搜索

Analysis of in vivo Cellulose Biosynthesis in Arabidopsis Cells by Spinning Disk Confocal Microscopy
使用转盘共聚焦显微镜分析拟南芥细胞中的纤维素生物合成   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Cellulose is a main component of plant cell walls. Tools to analyze cellulose mainly rely on analytical chemistry, which yields information about cellulose amounts and structure, but cannot be applied to intact tissues. Moreover, these methods measure total cellulose and cannot be used to assay cellulose synthesis per se. Live cell imaging of the catalytic subunits of the cellulose synthesis complex (CSC) conjugated to fluorescent proteins is an important tool to understand the dynamics of the cellulose biosynthesis process (Paredez et al., 2006). This method can be used in various genetic backgrounds (Sorek et al., 2014) or with different chemical inhibitors (Brabham and Debolt, 2012). Here we describe in detail the procedure to visualize the movement of CSCs at the plasma membrane. As the movement of CSCs is likely caused by glucan synthesis and extrusion into the cell wall, live cell analysis of CSC velocity provides a method to directly measure cellulose synthesis in vivo.

Keywords: Cellulose, Cell wall, Cellulose synthesis dynamics, Live cell imaging, Arabidopsis

Materials and Reagents

  1. Microscope slides (25 x 76 x 1.0 mm) and #1.5 cover glass (24 x 30 mm)
  2. Arabidopsis seedlings expressing functional fluorescent protein fusions to CESAs, the catalytic subunits of the CSC, such as GFP:CESA3 (Desprez et al., 2007), YFP:CESA6 (Paredez et al., 2006) or tdTomato:CESA6 (Sánchez-Rodríguez et al., 2012) under the control of their native promoters
  3. Household bleach (Clorox)
  4. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 71727 )
  5. Murashige and Skoog (MS) basal salts (Caisson Laboratories, catalog number: MSP01 )
  6. 2-(N-morpholino)ethanesulfonic acid (MES) (Sigma-Aldrich, catalog number: RES0113M-B103X )
  7. Sucrose (Fisher Scientific, catalog number: BP220 )
  8. Agar (Sigma-Aldrich, catalog number: RES10020-A102X )
  9. Vacuum Grease (Beckman Coulter)
  10. 0.5x Murashige and Skoog (MS) media (see Recipes)

Equipment

  1. Growth chamber to grow plant material (e.g., Percival Scientific, model: CU-36L5 )
  2. Square plates 90 x 90 x 15 mm
  3. Spinning disk confocal head (Yokogawa Electric Corporation) mounted on a motorized inverted microscope (e.g., Leica Microsystems, model: Leica DMI6000 or Zeiss, model: Zeiss Cell Observer SD ), equipped with 488 and/or 561 nm excitation lasers and a Photometrics QuantEM 512SC Camera

Software

  1. Software operating the confocal microscope (e.g., Metamorph, Molecular Devices)
  2. ImageJ (http://imagej.nih.gov/ij/)
  3. MultipleKymograph and WalkingAverage plugins for ImageJ (J. Reitdorf and A. Seitz, http://www.embl.de/eamnet/html/body_kymograph.html)
  4. Imaris (BitPlane)
  5. Excel (Microsoft)

Procedure

  1. Plant growth
    1. Sterilize Arabidopsis seeds in 30% (v/v) household bleach (final concentration ~1.5% w/v sodium hypochlorite) and 0.1% (w/v) SDS for 15 min and wash three times with sterile water.
    2. Stratify the seeds in sterile 0.15% (w/v) agar at 4 °C for at least three days in the dark and sow on 0.5x MS, 0.8% agar, 1% sucrose plates.
    3. After two hours of light exposure at room temperature, wrap the plates in aluminum foil and grow the seedlings vertically in the dark for 3 days at 22 °C.

  2. Sample preparation (Figure 1)
    1. Apply a thin layer of vacuum grease (in syringe) at four corners near the edges of a 25 x 75 x 1 mm slide (Figure 1A) to create a mounting chamber. The vacuum grease cushion stabilizes the seedling for sustained time-lapse imaging, minimizing compression and mechanical damage.
    2. Transfer an Arabidopsis etiolated seedling into the slide with 135 μl of water or liquid 0.5x MS media (Figure 1B) and mount with a #1.5 24 x 30 mm cover glass (Figure 1C). Manipulate the seedling as quickly as possible, or use a safer light such as green or red light, to minimize exposure to light.

  3. Confocal imaging
    1. Observe the seedling under a confocal microscope featuring a spinning disk head mounted on a motorized inverted microscope with a 100x/1.4 numerical aperture oil immersion objective.
    2. Use 488 nm or 561 nm excitation wavelength for the fluorescent markers GFP/YFP and tdTomato respectively; and band pass filters (525/50 nm for GFP, 535/50 for YFP, and 620/60 for tdTomato) for emission filtering. Perform imaging with 500-ms exposure times.
    3. Obtain CESA particle images from epidermal cells in the upper etiolated hypocotyl (Figure 2A) at the plasma membrane focal plane (Figure 2B), where cellulose synthesis takes place. The larger particles in the adjacent focal planes correspond to CESA particles in Golgi and cytoplasmic compartments (Figure 2C).
    4. Run a time-lapse imaging experiment with a time interval of 5 sec and duration of 5 min.
    5. During this time, vertical stage drift may occur, resulting in a shift from the plasma membrane focal plane. Make sure that images are acquired at the correct focal plane over time. This can be accomplished using a “live” preview mode every four or five frames to adjust the focus. Since this can increase photo bleaching of the sample, adjustments should be made only when necessary.
    6. To check that there is no horizontal stage drift in the time series, an average intensity projection can be created in ImageJ. Open the TIFF image stack in ImageJ and select the “Image → Stacks → Z project...” command. Select the “Average Intensity” option and click OK. In the newly generated time average image, CESA particles in the plasma membrane should appear as linear tracks. Background Golgi signal will appear as wide diffuse tracks and larger bright spots (Figure 3A).
    7.  Occasionally, a horizontal drift may occur due to movement of the seedling or movement of the confocal stage. This can be detected by applying Z project on the movie, as the tracks will smear (Figure 3B demonstrates a Z stack of such event). We prefer not to analyze this type of movies, although in mild cases, the StackReg ImageJ plugin can be used.

  4. Image processing
    1. Open the time-lapse series TIFF stack in ImageJ.
    2. As the images of the time-lapse series tend to be noisy and can vary in brightness depending on drift in the focal plane during acquisition, two filters can be applied in order to improve downstream analyses. The results of these operations are shown in Figure 4.
    3. First, select “Process → Enhance Contrast…” and set to “0.4%” saturated pixels. This saturation works well for our imaging. Select the option for “Process all X slices” and leave the remaining options unselected (Figure 4B).
    4. Noise can be greatly reduced by applying a walking average filter. Select “Plugins → Walking Average.” Enter “4” as the number of frames to average. Note that the resulting stack will contain 3 fewer frames than the initial stack (Figure 4C).
    5. Save the resulting stack and analyze particle velocity either by Kymograph in ImageJ or particle analysis in Imaris.

  5. Kymograph analysis in ImageJ
    1. Starting with the file generated by step D of “Image Processing,” create an average intensity projection as mentioned before.
    2. Use the straight line selection tool to trace a line that follows a CSC track in the time average image (Figure 5A).
    3. Switch back to the original time-lapse stack and select “Edit → Selection → Restore Selection.” The line drawn in step E2 should now be transferred to the time-lapse stack. (Figure 5B).
    4. Select “Plugins → MultipleKymograph.” Set the line width to “3”. This setting was shown to give the best result.
    5. A kymograph is generated depicting displacement along the defined path in the X dimension and time in the Y dimension (Figure 5C). Particles moving at a constant velocity thus appear as diagonal lines and their slope can be used to calculate the velocity of each particle in the kymograph.
    6. To easily determine the slope of a line, select “Analyze → Set Measurements…” and make sure that “Bounding Rectangle” is selected.
    7. On the kymograph, use the straight line tool to trace one of the diagonal lines corresponding to an individual CESA particle (Figure 5C).
    8. Press “M” to measure this line and the Results window will appear. Width and Height of the bounding box are the number of pixels corresponding to the displacement and time, respectively, that the particle has traveled.
    9. Calculating the velocity of each particle is straightforward, as each pixel in the Y dimension corresponds to the number of seconds between exposures in the time lapse, and each pixel in the X dimension has the same dimensions as the X and Y axes in the original image (This will vary depending on microscope and objective). For the path outlined in Figure 5C, where a frame was taken every 5 sec and each pixel in the original image corresponds to 135 nm:


        
  6. Imaris analysis of CSC velocity
    1. Imaris has a range of powerful tools for analyzing particle movement. A simple procedure for extracting plasma membrane CSC velocities while ignoring Golgi and other trafficking compartments is outlined here. These instructions are for Imaris 7.2 and may differ with later versions of the software.
    2. Ensure that the stack is saved as an 8 bit TIFF image after initial processing in ImageJ.
    3. Open the TIFF stack in Imaris “File → Open…”.
    4. Select “Image Processing → Swap Time and Z”.
    5. Select “Edit → Image Properties.” Set the voxel dimensions according to your microscope and objective. For example, with the microscope’s 100x objective used in this work, one pixel is 135 nm, so voxel size is 135 for X, 135 for Y and Z can be left as 1. Change the units to “nm.”
    6. In the same window, under the Time Point section, click “All Equidistant…” and enter the time interval used during acquisition. If the instructions at step C4 were followed, this is set to 5 sec. Click “OK.”
    7. Select “Surpass → Spots.” In the Spots tab, make sure that “Track Spots (over time)” is selected. In this example, the whole image will be processed, but a region of interest can be analyzed by selecting “Segment only a Region of Interest” and further deselecting “Process entire Image Finally”.
    8. Click the blue next arrow.
    9. Enter an estimated diameter for the spot detection. For this example, in our lab, 250 nm works well. You can measure several particles in Slice view to estimate particle size. Select “Background Subtraction.” Press the next button.
    10. Spots will be detected and will be marked with small squares as shown in Figure 6A. Quality thresholds can be adjusted at this point, but it is not critical to make sure that every spot is tracking a CESA particle. False positive spots can be filtered out by additional criteria later. Generally, adjust the threshold so that many spots are detected within cells, but few spots are detected in background regions. Click the next button.
    11. Click the next button, as there are no changes to be made on the Edit Spots screen.
    12. Select “Connected Components” and click next. This may take a couple of minutes.
    13. On the Classify Tracks screen, apply a filter of Track Duration above 60 sec. This will eliminate most of the fast moving trafficking compartments that are not associated with the plasma membrane. The remaining tracks are highlighted by color coded lines (Figure 6B).
    14. Click the green “finish” button.
    15. Click on the statistics tab, and then on the Detailed tab. Click the icon with multiple disks to export all statistics as a Microsoft Excel document.
    16. To calculate the velocity of the tracks, we take the Track Displacement Length and divide it by the Track Duration. The track speed that is calculated by Imaris is less accurate since at each time point the center of each particle is not determined reliably. Typically, 1,000 or more spots can be analyzed in a single movie.

Representative data



Figure 1. Sample preparation. A. Vacuum grease is applied at four corners in the slide. B. Arabidopsis seedling is transferred to the slide. C. Arabidopsis seedling is mounted with a cover glass.


Figure 2. GFP:CESA3 localization in etiolated hypocotyl cells. Imaging was performed on epidermal cells in the upper hypocotyl from etiolated seedlings (A). Adjacent focal planes of the same cell showing GFP:CESA3 particles at the plasma membrane indicated by arrowheads (B), and Golgi and cytoplasmic compartment at lower cell cortex (C). Scale bar: 10 μm


Figure 3. GFP-CESA3 time average images. A. An average intensity projection generated by ImageJ where CESA particles appear as linear tracks. B. An average intensity projection showing that the tracks smear when a horizontal stage drift takes place. Scale bar: 10 μm


Figure 4. Image Processing in ImageJ. A. One frame from the original unprocessed stack. B. The same frame after “Enhance Contrast.” C. The same frame after “Enhance Contrast” and “Walking Average”. Insets represent an enlargement of the boxed region from the image.


Figure 5. Defining a track for generation of a kymograph in ImageJ. A. Time average image used for selecting a linear track to analyze. B. The same selection transferred to the time-lapse stack. C. Kymograph analysis.


Figure 6. Analysis of particle movement using Imaris. A. Spots are detected and marked with small squares. B. Tracks after filtering based on duration of greater than 60 sec.

Notes

  1. Velocity of the CESA particles is variable depending on temperature and other factors; therefore comparative assays should be performed on the same day and under the same imaging conditions.
  2. No changes in complex motility were observed when seedlings were grown with or without sucrose in the plates.
  3. The N-terminal fusion of the fluorescent protein was required for all the constructs to be functional.
  4. All of these fluorescent protein fusions can be expressed either wild type or mutant backgrounds. It has been observed that GFP:CESA3 constructs are not able to rescue null alleles of CESA3, although hypomorphic alleles such as cesa3je5 are complemented (Desprez et al., 2007).
  5. Fluorescent marker YFP can also be excited at 514 nm if the laser is available.

Recipes

  1. 0.5x Murashige and Skoog (MS) media (1 L)
    Add 2.2 g MS salts
    Add 0.5 g MES
    Add 10 g sucrose
    Adjust to pH of 5.7 with 1 M KOH
    Add dH2O to 1 L
    Add 8 g agar for a solid media
    Autoclave at 121 °C for 30 min

Acknowledgments

This protocol was adapted from the previously published studies, Paredez et al. (2006). We gratefully thank Prof. Chris Somerville, Dr. Heidi Szemenyei and Dr. Charles T. Anderson for critical reading of the protocol. This work was carried out in the laboratory of Chris Somerville at UC Berkeley. TV was supported by an EMBO long-term fellowship (ALTF 711-2012) and by postdoctoral funding from the Philomathia Foundation. TY was also supported by a fellowship from the Philomathia Foundation. NS was the recipient of Postdoctoral Award No. FI-434-2010 from the Binational Agricultural Research and Development Fund.

References

  1. Brabham, C. and Debolt, S. (2012). Chemical genetics to examine cellulose biosynthesis. Front Plant Sci 3: 309.
  2. Desprez, T., Juraniec, M., Crowell, E. F., Jouy, H., Pochylova, Z., Parcy, F., Hofte, H., Gonneau, M. and Vernhettes, S. (2007). Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A 104(39): 15572-15577.
  3. Paredez, A. R., Somerville, C. R. and Ehrhardt, D. W. (2006). Visualization of cellulose synthase demonstrates functional association with microtubules. Science 312(5779): 1491-1495.
  4. Sanchez-Rodriguez, C., Bauer, S., Hematy, K., Saxe, F., Ibanez, A. B., Vodermaier, V., Konlechner, C., Sampathkumar, A., Ruggeberg, M., Aichinger, E., Neumetzler, L., Burgert, I., Somerville, C., Hauser, M. T. and Persson, S. (2012). Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis. Plant Cell 24(2): 589-607.
  5. Sorek, N., Sorek, H., Kijac, A., Szemenyei, H. J., Bauer, S., Hematy, K., Wemmer, D. E. and Somerville, C. R. (2014). The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane. J Biol Chem 289(50): 34911-34920.

简介

纤维素是植物细胞壁的主要成分。用于分析纤维素的工具主要依赖于分析化学,其产生关于纤维素量和结构的信息,但不能应用于完整组织。此外,这些方法测量总纤维素,并且不能用于测定纤维素合成本身。与荧光蛋白结合的纤维素合成复合物(CSC)的催化亚基的活细胞成像是理解纤维素生物合成过程的动力学的重要工具(Paredez等人,2006)。该方法可用于各种遗传背景(Sorek等人,2014)或使用不同的化学抑制剂(Brabham和Debolt,2012)。在这里我们详细描述的过程可视化CSCs在质膜的运动。由于CSC的运动可能是由葡聚糖合成和挤出进入细胞壁引起的,因此CSC速度的活细胞分析提供了一种直接测量体内纤维素合成的方法。

关键字

材料和试剂

  1. 显微镜载玻片(25×76×1.0mm)和#1.5盖玻片(24×30mm)
  2. 拟南芥表达与CESA的功能性荧光蛋白融合体的幼苗,CSC的催化亚基,例如GFP:CESA3(Desprez等人,2007 ),YFP:CESA6(Paredez等人,2006)或者tdTomato:CESA6(Sánchez-Rodríguezet al。 em>,2012)在他们的本地促销者的控制下
  3. 家用漂白剂(Clorox)
  4. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:71727)
  5. Murashige和Skoog(MS)基础盐(Caisson Laboratories,目录号:MSP01)
  6. 2-(N-吗啉代)乙磺酸(MES)(Sigma-Aldrich,目录号:RES0113M-B103X)
  7. 蔗糖(Fisher Scientific,目录号:BP220)
  8. 琼脂(Sigma-Aldrich,目录号:RES10020-A102X)
  9. 真空润滑脂(Beckman Coulter)
  10. 0.5x Murashige和Skoog(MS)培养基(参见配方)

设备

  1. 用于生长植物材料的生长室(例如,Percival Scientific,型号:CU-36L5)
  2. 方形板90 x 90 x 15毫米
  3. 安装在配备有488和/或561的电动倒置显微镜(例如,徕卡显微系统公司,型号:Leica DMI6000或Zeiss,型号:Zeiss Cell Observer SD)上的旋转盘共焦头(Yokogawa Electric Corporation) nm激发激光器和Photometrics QuantEM 512SC相机

软件

  1. 操作共聚焦显微镜的软件(例如,Metamorph,Molecular Devices)
  2. ImageJ( http://imagej.nih.gov/ij/
  3. 用于ImageJ的Multiple Kymograph和WalkingAverage插件(J.Reitdorf和A.Seitz, http://www。 embl.de/eamnet/html/body_kymograph.html
  4. Imaris(BitPlane)
  5. Excel(Microsoft)

程序

  1. 植物生长
    1. 在30%(v/v)家用漂白剂中(最终)消毒拟南芥种子 浓度?1.5%w/v次氯酸钠)和0.1%(w/v)SDS洗涤15分钟 min,用无菌水洗涤三次
    2. 分层种子 在无菌0.15%(w/v)琼脂中在4℃在黑暗中至少三天 并在0.5x MS,0.8%琼脂,1%蔗糖板上播种
    3. 两小时后 ?的室温下曝光,将板子包裹在铝箔中 ?并在22℃下在黑暗中垂直生长幼苗3天。

  2. 样品制备(图1)
    1. 在靠近四个角落处涂一薄层真空润滑脂(在注射器中) 25 x 75 x 1 mm滑块的边缘(图1A),以创建安装 室。真空润滑脂垫使幼苗稳定持续 ?延时成像,最小化压缩和机械损伤。
    2. 将拟南芥幼苗转移到含有135μl的载玻片中 ?的水或液体0.5x MS培养基(图1B),并用#1.5 24× ?30 mm玻璃盖(图1C)。操作幼苗尽快 可能,或使用更安全的光,如绿色或红色光,以最小化 曝光。

  3. 共焦成像
    1. 在具有旋转的共聚焦显微镜下观察幼苗 磁头安装在具有100x/1.4的电动倒置显微镜上 数值孔径油浸物镜。
    2. 使用488 nm或561 nm ?荧光标记GFP/YFP和tdTomato的激发波长 分别;和带通滤波器(GFP为525/50nm,YFP为535/50, 和620/60用于tdTomato)用于发射滤波。使用执行成像 500毫秒曝光时间。
    3. 从中获取CESA粒子图像 表皮细胞在上部的下胚轴(图2A) 质谱膜焦平面(图2B),其中纤维素合成 ?地点。相邻焦平面中的较大粒子对应于 CESA颗粒在高尔基体和细胞质区室(图2C)
    4. 运行时间推移成像实验,时间间隔为5秒,持续时间为5分钟。
    5. 在该时间期间,可能发生垂直级漂移,导致a 从等离子体膜焦平面偏移。确保图像是 随时间在正确的焦平面上获取。这可以实现 使用"实时"预览模式每四或五个帧进行调整 焦点。由于这可能增加样品的光漂白, 只有在必要时才应进行调整。
    6. 检查 在时间序列中没有水平阶段漂移,平均 可以在ImageJ中创建强度投影。打开TIFF图像堆栈 ?在ImageJ中选择"图像→堆栈→Z项目..."命令。 选择"平均强度"选项,然后单击"确定"。在新 生成时间平均图像,CESA颗粒在质膜中 应显示为线性轨道。背景高尔基体信号将显示为 宽漫射轨迹和较大的亮点(图3A)。
    7.  偶尔,水平漂移可能会由于移动而发生 幼苗或共焦阶段的运动。这可以通过检测 在电影上应用Z项目,因为轨道会涂抹(图3B 演示了这种事件的Z堆栈)。我们不想分析这个 类型的电影,虽然在轻微的情况下,StackReg ImageJ插件可以 使用。

  4. 图像处理
    1. 在ImageJ中打开延时系列TIFF堆栈。
    2. 作为图像 ?延时系列趋向于噪声并且可以在亮度上变化 取决于在采集期间焦平面的漂移,两个滤波器 可用于改善下游分析。结果 这些操作如图4所示。
    3. 首先,选择"Process→ 增强对比度...",并设置为"0.4%"饱和像素。这种饱和 适用于我们的成像。选择"处理所有X切片"选项, ?并保留其余选项未选择(图4B)。
    4. 噪声 可以通过应用步行平均滤波器大大减少。选择 "插件→行走平均"。输入"4"作为帧数 平均。请注意,生成的堆栈将包含少于3个帧 初始堆栈(图4C)。
    5. 保存生成的堆叠和分析粒子速度通过Kymograph在ImageJ或粒子分析在Imaris。

  5. ImageJ中的Kymograph分析
    1. 从"图像处理"的步骤D生成的文件开始,如前所述创建平均强度投影。
    2. 使用直线选择工具在时间平均图像(图5A)中跟踪跟随CSC轨迹的线。
    3. 切换回原始延时堆栈,然后选择"编辑→" 选择→恢复选择"。在步骤E2中绘制的线应为 转移到延时堆栈。 (图5B)。
    4. 选择"插件→多个Kymograph"。将线宽设置为"3"。显示此设置可获得最佳结果。
    5. 生成描述沿着所定义的位移的kymograph 路径在X维度和时间在Y维度(图5C)。 因此以恒定速度移动的颗粒表现为对角线 并且它们的斜率可以用于计算每个粒子的速度 在kymograph。
    6. 要轻松确定线的斜率,请选择 ?"分析→设置测量..."并确保"边界矩形" 已选择。
    7. 在kymograph上,使用直线工具 跟踪对应于单个CESA的对角线之一 颗粒(图5C)
    8. 按"M"测量此线和 将显示结果窗口。边框的宽度和高度为 ?对应于位移和时间的像素数, 分别是颗粒已经移动
    9. 计算 每个粒子的速度是直接的,如Y中的每个像素 尺寸对应于曝光之间的秒数 时间间隔,并且X维度中的每个像素具有相同的维度 ?原始图像中的X和Y轴(这将取决于 显微镜和物镜)。对于图5C中概述的路径,其中a 每5秒拍摄一帧,原始图像中的每个像素 对应于135nm:


        
  6. CSC速度的Imaris分析
    1. Imaris有一系列用于分析粒子运动的强大工具。一个 ?简单的提取质膜CSC速度的程序 忽略高尔基和其他贩运隔间在这里概述。 这些说明适用于Imaris 7.2,并且可能随后续版本而有所不同 ?的软件
    2. 确保堆栈在ImageJ中初始处理后保存为8位TIFF映像。
    3. 在Imaris"文件→打开..."中打开TIFF堆栈。
    4. 选择"图像处理→切换时间和Z"
    5. 选择"编辑→图像属性"。根据以下内容设置体素尺寸 ?到你的显微镜和物镜。例如,用显微镜 100x物镜在这项工作中使用,一个像素是135 nm,因此体素大小是 X为135,Y和Z为135可以保持为1.将单位改为"nm"
    6. 在同一窗口中的"时间点"部分下,单击"全部" 等距离..."并输入采集期间使用的时间间隔。如果 遵循步骤C4的指令,将其设置为5秒。点击 "OK。"
    7. 选择"Surpass→Spots"。在Spots选项卡中,确保 选择"跟踪点(随时间)"。在这个例子中,整个图像 将被处理,但是可以通过选择来分析感兴趣的区域 ?"仅分段感兴趣区域"并进一步取消选择"处理 整个图像最后"。
    8. 单击蓝色的下一个箭头。
    9. 输入 ?用于斑点检测的估计直径。对于这个例子,在我们的 实验室,250 nm工作良好。您可以在片视图中测量多个粒子 以估计粒度。选择"背景减法" 下一步按钮。
    10. 将检测到斑点,并将标记 小正方形,如图6A所示。质量阈值可以调整 在这一点上,但确保每个点是不重要 跟踪CESA颗粒。假阳性斑点可以通过滤除 附加条件。一般来说,调整阈值使许多 在细胞内检测到斑点,但在其中检测到少数斑点 背景区域。单击下一步按钮。
    11. 单击下一步按钮,因为在编辑空间屏幕上没有任何更改。
    12. 选择"连接的组件",然后单击下一步。这可能需要几分钟。
    13. 在分类轨道屏幕上,应用轨迹持续时间的过滤器 60秒以上。这将消除大多数快速移动的贩运 不与质膜相关的隔室。的 剩余的轨道由彩色编码线突出显示(图6B)
    14. 点击绿色的"完成"按钮。
    15. 单击统计选项卡,然后在详细选项卡上。点击 带有多个磁盘的图标将所有统计信息导出为Microsoft Excel文档。
    16. 要计算轨道的速度,我们采取 ?轨道位移长度,并除以轨迹持续时间。的 由Imaris计算的轨道速度不太准确,因为在每个 时间点不能可靠地确定每个颗粒的中心。 通常,可以在单个电影中分析1,000个或更多个点。

代表数据



图1.样品制备。A.在滑块的四个角上涂抹真空润滑脂。 B.拟南芥幼苗转移到载玻片上。 C.拟南芥幼苗用盖玻片安装。


图2.GFP:在钙化的下胚轴细胞中的CESA3定位。在来自化学培养的幼苗的上部下胚轴中的表皮细胞上进行成像(A)。相同细胞的相邻焦平面显示在箭头(B)所示的质膜处的GFP:CESA3颗粒,以及在较低细胞皮层(C)的高尔基体和细胞质区室。比例尺:10μm


图3.GFP-CESA3时间平均图像 A.由ImageJ产生的平均强度投影,其中CESA粒子表现为线性轨迹。 B.平均强度投影显示当水平舞台漂移发生时轨道拖尾。比例尺:10μm


图4. ImageJ中的图像处理 A.来自原始未处理堆栈的一个帧。 B."增强对比度"后面的相同帧。C."增强对比度"和"步行平均"后面的同一帧。插图表示从图像中放大的盒装区域。


图5.定义用于在ImageJ中生成kymograph的轨迹 A.用于选择要分析的线性轨迹的时间平均图像。 B.相同的选择转移到延时堆栈。 C. Kymograph分析。


图6.使用Imaris分析粒子运动。A.检测到斑点并用小方块标记。 B.基于大于60秒的持续时间过滤后的轨迹。

笔记

  1. CESA颗粒的速度根据温度和其他因素而变化;因此比较测定应在同一天并在相同的成像条件下进行。
  2. 当幼苗在有或没有蔗糖的板中生长时,没有观察到复合运动性的变化
  3. 所有构建体都需要N末端荧光蛋白融合才能发挥功能
  4. 所有这些荧光蛋白融合物可以表达野生型或突变背景。已经观察到,GFP:CESA3 构建体不能拯救CESA3的无效等位基因,虽然hypomorphic等位基因例如cesa3
  5. 如果激光器可用,荧光标记YFP也可以在514nm激发

食谱

  1. 0.5x Murashige和Skoog(MS)培养基(1L) 加入2.2g MS盐
    添加0.5 g MES
    加入10克蔗糖
    用1M KOH调节pH至5.7 将dH <2> O添加到1 L
    为固体培养基
    添加8克琼脂 在121℃高压灭菌30分钟

致谢

该协议改编自先前公开的研究,Paredez等人。(2006)。我们衷心感谢Chris Somerville教授,Heidi Szemenyei博士和Charles T. Anderson博士对协议的批判性阅读。这项工作是在加州大学伯克利分校的Chris Somerville实验室进行的。电视由EMBO长期奖学金(ALTF 711-2012)和Philomathia基金会的博士后资助支持。 TY还得到了Philomathia基金会的一个研究金的支持。 NS是博士后奖的获得者。来自二元农业研究和发展基金的FI-434-2010。

参考文献

  1. Brabham,C。和Debolt,S。(2012)。 化学遗传学检查纤维素生物合成。 前植物科学 3:309.
  2. Desprez,T.,Juraniec,M.,Crowell,E.F.,Jouy,H.,Pochylova,Z.,Parcy,F.,Hofte,H.,Gonneau,M.and Vernhettes, 在拟南芥中参与原代细胞壁合成的纤维素合酶复合物的组织 。 Proc Natl Acad Sci USA 104(39):15572-15577。
  3. Paredez,A.R.,Somerville,C.R.and Ehrhardt,D.W。(2006)。 纤维素合酶的可视化表现出与微管的功能性关联。 科学 312(5779):1491-1495。
  4. Sanchez-Rodriguez,C.,Bauer,S.,Hematy,K.,Saxe,F.,Ibanez,AB,Vodermaier,V.,Konlechner,C.,Sampathkumar,A.,Ruggeberg,M.,Aichinger, ,Neumetzler,L.,Burgert,I.,Somerville,C.,Hauser,MT和Persson,S。(2012)。 几丁质酶样1/pom-pom1及其同源物CTL2是对纤维素生物合成重要的葡聚糖相互作用蛋白拟南芥。植物细胞 24(2):589-607。
  5. Sorek,N.,Sorek,H.,Kijac,A.,Szemenyei,H.J.,Bauer,S.,Hematy,K.,Wemmer,D.E.and Somerville,C.R。(2014)。 拟南芥 COBRA蛋白有助于纤维素在质膜上结晶。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vellosillo, T., Yeats, T. and Sorek, N. (2015). Analysis of in vivo Cellulose Biosynthesis in Arabidopsis Cells by Spinning Disk Confocal Microscopy. Bio-protocol 5(19): e1617. DOI: 10.21769/BioProtoc.1617.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。