization in the mobilization of GFP-tagged proteins, we pretreated tissue samples either with latrunculin B, an inhibitor of actin polymerization, or with DMSO as control. The generated fluorescence recovery curves were used to obtain the percentage of maximum fluorescence recovery (MFR), which corresponds to the mobile fraction, and the half-time of maximum recovery (t1/2) values.

Materials and Reagents

  1. 3-4 weeks old Nicotiana benthamiana plants
  2. Agrobacterium tumefaciens (A. tumefaciens) C58C1 strain or similar transformed with the binary vector pMOG800 harboring the protein coding sequence. In our case they were:
    1. pMGFP-p7B encoding GFP-fused Melon necrotic spot virus (MNSV) p7B
    2. pMGFP-p7B [D7AP10A] encoding GFP-fused MNSV p7B carrying D7AP10A mutation
    3. pMGFP-p7B [K49A] encoding GFP-fused MNSV p7B carrying K49A mutation
    4. pMGFP-KDEL encoding a GFP engineered to be targeted to the ER lumen
  3. Yeast extract (Difco, catalog number: 212750 )
  4. Tryptone (Difco, catalog number: 211705 )
  5. Sodium chloride (NaCl) (Panreac Applichem, catalog number: 131659 )
  6. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  7. MES (Sigma-Aldrich, catalog number: M8250 )
  8. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M9272 )
  9. Antibiotics
    1. Kanamycin (50 mg/ml) (Sigma-Aldrich, catalog number: K4000 )
    2. Rifampicin (50 mg/ml) (Duchefa Biochemie, catalog number: R0146 )
  10. Mowiol® 4-88 (Sigma-Aldrich, catalog number: 81381 )
  11. Dabco® 33-LV (Sigma-Aldrich, catalog number: 290734 )
  12. Tris base (Roche Diagnostics, catalog number: 03118142001 )
  13. Tris-HCl (1 M, pH 5.6)
  14. Immersion oil Immersol 518 F (ZEISS, catalog number: 444960-0000-000 )
  15. Latrunculin B (Sigma-Aldrich, catalog number: L5288 )
  16. 25 µM Latrunculin B solution in 10 mM DMSO
  17. LB medium (see Recipes)
  18. Agrobacterium infiltration buffer (see Recipes)
  19. Mowiol mounting medium (see Recipes)

Equipment

  1. 28 °C growing chamber
  2. 15 ml culture tubes
  3. Swinging centrifuge rotor for 15 ml tubes
  4. 1.5 ml tubes (standard Eppendorf tubes or similar)
  5. BioPhotometer plus (Eppendorf)
  6. 1 ml syringes without needle
  7. Plant growing chamber
  8. Fine paddle forceps
  9. Petri culture dishes 35 x 10 mm
  10. Microscope slides 76 x 26 mm (Menzel-Gläser, catalog number: AA00000112E )
  11. Microscope cover slips 24 x 24 mm Nr. 1 (Menzel-Gläser, catalog number: BB024024A1 )
  12. Adhesive one-sided tape
  13. A cork borer (one centimeter diameter) or similar cutting device such as a scalpel blade.
  14. An inverted confocal microscope (ZEISS, model: LSM 780)
  15. Plan-Apochromat 63x/1.40 oil objective

Software

  1. Graphpad prism software (http://www.graphpad.com)
  2. MS Excel

Procedure

  1. Transient expression of proteins by agro-infiltration
    1. Inoculate a single colony of transformed A. tumefaciens in 5 ml of LB medium with Kanamycin (50 µg/ml) and Rifampicin (50 µg/ml). Incubate the culture (12 h or more) at 28-30 °C with vigorous shaking.
    2. Measure the A600 of the culture.
    3. Collect the bacteria by slow-speed centrifugation (2,000 x g, 15 min) in a swinging rotor. Remove the supernatant.
    4. Resuspend the pellet with Agrobacterium infiltration buffer. The final A600 should be adjusted to 0.2.
    5. Leave at room temperature for 2 h before infiltration.
    6. Infiltrate the cultures with 1 ml syringe into the lower side of the leaves. Use 3-4 week-old N. benthamiana plants and avoid cotyledons. Use your fingertip to apply gentle counter pressure to the other side of the leaf. A fully infiltrated part of the leaf gives a water-soaked appearance.
    7. Keep the plants in growth chambers in 16 h light at 25 °C and 8 h dark at 22 °C.

  2. Microscope slide preparation
    1. To minimize transient overexpression artifacts in the FRAP experiments plants were used 36 h after infiltration.
    2. Cut a small disk of the infiltrated leaf with a cork borer or similar device. Try to avoid the leaf veins to eliminate any potential irregularity or significant depression in the sample.
    3. In the case of drug treatment, immerse tissue samples into latrunculin B (1 ml of a 25 µM solution) in a small petri culture dish (35 x 10 mm) for 1h. Perform non-treated control by tissue immersion into 10 mM DMSO.
    4. Using fine paddle forceps place the leaf disk in a microscope slide with the back side facing up and mount in Mowiol mounting medium.
    5. Add a coverslip and stick them to the slide with adhesive one-sided tape.
    6. Place a drop of immersion oil on the cover slip over the leaf disk.

  3. Fluorescence recovery after photobleaching (FRAP) assay
    1. FRAP experiments were performed on an inverted Zeiss LSM 780 confocal microscope using the LSM FRAP Module from Zen 2011 software.
    2. Switch on the laser lines for imaging the sample.
    3. Find epidermic cells showing moderated expression levels and homogenous distribution through the ER of the GFP-tagged proteins with the Plan-Apochromat 40x/1.4 oil objective and then switch to the Plan-Apochromat 63x/1.40 oil objective to capture images.
    4. Select “range indicator”. Now image turn into one of two-color (red and blue) (Figure 1). The red areas indicated saturation of the detector due to high fluorescence intensity. Similarly, the blue area either represents no fluorescence or intensity lower than the detector can sense. Only a few pixels have to be saturated (in red), if not, adjust the pinhole, laser intensity, or detector gain.


      Figure 1. Laser confocal scan in green A and range indicator mode B.
      The saturated areas in red are indicated.

    5. Use 5x optical zoom and 256 x 256 pixel resolution to take a single image of a short piece of the endoplasmic reticulum (for this purpose use “Snap” function). This zoom factor should stay the same between samples.
    6. Click the bleaching option on the acquisition tab. Time series and region options will be automatically selected.
    7. To define the regions of interest (ROI) which should be bleached select the circular shape in the regions menu and draw four circular ROIs of 1.7 μm diameter in fluorescent regions of the sample. Select a flat membrane region of the ER significantly larger than the bleach ROIs. Three of ROIs will be bleached and the fourth one will be used as the non-bleached reference to correct the FRAP curves for photofading caused by imaging. Locate the reference ROI as far apart as possible from the bleached ROIs. Ensure that a region which is background is in your image and draw a fifth circular ROI inside. In fast recovery rates, it is important to use small ROIs to decrease at maximum the time interval between photobleaching and imaging.


      Figure 2. Spatial distribution of the región of interest (ROIs).
      Reference ROI must be located away from bleached ROIs to be not affected by the laser bleach. Background ROI should be in a non-fluorescent area (blue pixels in range indicator mode).

    8. To setup bleaching protocol select start bleaching after 3 scans and use 100 iterations of the bleach laser over the bleach region. Do not use zoom bleach. In excitation of bleach, select the 488 nm line of the Argon laser (25% power, 100% transmission). After bleaching, try to reduce the ROI intensity between 50-75% without photodamage to the cell. After bleaching collect images at low laser power (25% power, 2% transmission).
    9. Open the time series menu and specify the total number of images in the final data set (before and after bleaching) by setting the number of cycles to 100. This results in an interval between the beginning of each scan of approximately 191 ms. To further increase the rate of data acquisition, use bidirectional scanning.
    10. Start FRAP experiment by pressing “Start Experiment button” and examine the FRAP curve on the computer. ROI reference intensity should remain constant or be slightly reduced due to photofading during image acquisition Repeat FRAP on different cells and days to ensure reproducibility. To allow comparison between different cells and proteins, the detector gain can be readjusted but all other settings must be kept the same.

  4. FRAP data processing
    1. FRAP data generated using the LSM systems can be displayed and analyzed with the confocal operating software. Alternatively, raw fluorescence intensity values from bleached, background and reference ROIs can be exported to Microsoft Excel to be double normalized (Phair et al., 2004).
    2. Calculate the photobleaching rate (R) by comparing the intensity of the fluorescence of the reference ROI before (RF0, average intensity of the three scans taken before bleaching) and at each scan after photobleaching (RFn) Rn = Fn/F0.
    3. Normalize the fluorescence intensity of the bleached ROIs (BleachedFn) as follows: NF = (BleachedFn - BackgroundFn)/r.
    4. Copy the data to a program such as GraphPad Prism and curve fit the fluorescence by using non-linear regression and the exponential one-phase association model. The mobile fraction (Mf) that corresponds to the plateau value and half-life 1/2, the time it takes for fluorescence intensity to reach half the maximum of the plateau level are obtained by the software.

Notes

  1. Every FRAP protocol need to be adapted depending on the nature of sample and preparation. FRAP setup shown herein is merely orientative and should not be considered as guarantee of good results. Then, it is very likely your first session efforts will be focused in optimizing photobleaching and data acquisition (Carisey et al., 2011).

Recipes

  1. LB medium
    Mix 5 g of yeast extract with 10 g of tryptone, 10 g of NaCl
    Add dH2O to 1,000 ml
  2. Agrobacterium infiltration buffer
    10 mM MES (pH 5.6)
    10 mM MgCl2
    150 µM acetosyringone
  3. Mowiol mounting medium
    1. Add 9.6 ml of Mowiol 4-88 to 24 ml of glycerol and 24 ml of dH2O.
    2. Stir to mix and leave for several hours at room temperature.
    3. Add 9.6 ml of 1 M Tris-Cl (pH 8.0) and 38.4 ml of dH2O heat to 50 °C for 10 min with occasional mixing.
    4. After the Mowiol dissolves (approximately 3 h), clarify by centrifugation at 5,000 x g for 15 min.
    5. For fluorescence detection, add Dabco to 2.5% to reduce fading.
    6. Aliquot in 1.5 ml tubes and stored at -20 °C.

Acknowledgments

This work was funded by grant BIO2011-25018 from the Spanish Ministerio de Economia y Competitividad and by Prometeo Program GV2011/003 from the Generalitat Valenciana. J.A.N. and M.S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Educacion y Ciencia of Spain.

References

  1. Carisey, A., Stroud, M., Tsang, R. and Ballestrem, C. (2011). Fluorescence recovery after photobleaching. Methods Mol Biol 769: 387-402.
  2. Phair, R. D., Gorski, S. A. and Misteli, T. (2004). Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Methods Enzymol 375: 393-414.
  3. Serra-Soriano, M., Pallas, V. and Navarro, J. A. (2014). A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. Plant J 77(6): 863-879.

简介

在该协议中,我们使用光漂白(FRAP)后的荧光恢复来测量一些突变和药物治疗对于将绿色荧光蛋白(GFP) - 融合的病毒跨膜蛋白移动到内质网膜中的影响(Serra-Soriano, et al。,2014)。感兴趣的蛋白质通过农杆菌浸润在烟草(Nicotiana benthamiana)(本塞姆氏烟草)表皮细胞中瞬时表达。为了使瞬时过表达伪像最小化,使用倒置Zeiss LSM 780共聚焦显微镜在36hpi收集荧光强度值。只有表现出中等表达水平和通过GFP标记的蛋白的ER的均匀分布的表皮细胞用于进一步的实验。为了检查肌动蛋白聚合在GFP标记的蛋白质的动员中的作用,我们用拉特管素B,肌动蛋白聚合的抑制剂或用DMSO作为对照预处理组织样品。使用所产生的荧光恢复曲线来获得对应于流动级分的最大荧光回收率(MFR)的百分比和最大回收的半衰期(t 1/2)/sub>)值。

材料和试剂

  1. 3-4周龄的Nicotiana benthamiana植物
  2. 用包含蛋白质编码序列的二元载体pMOG800转化的根癌土壤杆菌(根瘤土壤杆菌)C58C1菌株或类似菌株。 在我们的案例中,他们是:
    1. 编码GFP融合的Melon坏死斑点病毒(MNSV)p7B的pMGFP-p7B
    2. pMGFP-p7B [D sub 7 AP 10 A]编码携带D sub 7 AP 10 A的GFP融合MNSV p7B 突变
    3. 编码具有K A突变的GFP融合MNSV p7B的pMGFP-p7B [K <49> A]
    4. 编码GFP的pMGFP-KDEL,其被设计成靶向于ER流体
  3. 酵母提取物(Difco,目录号:212750)
  4. 胰蛋白胨(Difco,目录号:211705)
  5. 氯化钠(NaCl)(Panreac Applichem,目录号:131659)
  6. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  7. MES(Sigma-Aldrich,目录号:M8250)
  8. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M9272)
  9. 抗生素
    1. 卡那霉素(50mg/ml)(Sigma-Aldrich,目录号:K4000)
    2. 利福平(50mg/ml)(Duchefa Biochemie,目录号:R0146)
  10. Mowiol 4-88(Sigma-Aldrich,目录号:81381)
  11. Dabco 33-LV(Sigma-Aldrich,目录号:290734)
  12. Tris碱(Roche Diagnostics,目录号:03118142001)
  13. Tris-HCl(1M,pH 5.6)
  14. 浸渍油Immersol 518F(ZEISS,目录号:444960-0000-000)
  15. Latrunculin B(Sigma-Aldrich,目录号:L5288)
  16. 25μM在10mM DMSO中的Latrunculin B溶液
  17. LB介质(见配方)
  18. 土壤杆菌渗透缓冲液(参见Recipes)
  19. Mowiol安装介质(参见配方)

设备

  1. 28℃生长室
  2. 15 ml培养管
  3. 15 ml离心管旋转离心机转子
  4. 1.5ml管(标准Eppendorf管或类似物)
  5. BioPhotometer plus(Eppendorf)
  6. 1 ml无针头的注射器
  7. 植物生长室
  8. 细桨钳
  9. 培养皿35×10毫米
  10. 显微镜载片76×26mm(Menzel-Glaser,目录号:AA00000112E)
  11. 显微镜盖玻片24 x 24毫米 1(Menzel-Glüser,目录号:BB024024A1)
  12. 单面胶带
  13. 软木钻孔器(直径一厘米)或类似切割装置,如手术刀刀片
  14. 倒置共焦显微镜(ZEISS,型号:LSM 780)
  15. Plan-Apochromat 63x/1.40油物镜

软件

  1. Graphpad棱镜软件( http://www.graphpad.com
  2. MS Excel

程序

  1. 通过农杆菌渗透的蛋白质的瞬时表达
    1. 接种转化的单个集落A.在含有卡那霉素(50μg/ml)和利福平(50μg/ml)的5ml LB培养基中培养。在28-30°C下剧烈振荡孵育培养物(12小时或更长时间)
    2. 测量培养物的A 600
    3. 在摇摆转子中通过慢速离心(2,000×g/min,15分钟)收集细菌。取出上清液。
    4. 用土壤杆菌浸润缓冲液重悬沉淀。最后的A 600 应调整为0.2。
    5. 在室温下放置2小时,然后浸润
    6. 用1ml注射器将培养物浸润到叶子的下侧。使用3-4周龄的 N。本生植物,并避免子叶。使用指尖在叶片的另一侧施加温和的反压力。叶子的一个完全缩小的部分产生水浸泡的外观
    7. 保持植物在生长室在16小时光照在25°C和8小时黑暗在22°C

  2. 显微镜载片准备
    1. 为了使FRAP实验中的瞬时过表达伪像最小化,在浸润后36小时使用植物
    2. 用软木钻或类似装置切下一片小叶的渗入叶。 尽量避免叶脉,以消除样品中的任何潜在的不规则或显着的凹陷
    3. 在药物治疗的情况下,将组织样品浸入小培养皿(35×10mm)中的latrunculin B(1ml的25μM溶液)1小时。 通过组织浸入10mM DMSO进行未处理的对照
    4. 使用细桨钳将叶盘放在显微镜载玻片上,背面朝上,并安装在Mowiol安装介质中。
    5. 添加盖玻片,并用胶带单面胶带将它们粘在幻灯片上。
    6. 将一滴浸油放在叶盘上的盖玻片上。

  3. 光漂白后的荧光恢复(FRAP)测定
    1. 使用来自Zen 2011软件的LSM FRAP模块在倒置的Zeiss LSM 780共聚焦显微镜上进行FRAP实验。
    2. 打开激光线以对样品成像。
    3. 使用Plan-Apochromat 40x/1.4油物镜找到表现出中等表达水平和均匀分布的GFP标记蛋白的表皮细胞,然后切换到Plan-Apochromat 63x/1.40油物镜捕获图像。
    4. 选择"量程指示器"。现在图像变成双色(红色和蓝色)之一(图1)。由于高荧光强度,红色区域指示检测器饱和。类似地,蓝色区域表示没有荧光或低于检测器可以感测的强度。只有几个像素必须饱和(红色),如果不是,调整针孔,激光强度或探测器增益。


      图1.激光共聚焦扫描为绿色A和范围指示模式B. 显示红色的饱和区域。

    5. 使用5倍光学变焦和256×256像素分辨率拍摄一片短的内质网的单个图像(为此目的使用"捕捉"功能)。此缩放因子在样本之间应保持不变。
    6. 单击采集选项卡上的漂白选项。将自动选择时间序列和区域选项。
    7. 为了定义应该漂白的感兴趣区域(ROI),在区域菜单中选择圆形形状,并在样品的荧光区域中绘制四个直径为1.7μm的圆形ROI。选择ER的平坦膜区域显着大于漂白ROI。三个ROI将被漂白,第四个将被用作非漂白参考以校正由成像引起的光衰减的FRAP曲线。将参考投资回报率尽可能远离漂白的投资回报率。确保背景区域在您的图像中,并在其中绘制第五个圆形ROI。在快速恢复率,重要的是使用小的投资回报率,最大减少漂白和成像之间的时间间隔。


      图2.感兴趣区域(ROI)的空间分布。参考ROI必须远离漂白的ROI,以免受激光漂白的影响。背景ROI应位于非荧光区域(范围指示器模式中的蓝色像素)。

    8. 要设置漂白方案,请选择在3次扫描后开始漂白,并在漂白区域上使用100次漂白激光。不要使用缩放漂白。在漂白剂的激发中,选择氩激光器的488nm线(25%功率,100%透射率)。漂白后,尝试减少50-75%之间的ROI强度,而没有光损伤细胞。漂白后以低激光功率(25%功率,2%透射率)收集图像。
    9. 打开时间系列菜单,通过将循环次数设置为100,指定最终数据集中的图像总数(漂白前后)。这将导致每次扫描开始之间的间隔约为191 ms。要进一步提高数据采集率,请使用双向扫描
    10. 通过按"开始实验按钮"开始FRAP实验,并检查计算机上的FRAP曲线。 ROI参考强度应保持不变或由于图像采集期间的光褪色而略微降低。在不同细胞和天中重复FRAP以确保重复性。为了允许不同细胞和蛋白质之间的比较,可以重新调整检测器增益,但所有其他设置必须保持不变。

  4. FRAP数据处理
    1. 使用LSM系统生成的FRAP数据可以用共聚焦操作软件显示和分析。或者,来自漂白,背景和参考ROI的原始荧光强度值可以导出到Microsoft Excel中以进行双归一化(Phair等人,2004)。
    2. 通过比较在漂白之前(RF 0)之前的参考ROI的荧光强度,在漂白之前进行的三次扫描的平均强度)计算光漂白率(R) 光漂白后的扫描(RF n )R sub n = F sub n/f sub sub 0 。
    3. 将漂白的ROI(漂白的F sub)的荧光强度归一化如下:NF =(漂白的F sub -F背景)/r。 />
    4. 将数据复制到程序如GraphPad Prism和曲线拟合荧光通过使用非线性回归和指数单相关联模型。对应于平台值和半衰期的移动分数(Mf) 1/2 ,通过软件获得荧光强度达到平稳水平的最大值的一半所花费的时间。

笔记

  1. 每个FRAP协议需要根据样品和准备的性质进行调整。本文所示的FRAP设置仅是定向的,并且不应被认为是良好结果的保证。然后,很可能 您的第一次会议的工作将集中在优化光漂白和数据采集(Carisey等人,2011年)。

食谱

  1. LB培养基
    将5g酵母提取物与10g胰蛋白胨,10g NaCl混合 将dH <2> O添加至1,000 ml
  2. 土壤杆菌浸润缓冲液
    10mM MES(pH 5.6)
    10mM MgCl 2/
    150μM乙酰丁香酮
  3. Mowiol安装介质
    1. 将9.6ml Mowiol 4-88加入24ml甘油和24ml dH 2 O中。
    2. 搅拌混合并在室温下放置数小时。
    3. 添加9.6ml的1M Tris-Cl(pH 8.0)和38.4ml的dH 2 O加热至50℃,持续10分钟,偶尔混合。
    4. 在Mowiol溶解(约3小时)后,通过在5,000xg离心15分钟澄清。
    5. 对于荧光检测,添加Dabco到2.5%以减少褪色。
    6. 在1.5ml管中等分并储存在-20℃

致谢

这项工作由来自西班牙经济部长竞争力奖项BIO2011-25018和来自Valenciana Generalitat的Prometeo计划GV2011/003资助。 J.A.N. 和M.S. 是西班牙的教育部长西西亚的博士后合同和博士研究生的接受者。

参考文献

  1. Carisey,A.,Stroud,M.,Tsang,R。和Ballestrem,C。(2011)。 光漂白后的荧光恢复 方法Mol Biol 769: 387-402。
  2. Phair,R.D.,Gorski,S.A.和Misteli,T。(2004)。 使用光漂白显微镜测量体内动态蛋白与染色质结合的强度。 Methods Enzymol 375:393-414。
  3. Serra-Soriano,M.,Pallas,V。和Navarro,J.A。(2014)。 通过早期分泌途径转运病毒膜蛋白的模型:最小序列和内质网侧向 移动性要求。 Plant J 77(6):863-879
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引用:Navarro, J. A., Serra-Soriano, M. and Pallás, V. (2014). Fluorescence Recovery after Photobleaching (FRAP) Assay to Measure the Dynamics of Fluorescence Tagged Proteins in Endoplasmic Reticulum Membranes of Plant Cells. Bio-protocol 4(20): e1268. DOI: 10.21769/BioProtoc.1268.
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