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Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells
双光子光活化法测定植物根细胞中的组蛋白交换动力学   

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Abstract

Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al., 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.

Keywords: Histone exchange(Histone交换), Two-Photon Microscopy(双光子显微镜), FRAP(FRAP), Plant stem cells(植物干细胞)

Materials and Reagents

  1. Sterile 9 cm square Petri dishes for plant growth media
  2. Cover slips No 1.5 (VWR International, catalog number: 631-0125 )
  3. Microscope slides with frosted end (VWR International, Superfrost®, catalog number: 631-0114 )
  4. Secure Seal Adhesive Sheets (0.12 mm thick) (Grace Bio-Labs, Inc., catalog number: 620001 )
  5. Arabidopsis lines expressing a protein of interest tagged with PA-GFP (Patterson and Lippincott-Schwartz, 2002)
    Note: In our case, for measuring histone exchange dynamics in the root stem cells we placed the H2B-PAGFP construct under the control of a root cell-specific promoter (pSCR) (Rosa et al., 2014), which derives expression simultaneously in quiescent centre cells, endodermis/cortex initials, and root endodermis. This setup allowed us to compare the dynamics of a histone protein in pluripotent stem cells as they progress into more differentiated states. In this protocol we will not mention particular cells or tissues as the procedure can be applied to any cell type in the root.
  6. Murashige & Skoog Basal Medium with Vitamins (any equivalent source would be suitable) (PhytoTechnology Laboratories®, catalog number: M519 )
  7. 10% bleach (contains 5-10% sodium hypochlorite) in dH2O (Vortex, Procter & Gamble)
  8. NH4NO3 (pH 5.8) (ForMediumTM)
  9. Sucrose (Sigma-Aldrich)
  10. Phytagel (Sigma-Aldrich, catalog number: P8169 )
  11. Murashige and Skoog medium (see Recipes)

Equipment

  1. Plant growth chamber
  2. Two-photon photoactivation experiments were performed on an Ultima two-photon laser scanning microscope (Buker Coopration, Prairie Technologies, model: Ultima 2-Photon Microscope )
    Note: Live images were acquired using Olympus 60x 0.9 NA water immersion objectives at 512 x 512 resolution (0.203 μm/pixel) and 1 μm focal steps.
  3. Laminar flow cabinet

Software

  1. ImageJ software
  2. MS Excel
  3. Graphpad prism software

Procedure

  1. Plant growth and microscope slide preparation
    1. Surface sterilize seeds in 5% v/v sodium hypochlorite for 5 min and rinse three times in sterile distilled water.
    2. Plate the seeds on to Murashige and Skoog medium (pH 5.8) supplemented with 1% w/v sucrose and 0.5% w/v Phytagel.
    3. Stratify the seeds by incubating for 2 days at 4 °C in darkness and then grown at 25 °C in continuous light in vertically oriented Petri dishes for approximately 3-5 days.
    4. Slide preparation: Cut small square frames of Secure Seal Adhesive with similar dimensions to the cover slip and stick it to the slide (this will avoid squashing the roots between the slide and cover slip and prevents the evaporation of mounting media during imaging) (Figure 1).
    5. Cut root tips from Arabidopsis seedlings whilst still on plates and place onto the microscope slide containing a drop of water. Alternatively, if seedlings are not too big, the whole seedling can be mounted on the slide. We mounted typically 1-2 roots per slide, but used only one of them for the 1 h time course, as we tried to avoid having the material too long on the slide prior to the experiment.
    6. Add a coverslip and stick it to the slide with the adhesive tape.


      Figure 1. A. Schematic of microscope slide preparation. Arabidopsis root tips (asterisk) were cut and placed onto slides. B. Images showing selection of regions for photoactivation (labeled from 1 to 9) in roots expressing H2B-PAGFP. Before photoactivation nuclei show a very weak signal due to the faint pre-activation (see step B4). Scale bar = 5 μm.

  2. Imaging
    1. Two-photon photoactivation experiments were performed on an Ultima two-photon laserscanning microscope. Images were acquired using an Olympus 60x 0.9 NA water immersion objective.
    2. Use 2.5x optical zoom and 512 x 512 resolution.
    3. Set up Z-steps of 1 μm.
    4. The absence of fluorescence prior to photoactivation makes it is impossible to identify the regions of interest, therefore an initial pre-activation is necessary. This faint pre-activation is achieved by scanning the root tip at 850nm wavelength, 50% power and with a pixel time of 5.6 μs. One or two scans were necessary depending on the root (Figure 1B).
    5. For photoactivation define simultaneously several regions-of-interest (ROI) covering half of each nucleus in the different Z- positions. Pulse the ROIs with a 710 nm laser light at 30% power with a pixel time of 8.8 μs. Repeat this cycle two times, and then set up normal time series.
    6. For PA-GFP time series imaging use a 925 nm laser line at 50% power, 8.8 μs/pixel, with one acquisition (one z-stack, same z-step) every 60 sec for 1 h.

  3. Image processing
    1. Image analysis was performed using Image J software.
    2. Open the hyperstack (containing z and t dimensions) and apply a Z-stack maximum projection.
    3. It is expected that cell movements and/or drift will occur during image acquisition. These movements can be corrected and/or eliminated by aligning consecutive frames using StackReg plug-in of Image J. This plugin aligns successive image frames by a cross-correlation procedure, which works easily and without operator intervention, as long as movements are not too large. More details, including screen shots are available on the developers’ website. (http://bigwww.epfl.ch/thevenaz/stackreg/)
    4. Once images are aligned using the “ROI manager” tool from Image J to define three regions of interest: Photoactivation Region; Total Nucleus and Background (Figure 2A). Both the Photoactivation and Total Nucleus Regions are drawn by hand based on contrast of fluorescence. Because mean intensities are measured the exact outline of each area is not critical for the result.
    5. Once the three regions are loaded onto the “ROI manager” window click “Multi Measure” and a results table will be provided with the mean intensity values for each region at the different time points. These measurements will be used later for normalizing the data.

  4. Data analysis
    1. Export the raw intensity measurements to Microsoft Excel to perform a double normalization (Phair et al., 2004). This method normalizes the initial fluorescence to “1” and corrects for bleaching during imaging (Figure 2).


      Figure 2. Determination of relative fluorescence signal in the ROI region, and correction for background and bleaching during imaging

    2. It is also important to normalize for the proportion of the nucleus that is activated (Figure 3). In our case because half of the nucleus was activated the fluorescence decay could only reach at most half of the initial fluorescence. We therefore took this into account by additionally normalizing as follows: I (norm) = (Ft-F0/2)/(F0/2), where Ft is the fluorescence intensity at each time point (t) and F0 is the initial fluorescence intensity after photoactivation.


      Figure 3. Correction for the proportion of the nucleus activated

    3. Copy the normalized data to a program such as GraphPad Prism and perform a curve fitting analysis. Use a non-linear regression and to a single exponential decay function (Figure 2C). The mobile fraction (Mf) and half-life values (the time it takes for fluorescence intensity to reach half the maximum of the plateau level) are obtained by the software.
    4. Present data as the average ± SEM and assess the statistical significance by t-test.
      Note: The examples regarding decays at different developmental stages can be found in our publication (Rosa et al., 2014).


      Figure 4. Example of a photoactivation experiment. A. Establishment of three ROIs for analysis (Photoactivation region, Total nucleus and background). B. Representative fluorescence changes in a nucleus from the endodermis expressing H2B-PAGFP. Half of the nucleus was photoactivated (710 nm laser line) and the loss of fluorescence within the activated area was measured. C. Decay curve for nucleus depicted in B. Data corresponded to fluorescent intensities at the photoactivated region (1) after normalisation. In red, non-linear regression using a single-exponential function.

Recipes

  1. Murashige and Skoog medium
    0.025 mg/L CoCl2.6H2O
    0.025 mg/L CuSO4.5H2O
    36.7 mg/L Na Fe-EDTA
    6.2 mg/L H3BO3
    0.83 mg/L KI
    16.9 mg/L MnSO4.2H2O
    0.25 mg/L Na2MoO4.2H2O
    8.6 mg/L ZnSO4.7H2O
    332.02 mg/L CaCl2.2H2O
    170 mg/L KH2PO4
    1,900 mg/L KNO3
    180.5 mg/L MgSO4.7H2O
    1,650 mg/L NH4NO3 (PH 5.8)
    1% sucrose
    0.5% phytagel

Acknowledgments

We thank Nuno Moreno for the help with the multiphoton microscope at Instituto Gulbenkian de Ciência, Portugal. This work was supported by grant (SFRH/BD/23202/2005) from the Portuguese Fundação para a Ciência e a Tecnologia, by the Biotechnology, and Biological Sciences Research Council of the UK (grant BB/D011892/1 and BB/J004588/1) and the John Innes Foundation.

References

  1. Patterson, G. H. and Lippincott-Schwartz, J. (2002). A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297(5588): 1873-1877.
  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. Rosa, S., Ntoukakis, V., Ohmido, N., Pendle, A., Abranches, R. and Shaw, P. (2014). Cell differentiation and development in Arabidopsis are associated with changes in histone dynamics at the single-cell level. Plant Cell 26(12): 4821-4833.

简介

染色质结合蛋白在染色质结构和基因表达中起着至关重要的作用。染色质蛋白的直接结合维持和调节转录状态。因此,重要的是在细胞核的自然环境内研究这些蛋白质在体内的结合特性。光漂白,光活化和光转化(光电转换)可以提供非侵入性实验方法来研究活细胞和生物体的动态性质。我们使用光活化来确定根的植物干细胞中组蛋白H2B的交换动力学(Rosa等人,2014)。根的干细胞位于组织的中间,这使得不可能在共聚焦显微镜中用常规激光照射进行足够小和明确的亚细胞区域的光活化,主要是因为散射和折射效应在根组织分散焦斑并导致太大的区域的光活化。因此,我们使用双光子激活,其具有更好的固有分辨率的照明区域。这是因为激活依赖于两个或更多个光子的同时吸收,这又依赖于强度的平方(或更高的功率) - 更尖锐的峰。在本协议中,我们将描述进行双光子光活化实验和相应的图像分析的实验程序。该协议可以用于标记有在根组织中表达的可光活化GFP(PA-GFP)的核蛋白。

关键字:Histone交换, 双光子显微镜, FRAP, 植物干细胞

材料和试剂

  1. 无菌9厘米正方形植物培养基培养皿
  2. 盖玻片No 1.5(VWR International,目录号:631-0125)
  3. 带有磨砂端的显微镜载玻片(VWR International,Superfrost ,目录号:631-0114)
  4. 安全密封粘合片(0.12mm厚)(Grace Bio-Labs,Inc.,目录号:620001)
  5. 表达用PA-GFP标记的感兴趣蛋白质的拟南芥品系(Patterson和Lippincott-Schwartz,2002)
    注意:在我们的情况下,为了测量根干细胞中的组蛋白交换动力学,我们将H2B-PAGFP构建体置于根细胞特异性启动子(pSCR)(Rosa等人,2014)的控制下,其导出表达同时在静止中心细胞,内皮层/皮层初始和根内皮层。这种设置允许我们比较多能干细胞中的组蛋白蛋白质的动力学,因为它们进入更多的分化状态。在此协议中,我们不会提及特定的细胞或组织,因为该程序可应用于根中的任何细胞类型。
  6. Murashige&具有维生素的Skoog基础培养基(任何等效来源将是合适的)(PhytoTechnology Laboratories ,目录号:M519)
  7. 在dH 2 O(Vortex,Procter& Gamble)中的10%漂白剂(含有5-10%次氯酸钠)

  8. (pH 5.8)(forMedium TM
  9. 蔗糖(Sigma-Aldrich)
  10. Phytagel(Sigma-Aldrich,目录号:P8169)
  11. Murashige和Skoog培养基(见配方)

设备

  1. 植物生长室
  2. 在Ultima双光子激光扫描显微镜(Buker Coopration,Prairie Technologies,型号:Ultima 2-Photon显微镜)上进行双光子光活化实验。
    注意:使用Olympus 60x 0.9 NA水浸物镜以512 x 512分辨率(0.203μm/像素)和1μm聚焦台阶获取实时图像。
  3. 层流柜

软件

  1. ImageJ软件
  2. MS Excel
  3. Graphpad棱镜软件

程序

  1. 植物生长和显微镜载玻片准备
    1. 表面消毒种子在5%v/v次氯酸钠中5分钟,并在无菌蒸馏水中漂洗三次。
    2. 将种子置于补充有1%w/v蔗糖和0.5%w/v Phytagel的Murashige和Skoog培养基(pH5.8)上。
    3. 通过在4℃下在黑暗中孵育2天来分层种子 然后在25℃下在垂直取向的Petri中在连续光下生长 菜肴约3-5天。
    4. 载玻片准备:切小 方形框架的安全密封胶具有相似的尺寸 盖滑动并粘贴到幻灯片(这将避免挤压 根部之间的滑动和盖滑动并防止蒸发 在成像期间安装介质)(图1)
    5. 从拟南芥苗切根尖,同时仍然在板上,并放置在 显微镜幻灯片包含一滴水。或者,如果幼苗 ?不是太大,整个幼苗可以安装在幻灯片上。我们 每个幻灯片通常安装1-2根,但只使用其中一个 ?1小时的时间过程,因为我们试图避免材料太长 实验前的幻灯片
    6. 添加盖玻片,并用胶带将其粘贴到幻灯片。


      图1。 A.显微镜载片准备示意图。 拟南芥根尖(星号),并置于载玻片上。 B.显示的图像 在根中选择用于光激活的区域(从1到9标记) 表达H2B-PAGFP。在光激活之前核显示非常弱 信号(参见步骤B4)。比例尺=5μm

  2. 成像
    1. 在Ultima上进行双光子光活化实验 双光子激光扫描显微镜。使用a。获得图像 Olympus 60x 0.9 NA水浸物镜
    2. 使用2.5倍光学变焦和512×512分辨率
    3. 设置1μm的Z级。
    4. 在光活化之前不存在荧光使得它是 不可能识别感兴趣的区域,因此是初始的 预激活是必要的。这种微弱的预激活是通过 在850nm波长,50%功率和一个像素下扫描根尖 时间为5.6μs。根据根目录,需要进行一次或两次扫描 (图1B)。
    5. 对于光活化同时定义几个 感兴趣区域(ROI)覆盖不同的每个核的一半 ?Z位置。使用710nm激光以30%功率脉冲ROI 像素时间为8.8μs。重复此循环两次,然后设置 正常时间序列。
    6. 对于PA-GFP时间序列成像使用925 nm激光线以50%功率,8.8μs/像素,具有一个采集(一个 z-堆叠,相同的z-步骤)每60秒1小时。

  3. 图像处理
    1. 使用Image J软件进行图像分析
    2. 打开超栈(包含 z 和 t 维度)并应用Z-stack最大投影。
    3. 预期细胞运动和/或漂移将发生在期间 图像采集。这些运动可以通过校正和/或消除 ?使用Image J的StackReg插件对齐连续的帧 插件通过互相关过程对连续图像帧进行对齐, 它工作容易,无需操作员干预,只要 运动不会太大。更多详细信息,包括屏幕截图 可在开发者的网站上。 ( http://bigwww.epfl.ch/thevenaz/stackreg/
    4. 一旦图像 alignment使用来自Image J的"ROI管理器"工具定义三个区域 感兴趣的是:Photoactivation Region;总核和背景 (图2A)。光活化和总核区域都是 基于荧光的对比度手工绘制。因为平均 强度被测量,每个区域的确切轮廓不是关键的 结果。
    5. 一旦三个区域被加载到"ROI"上 管理器"窗口单击"多测量",结果表将 提供有在不同的每个区域的平均强度值 ?时间点。这些测量将在以后用于归一化 数据。

  4. 数据分析
    1. 将原始强度测量导出到Microsoft Excel以执行 双归一化(Phair等人,2004)。此方法规范化 初始荧光变为"1",并校正成像期间的漂白 (图2)。


      图2.相对荧光信号的测定 在ROI区域,以及背景和漂白校正 成像

    2. 对比例进行归一化也很重要 ?的被激活的细胞核(图3)。在我们的情况下因为一半 的核被激活,荧光衰减只能达到 大部分的初始荧光。因此,我们把它 通过进一步归一化如下:I(norm)= (F sub -F sub)/2)/(F sub 0/2),其中F sub是第荧光强度 点(t)和F 0 0是后的初始荧光强度 光活化

      图3.校正激活的核的比例

    3. 将标准化的数据复制到程序,如GraphPad Prism和 执行曲线拟合分析。使用非线性回归和a 单指数衰减函数(图2C)。流动分数(Mf) 和半衰期值(荧光强度所需的时间 达到平稳水平的最大值的一半) 软件。
    4. 现有数据为平均值±SEM,并通过t检验评估统计学显着性 注意:关于不同发育阶段衰变的例子可以在我们的出版物中找到(Rosa et al。,2014)。


      图4.光敏化实验的示例 A.建立 三个ROI用于分析(光活化区,总核和 背景)。核中的代表性荧光变化 内皮细胞表达H2B-PAGFP。一半的核 光活化(710nm激光线)和其内的荧光损失 测量活化面积。 C.描绘的核的衰变曲线 B.数据对应于光活化的荧光强度 区域(1)。在红色,非线性回归中使用a 单指数函数。

食谱

  1. Murashige和Skoog培养基
    0.025mg/L CoCl 2 。 6H 2 O 0.025mg/L CuSO 4 5H O 36.7mg/L Na Fe-EDTA
    6.2mg/L H sub 3 BO 3 sub 0.83mg/L KI
    16.9mg/L MnSO 4 。 2H 2 0.25mg/L Na 2 SO 4 MoO 4+。 2H 2 O 8.6mg/L ZnSO 4·7H 2 O 7H/SiO 2。 332.02mg/L CaCl 2 。 2H 2 O
    170mg/L KH 2 PO 4 sub/
    1,900mg/L KNO 3
    180.5mg/L MgSO 4 .7H 2 O 2 1,650mg/L NH 4 NO 3(PH 5.8)
    1%蔗糖 0.5%植物凝集素

致谢

我们感谢Nuno Moreno对葡萄牙Gulbenkian deCiênciaInstituto的多光子显微镜的帮助。这项工作得到了英国生物技术和生物科学研究委员会(授予BB/D011892/1和BB/J004588/1)授权(SFRH/BD/23202/2005)由葡萄牙语基础技术委员会)和约翰·因尼斯基金会。

参考文献

  1. Patterson,G.H。和Lippincott-Schwartz,J。(2002)。 一种可光活化的GFP,用于选择性地标记蛋白质和细胞。 科学< em> 297(5588):1873-1877。
  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. Rosa,S.,Ntoukakis,V.,Ohmido,N.,Pendle,A.,Abranches,R。和Shaw,P。 拟南芥中的细胞分化和发育与组蛋白动力学的变化有关 单核细胞水平。 植物细胞 26(12):4821-4833。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rosa, S. and Shaw, P. (2015). Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells. Bio-protocol 5(20): e1628. DOI: 10.21769/BioProtoc.1628.
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