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FRAP Analysis of LET-23::GFP in the Vulval Epithelial Cells of Living Caenorhabditis elegans Larvae
荧光漂白恢复(FRAP)分析活秀丽隐杆线虫幼体外阴上皮细胞中的LET-23::GFP   

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Abstract

The Caenorhabditis elegans (C. elegans) vulva is a well-established system to study organ development as the molecular mechanisms that govern its formation are conserved in animals. Of special interest is the EGFR/RAS/MAPK signaling pathway that is required for fate acquisition and morphogenesis of the vulva. let-23 encodes the sole homologue of the epidermal growth factor receptor (EGFR), is expressed at the plasma membrane of the vulval precursor cells (VPCs) and is activated by LIN-3 EGF at the end of the L3 larval stage to initiate vulva development. LET-23 activity can be modulated through altering its subcellular and plasma membrane localization. To study the trafficking of EGF receptor in a living organism, we created a functional LET-23::GFP translational reporter worm line (Haag et al., 2014) and quantified the mobile fraction of LET-23::GFP at the basolateral membrane of the VPCs by fluorescence recovery after photobleaching (FRAP). Here we describe the protocol for LET-23::GFP FRAP at the basolateral membrane of the VPCs and the data analysis using FIJI (ImageJ).

Materials and Reagents

  1. C. elegans strain zhIs038 [let-23::gfp; unc-119(+)] (Haag et al., 2014)
    Note: For information on maintenance of C. elegans: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html.
  2. 4% agarose pads (for an example see http://www.wormatlas.org/agarpad.html by Monica Driscoll)
  3. M9 solution with 20 mM tetramisole hydrochloride (anesthetic)
  4. M9 buffer for C. elegans (see Recipes)

Equipment

  1. Standard microscopy slides and 18 x 18 cover slips
  2. Zeiss LSM710 confocal microscope equipped with 458/488/514 nm argon laser
  3. Computer with FIJI (ImageJ. National Institutes of Health) and appropriate Plugins for FRAP analysis (see below)

Software

  1. LSM710 ZEN software (ZEISS)
  2. FIJI (Image J) (National Institutes of Health)

Procedure

This protocol describes FRAP of LET-23::GFP at the basal membrane of the VPCs, thus the “bleach region” is a part of the basal membrane. The protocol may be applied to lateral and apical membranes as well.

  1. Microscopy
    1. Prepare an agarose pad using 4% melted agarose. Cut the edges of the pad to obtain a square of around 1 cm x 1 cm (Figure 1a). The agarose square should be smaller than the area of the cover slip (18 mm x 18 mm). Add 3 μl of 20 mM tetramisol and pick 20-30 worms from late L3 stage into the tetramisol drop. Carefully place the coverslip on the top of the pad and add M9 buffer with a pipette to fill in the space between the agar and the borders of the cover slip. Under these conditions animals survive for more than 1 h, but do not analyze animals that have been left more than 45 min on the slide.
      Notes:
      1. It is not possible to identify worms at the 4-cell stage (Pn.pxx stage) with the dissection scope. Pick several worms and confirm the 4-cell stage on the confocal microscope using weak fluorescence light to avoid photobleaching.
      2. Animals at the 4-cell stage of vulval development survive longer under the confocal scope.
      3. It is fundamental that the animals are not moving on the slide. Weak horizontal or vertical drifts can be digitally corrected (see below), but animals that move too much during the acquisition cannot be analyzed.
    2. On the confocal scope: Identify the ventral region of the animal where the vulval cells are located under DIC (differential interference contrast) or transmission light. Use the 63x /1.4 NA oil lens and change to UV fluorescence to identify the VPCs expressing LET-23::GFP. Center the field in the VPCs.
    3. In the Zeiss LSM710 ZEN software, use the “Channel color lookup table” to correct the saturation for each animal using the “Master Gain” function.
    4. Crop/Zoom 4x and rotate image to place the VPC horizontally in the viewing field. It’s important to keep the 4x zoom throughout all experiments.
    5. Perform a “Snap image” (Figure 1b).
    6. Bleaching settings:
      1. On the “Bleaching” window select the 488 nm argon laser with 85% laser power.
      2. On the “Start bleaching after # scan” option select “2”, and in the “iterations” options select “5” times bleach repeat.
      3. On the “Regions” window click on “Acquisition” for region #1 and “Bleach” for region #2. Select with the “rectangle” function from the “Regions” window the four VPCs and define this region as “Acquisition” and “Analysis” (Figure 1b).
      4. Select an area of the basal or lateral membrane to be photo bleached with the “rectangle” function and define this region as “Bleach” and “Analysis” (Figure 1b).
      5. Under these settings approximately 70% of the signal is photo bleached. Record the fluorescence recovery for 296 sec, taking a frame every 8 sec (“Time Series” window) with a 488 nm laser excitation, at 20% power intensity, a pinhole equivalent to 2.12 Airey units, a frame size of 256 x 256 dpi and speed (Pixel Dwell) of 3.15 microsecond (Figure 1 b-c).
        Note: Perform a few bleach experiments and analyze the data (see below) to determine if photo bleach and recovery occurs under these conditions. If photo bleach is not efficient, avoid increasing the percentage of laser power; instead increase the iterations to bleach several times repeatedly. If recovery does not occur, check for integrity of the animals and reduce the time they are mounted on the slide.
    7. Save the experiment in the .lsm format.

  2. Data handling and analysis
    In FIJI (ImageJ)
    1. Install the following Plug-Ins:
      StackReg: http://bigwww.epfl.ch/thevenaz/stackreg/ (written by Philippe Thévenaz)
      TurboReg: http://bigwww.epfl.ch/thevenaz/turboreg/ (written by Philippe Thévenaz)
      Edge Detection:
      http://imagejdocu.tudor.lu/doku.php?id=plugin:filter:edge_detection:start (written by Thomas Boudier and repackaged by Joris Meys)
      FRAP Norm:
      http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:frap_normalization:start (written by Joris Meys) (Phair et al., 2004)
      The “StackReg” and “TurboReg” are used to align the images as worms usually move a little during the recording.
    2. Open the .lsm files in FIJI and perform the following:
      Plugins > StackReg > Transformation > Rigid body > ok
      Plugins > FRAP Analysis > FRAP Norm
      Two windows appear: “Pre Bleach” and “Post Bleach” Window (ignore these windows).
      In Window FRAP Analysis > Settings change the following: Pre Bleaching slice: 2 and Post Bleaching Slice: 3. Time units between slides: 8 (sec) > ok.
      Note: In our setup the first picture is the “snapshot”. Therefore the Pre Bleached slice is “2” and the Post bleach is “3”.
      In Frap Analysis window: > Measurements > Automatic (Figure 1d).
    3. Select the rectangle selection tool.
      1. Go to the image window and select the post bleach image.
      2. Select the FRAP region with the rectangle selection tool. Choose a rectangle size that covers all bleached area (Figure 1e). Use the pre-bleach and post-bleach pictures as reference.
      3. In the FRAP Analysis window > FRAP Region> Set ROI > Apply to Image > Measure.
        A “Measurement Window” displays values of the images over time (this information will not be used, but keep it open).
        With the rectangle tool, select a part of the picture outside of the fluorescent area (Figure 1e’) (Background) and repeat the process in the FRAP Analysis window, this time as: Background > Set ROI > Apply to Image > Measure (Figure 1d).
        With the rectangle tool select the 4 cells (Figure 1e’’) and in FRAP Analysis window click on:
        Whole Cell > Set ROI > Apply to Image > Measure (Figure 1d).
        Go back to the Fluorescence image and select with the rectangle tool a region of the membrane that was not bleached. For example, the basal membrane of the cell adjacent to the bleached cell (Figure 1e’’’). In FRAP Analysis window repeat the above process for > Reference > Set ROI > Apply to Image > Measure.
      4. Click “Normalize” to obtain the normalization results.
      5. This procedure is used to doubly normalize the data: with the Reference region (nonbleached membrane) and with the total fluorescence of the 4 VPCs in the single section.
      6. Copy all results to a data analysis software (e.g. Excel) and keep the columns “Time” and
      7. “Normalized” for the analysis. Plot the data: Fluorescence intensity (normalized) vs. time (sec) (Figure 1f).
      8. The plots can be used to estimate the immobile and mobile fractions of the analyzed molecule. The mobile fraction is the fraction of fluorescence that is recovered and is represented by the difference between the initial and final fluorescence. FRAP-bleached molecules that are immobile do not permit mobilization of incoming proteins into those sites at the membrane and represent the immobile fraction. For a comprehensive explanation see White and Stelzer (1999).


        Figure 1. Confocal set up for bleach and recovery measurements and data analysis. a. Set up of the agar pad on the microscopy slide. For optimal immobilization of the animal, use agar pads that are smaller than the cover slip with 3 µl of tetramisole. b. Screenshots of the ZEN software. Bleach and recovering settings: Red rectangle “1” is the acquisition area (VPCs). Green rectangle “2” is the bleaching area (basal membrane). c. Images acquired before and after photobleaching of the basal membrane of the VPCs over 240 sec. d. Reference settings for the FRAP analysis plugin from FIJI. e. Selection of areas to normalize in the FRAP analysis plugin. f. Example of a FRAP curve of LET-23::GFP and its parameters (n= 40 animals).

Notes

  1. FRAP approximately 30 animals per experiment. In some cases the animals move too much or die during the experiment. Some of the worms may not recover or stop recovering in the first minute. Before each measurement briefly determine the morphological integrity of the animal. Animals that have been left too long on the pad look less turgid and accumulate cavity-like structures in the head. Some worms may also “move” in the z-section as they collapse. Do not include these data for the analysis. We end up with around 20 animals that we use for the measurements.
  2. Only after the data is normalized and plotted it is possible to give an estimate of the recovery of the bleached protein. Before performing large experiments, perform a couple of pilot photo-bleaches and post-bleach acquisitions to determine if the settings are appropriate or if the animals are still alive under these conditions.
  3. Do not compromise speed for resolution: Speed of image acquisition is crucial for the FRAP analysis.
    Microscope lens: Bleaching and acquisition with the 63x /1.4 NA oil lens give reproducible results in wild-type animals expressing LET-23::GFP. It might be necessary to use the 40x lens to collect more signals for mutant backgrounds or chemical treatments where LET-23::GPF expression may reduce.

Recipes

  1. M9 buffer for C. elegans
    3.0 g KH2PO4
    6.0 g Na2HPO4
    0.5 g NaCl
    1.0 g NH4Cl
    Bring to 1 L with H2O

Acknowledgments

Funding: This work was supported by a grant from the Swiss National Science Foundation (no. 31003A-146131) to AHaj and by the Kanton of Zurich.

References

  1. Haag, A., Gutierrez, P., Buhler, A., Walser, M., Yang, Q., Langouet, M., Kradolfer, D., Frohli, E., Herrmann, C. J., Hajnal, A. and Escobar-Restrepo, J. M. (2014). An in vivo EGF receptor localization screen in C. elegans Identifies the Ezrin homolog ERM-1 as a temporal regulator of signaling. PLoS Genet 10(5): e1004341.
  2. Phair, R. D., Gorski, S. A. and Misteli, T. (2004). Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Method Enzymol 375: 393-414.
  3. White, J. and Stelzer, E. (1999). Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol 9(2): 61-65.

简介

秀丽隐杆线虫( C. elegans )外阴是研究器官发育的公认的系统,因为控制其形成的分子机制在动物中是保守的。特别感兴趣的是外植体的命运获取和形态发生所需的EGFR/RAS/MAPK信号传导途径。 let-23 编码表皮生长因子受体(EGFR)的唯一同源物,在外阴前体细胞(VPC)的质膜处表达,并在LIN-3 EGF结束时被激活L3幼虫阶段以启动外阴发育。 LET-23活性可以通过改变其亚细胞和质膜定位来调节。为了研究活生物体中EGF受体的运输,我们创建了功能性LET-23 :: GFP翻译报道子螺旋系(Haag等人,2014),并且量化LET-23的移动部分:: GFP在荧光漂白后的荧光恢复(FRAP)在VPC的基底外侧膜。在这里我们描述LET-23 :: GFP FRAP在VPC基底外膜的协议和使用FIJI(ImageJ)的数据分析。

材料和试剂

  1. C。 elegans strain zhIs038 [let-23 :: gfp; unc-119(+)] (Haag ,2014)
    注意:有关维持线虫的信息: http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html。
  2. 4%琼脂糖垫(示例参见 http://www.wormatlas.org/agarpad.html由Monica Driscoll)
  3. M9溶液与20mM盐酸四甲基噻唑(麻醉)
  4. m9缓冲区 (参见配方)

设备

  1. 标准显微镜载玻片和18 x 18盖玻片
  2. 装有458/488/514nm氩激光的Zeiss LSM710共聚焦显微镜
  3. 计算机与FIJI(ImageJ。国家卫生研究所)和适当的插件进行FRAP分析(见下文)

软件

  1. LSM710 ZEN软件(ZEISS)
  2. FIJI(图像J)(国立卫生研究院)

程序

该协议描述了在VPC的基底膜处的LET-23 :: GFP的FRAP,因此"漂白区域"是基底膜的一部分。 该方案也可以应用于侧膜和顶膜。

  1. 显微镜
    1. 使用4%熔化的琼脂糖制备琼脂糖垫。 切割焊盘的边缘   以获得约1cm×1cm的正方形(图1a)。 琼脂糖 方形应小于盖玻片的面积(18mm×18mm) mm)。 加入3μl的20 mM tetramisol和从晚期L3挑选20-30蠕虫 阶段进入tetramisol滴。 小心地将盖玻片放在顶部   的垫,并用移液管添加M9缓冲液填充空间 在琼脂和盖玻片的边界之间。 在这些 条件动物生存1小时以上,但不分析动物   在幻灯片上留下了超过45分钟。
      注意:
      1. 在4细胞阶段(Pn.pxx阶段)不可能鉴定蠕虫, 与解剖范围。 选择几个蠕虫,确认4细胞 阶段在共聚焦显微镜上使用弱荧光来避免 漂白。
      2. 在阴道发育的4细胞期的动物在共焦范围内存活更长时间。
      3. 根本的是动物不在幻灯片上移动。 弱   水平或垂直漂移可以数字校正(见下文), 但是在收购期间移动太多的动物不能 分析。
    2. 在共焦范围:确定腹侧区域 外阴细胞位于DIC下的动物(差异 干涉对比度)或透射光。 使用63x/1.4 NA油 透镜并改变为UV荧光以鉴定表达的VPC LET-23 :: GFP。 将字段居中在VPC中。
    3. 在蔡司LSM710 ZEN软件,使用"通道颜色查找表"进行校正 饱和度为每个动物使用"主增益"功能
    4. 裁剪/缩放4x并旋转图像以将VPC水平放置 视野。 重要的是保持4倍放大 实验
    5. 执行"捕捉映像"(图1b)。
    6. 漂白设置:
      1. 在"漂白"窗口中选择488nm氩激光器,激光功率为85%。
      2. 在"扫描后开始漂白"选项选择"2",并在"迭代"选项中选择"5"次漂白重复。
      3. 在"地区"窗口上,单击区域#1和区域"获取" 区域#2的"漂白"。使用"矩形"功能从中选择 "地区"窗口的四个VPC,并将此区域定义为"采集" 和"分析"(图1b)。
      4. 选择基础区域或 侧面膜用"矩形"功能进行照片漂白 将此区域定义为"漂白"和"分析"(图1b)。
      5. 下  这些设置大约70%的信号被光漂白。 记录荧光恢复296秒,每8秒一帧  ("时间序列"窗口),具有488nm激光激发,功率为20% 强度,针孔相当于2.12 Airey单位,帧大小为256  x 256 dpi和3.15微秒的速度(像素驻留)(图1b-c)。
        注意:进行一些漂白实验并分析数据(见下文) 以确定在这些条件下是否发生光漂白和恢复。  如果照片漂白效率不高,避免增加的百分比 激光功率; 而是增加迭代几次以漂白 反复。 如果恢复没有发生,检查的完整性 动物,并减少他们在幻灯片上的时间。
    7. 将实验保存为.lsm格式。

  2. 数据处理和分析
    在FIJI(ImageJ)
    1. 安装以下插件:
      StackReg: http://bigwww.epfl.ch/thevenaz/stackreg/(Philippe写的 Thévenaz)
      TurboReg: http://bigwww.epfl.ch/thevenaz/turboreg/(Philippe写) Thévenaz)
      边缘检测:
      http://imagejdocu.tudor.lu/doku.php?id = plugin:filter:edge_detection:start (由Thomas Boudier撰写并由Joris Meys重新打包)
      FRAP规范:
      http://imagejdocu.tudor.lu/doku.php?id = plugin:analysis:frap_normalization:start (由Joris Meys写)(Phair等人,2004)
      "StackReg"和"TurboReg"用于对齐图像,因为蠕虫通常在记录期间移动一点。
    2. 在FIJI中打开.lsm文件,然后执行以下操作:
      插件> StackReg>变换>刚体>确定
      插件> FRAP分析> FRAP标准
      出现两个窗口:"Pre Bleach"和"Post Bleach"窗口(忽略这些窗口)。
      在窗口FRAP分析>设置更改以下内容:前 漂白切片:2和后漂白切片:3.时间单位之间 载玻片:8(sec)确定。
      注意:在我们的设置中,第一张图片是"快照"。因此,预漂白切片为"2",后漂白为"3"。
      在Frap分析窗口:> 测量> 自动(图1d)。
    3. 选择矩形选择工具。
      1. 转到图像窗口并选择漂白后图像。
      2. 使用矩形选择工具选择FRAP区域。 选择一个 矩形大小覆盖所有漂白区域(图1e)。 使用 预漂白和漂白后图片作为参考。
      3. 在FRAP分析窗口> FRAP区域> 设置ROI> 应用于图片> 测量。
        "测量窗口"显示随时间变化的图像的值(该信息将不被使用,但保持打开) 使用矩形工具,选择图片的一部分 荧光区(图1e')(背景)并重复该过程   FRAP分析窗口,此时为:Background> 设置ROI> 应用 到图像>测量(图1d)。
        使用矩形工具选择4个单元格(图1e''),然后在FRAP分析窗口中单击:
        全细胞>设置ROI>应用于图片>测量(图1d)。
        返回到荧光图像,并使用矩形工具a选择 没有被漂白的膜的区域。例如,基础 膜与漂白的细胞相邻(图1e"')。在 FRAP分析窗口重复上述过程,参考> 设置ROI>应用于图片>测量。
      4. 点击"归一化"获得归一化结果。
      5. 此过程用于双重规范化数据:使用 参考区(非漂白膜)和总荧光 的单个部分中的4个VPC
      6. 将所有结果复制到数据分析软件(例如 Excel),并保留栏"时间"和
      7. "归一化"用于分析。绘制数据:荧光强度(标准化) vs 。时间(秒)(图1f)。
      8. 这些图可用于估计不动和流动分数 的分析。流动分数是的分数 荧光被恢复并且由差异表示 之间的初始和最终荧光。 FRAP漂白分子  是不动的,不允许将进入的蛋白质动员到那些中  位点,并代表不动的分数。为一个 综合解释参见White and Stelzer(1999)。


        图1。 共聚焦设置漂白和恢复测量和数据分析。。在显微镜载玻片上设置琼脂垫。最佳 固定的动物,使用琼脂垫小于 盖玻片用3μl四甲氧基苯。 b。 ZEN software的屏幕截图。  漂白和恢复设置:红色矩形"1"是采集 区域(VPC)。 绿色矩形"2"是漂白区(基底膜)。   C。 在基底的漂白之前和之后获得的图像 膜的VPC超过240秒。 d。 FRAP的参考设置 分析插件。 e。 选择区域进行归一化处理 FRAP分析插件。 F。 LET-23 :: GFP及其FRAP曲线的实例 参数(n = 40只动物)。

笔记

  1. FRAP每个实验约30只动物。 在某些情况下,动物在实验过程中移动太多或死亡。 一些蠕虫可能无法恢复或停止恢复在第一分钟。 在每次测量之前简要确定动物的形态完整性。 已经留在垫上太长的动物看起来更少的鼓动,并在头部积累腔状结构。 一些蠕虫在塌缩时也可能在z截面中"移动"。 做 不包括这些数据进行分析。我们最终有大约20只动物,我们用于测量
  2. 只有在数据被归一化和绘图之后,才可以估计漂白蛋白的回收率。在进行大型实验之前,进行几次试验光漂白和漂白后采集,以确定设置是否合适,或者在这些条件下动物是否还活着。
  3. 不要牺牲分辨率的速度:图像采集的速度对于FRAP分析至关重要。
    显微镜透镜:用63x/1.4 NA油透镜漂白和获得在表达LET-23 :: GFP的野生型动物中提供可重复的结果。可能有必要使用40x透镜收集更多的信号用于突变体背景或化学处理,其中LET-23 :: GPF表达可能减少。

食谱

  1. M9缓冲液。 elegans
    3.0克KH 2 PO 4 4/
    6.0g Na 2 HPO 4
    0.5克NaCl
    1.0g NH 4 Cl/h 用H sub 2 O将其带到1 L

致谢

资助:这项工作得到瑞士国家科学基金会(no.31003A-146131)给予AHaj和苏黎世Kanton的资助。

参考文献

  1. Haag,A.,Gutierrez,P.,Buhler,A.,Walser,M.,Yang,Q.,Langouet,M.,Kradolfer,D.,Frohli,E.,Herrmann,CJ,Hajnal, -Restrepo,JM(2014)。 体内 EGF受体定位屏幕。 elegans 标识Ezrin同源物ERM-1作为信号的时间调节剂。 PLoS Genet 10(5): e1004341。
  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。 Method Enzymol 375:393-414。
  3. White,J。和Stelzer,E。(1999)。 光漂白GFP显示活细胞内的蛋白质动力学。 趋势细胞生物学< em> 9(2):61-65。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Walser, M., Hajnal, A. and Escobar-Restrepo, J. M. (2015). FRAP Analysis of LET-23::GFP in the Vulval Epithelial Cells of Living Caenorhabditis elegans Larvae. Bio-protocol 5(11): e1489. DOI: 10.21769/BioProtoc.1489.
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