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Measurement of the Number of Peroxisomes
过氧化物酶体数量的测量   

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Abstract

This is the detailed protocol for the measurement of the number of peroxisomes described by Shibata et al. (2013). It is difficult to count the number of organelles in a cell because of the thickness of plant leaves. To overcome this challenge, protoplasts were isolated from leaves, and the number of peroxisomes per protoplast was counted. This method can be applied to other organelles such as mitochondria that are labeled with GFP or its derivatives.

Keywords: Peroxisome(过氧化物酶体), Peroxisome-targeted GFP(GFP的过氧化物酶体靶向性), Protoplast(原生质体), Number of organelles(细胞器数量)

Material and Reagents

  1. Arabidopsis plant expressing peroxisome-targeted GFP (Mano et al., 2002)
  2. Cellulase Onozuka R-10 (Yakult Honsha)
  3. Macerozyme R-10 (Yakult Honsha)
  4. MES
  5. Mannitol
  6. Enzyme solution (see Recipes)
  7. Wash solution (see Recipes)

Equipment

  1. Time tape (Time Med)
  2. Mending tape (3M)
  3. Aluminum foil
  4. 20 ml beaker
  5. Shaker
  6. Pasteur pipette
  7. Test tube
  8. Swing-out rotor centrifuge machine
  9. Confocal laser scanning microscope (Zeiss, model: LSM510 META )
  10. MAS-coated glass slide (super frost) (Matsunami Glass, catalog number: S9441 )
  11. Cover slip (24 x 60 No.1, 0.12-0.17 mm) (Matsunami Glass, catalog number: C024601 )

Software

  1. NIH ImageJ software 1.46 for Windows (http://imagej.nih.gov/ij)
    Note: ImageJ is also available on Mac OS X and Linux.

Procedure

  1. Protoplast isolation
    There are several methods for protoplast isolation. The following method is based on the Tape-Sandwich method (Wu et al., 2009) with slight modifications.
    1. Collect the sixth and seventh leaves of 3-week-old Arabidopsis plants. Four leaves are enough for this experiment.
    2. Remove the leaf epidermis by peeling using two types of tape, Time tape and mending tape. Put a Time tape and a Mending tape on the upper side and the underside of a leaf, respectively. Gently remove the Mending tape to peel the epidermis of a leaf. Please see Wu et al. (2009) for more detail.
      Note: Once you find a compatible tape-set, you can peel the epidermis easily and cleanly. However, there are inconvenient lots even in same products.
    3. Put the Time tape with the leaf into a beaker with 5 ml of enzyme solution (see Recipes below).
    4. Wrap the beaker in aluminum foil to avoid light damage and gently shake (~40 rpm) for 30 min at room temperature. During this step, cell walls are digested with enzyme solution, and protoplasts are isolated from the leaves.
    5. Gently transfer the protoplast-containing solution into a test tube by decantation.
    6. Spin down at 100 x g for 1 min at room temperature using a swing-out rotor.
    7. Discard the supernatant and gently add 2 ml of wash solution (see Recipes below).
    8. Spin down at 100 x g for 1 min at room temperature using a swing-out rotor.
    9. Repeat the steps A7-8 once again.
    10. Discard the supernatant and gently add 1 ml of wash solution.

  2. Acquisition of protoplast images by microscope
    1. Carefully put the protoplast solution on a MAS-coated glass slide using Pasteur pipette.
    2. Gently put a cover slip on the slide.
    3. Mount prepared specimen on microscope and focus. Look for protoplasts which are 40- 55 µm in diameter.
    4. Acquire the Z-series images of a protoplast from the upper to the lower (or middle) position.
      Our condition using LSM510 is as follows: Argon laser 488 nm, Emission filter BP505-550, 40x dry objective (Plan-Neofluar 40x/0.75), 1 x zoom, pixel size 0.22 µm. The step size between each focal plane is 0.887 µm. To obtain images quickly, minimize the scanning area using “ROI”.
    5. Execute Z-projection to compile the acquired sequential images in one image by operating software of LSM510 META.
      Note: The observation of protoplasts should be finished within one hour after the isolation, since isolated protoplasts become quickly unhealthy and easily burst. Z projection can also be done in ImageJ: Image>Stacks>Z project>Maximum intensity projection.

  3. Peroxisome counting
    1. Launch an ImageJ software and open the Z-projection image.
    2. Calculate the diameter of each protoplast using the menu of Analyze>Measure.
      Note: You need to carry out “Set scale” before calculation. Click “Straight” tool and trace the scale bar of opened image. Go Analyze>Set Scale, input the distance in the box “Known distance”, check Global, and click OK to close the window.
    3. Go Plugins>Analyze>Cell Counter and count up peroxisomes manually (Figure 1).


      Figure 1. Peroxisome counting using ImageJ

      Note: For statistical analysis, we counted 15-20 protoplasts. See Table 1 and 2. It is necessary to confirm that the average of diameters of protoplasts between the control and your targets are nearly equal. In the case of table 1, a protoplast with small diameter (sample 1) was eliminated from the analysis due to too small diameter. Carefully discriminate between organelles and noises because signals from the bottom side are low, and some of them are observed as a connected signal. To minimize the subjectivity of people, it is recommended that multiple people independently count peroxisomes against same data as a blind-test.

      Table 1. Diameters and numbers of peroxisome in a protoplast

      Diameter
      Number
      sample 1*1
      29.8
      31
      sample 2
      36.1
      29
      sample 3
      36.6
      25
      sample 4
      37.7
      47
      sample 5
      40.4
      42
      sample 6
      42.0
      40
      sample 7
      43.5
      59
      sample 8
      44.0
      42
      sample 9
      45.0
      58
      sample 10
      45.5
      49
      sample 11
      45.9
      54
      sample 12
      45.9
      81
      sample 13
      46.0
      45
      sample 14
      47.0
      52
      sample 15
      47.3
      33
      sample 16
      47.6
      66
      sample 17
      47.9
      52
      *1A protoplast with small diameter, such as sample 1, was eliminated from the analysis due to too small diameter.

      Table 2. The average of diameters and numbers of peroxisome in a protoplast

      Diameter (D)
      Number
      n
      35<D<50
      43.6
      48.4
      16
      SD
      5.1
      14.2

      SE
      1.3   
      3.6


Recipes

  1. Enzyme solution
    1% (v/w) cellulase Onozuka R-10
    0.25% (v/w) macerozyme R-10
    0.4 M mannitol
    20 mM MES (pH 5.7)
  2. Wash solution
    0.4 M mannitol
    20 mM MES (pH 5.7)

Acknowledgments

This is the detailed protocol for the measurement of the number of peroxisomes described by Shibata et al. (2013).

References

  1. Mano, S., Nakamori, C., Hayashi, M., Kato, A., Kondo, M. and Nishimura, M. (2002). Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement. Plant Cell Physiol 43(3): 331-341.
  2. Shibata, M., Oikawa, K., Yoshimoto, K., Kondo, M., Mano, S., Yamada, K., Hayashi, M., Sakamoto, W., Ohsumi, Y. and Nishimura, M. (2013). Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis. Plant Cell 25(12): 4967-4983.
  3. Wu, F. H., Shen, S. C., Lee, L. Y., Lee, S. H., Chan, M. T. and Lin, C. S. (2009). Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods 5: 16.

简介

这是用于测量由Shibata等人(2013)描述的过氧化物酶体的数量的详细方案。 由于植物叶片的厚度,很难计数细胞中的细胞器的数目。 为了克服这种挑战,从叶中分离原生质体,并计数每个原生质体的过氧化物酶体的数目。 该方法可应用于用GFP或其衍生物标记的其他细胞器,例如线粒体。

关键字:过氧化物酶体, GFP的过氧化物酶体靶向性, 原生质体, 细胞器数量

材料和试剂

  1. 植物表达过氧化物酶体靶向的GFP(Mano等人,2002)
  2. 纤维素酶Onozuka R-10(Yakult Honsha)
  3. Macerozyme R-10(Yakult Honsha)
  4. MES
  5. 甘露醇
  6. 酶溶液(见配方)
  7. 洗涤溶液(见配方)

设备

  1. 时间磁带(Time Med)
  2. 修补胶带(3M)
  3. 铝箔
  4. 20ml烧杯
  5. 振动器
  6. 巴斯德移液器
  7. 试管
  8. 旋转式转子离心机
  9. 共焦激光扫描显微镜(Zeiss,型号:LSM510 META)
  10. MAS涂层玻璃载片(超级霜)(Matsunami Glass,目录号:S9441)
  11. 盖玻片(24×60 No.1,0.12-0.17mm)(Matsunami Glass,目录号:C024601)

软件

  1. 用于Windows的NIH ImageJ软件1.46( http://imagej.nih.gov/ij
    注意:ImageJ也可在Mac OS X和Linux上使用。

程序

  1. 原生质体分离
    有几种原生质体分离的方法。以下方法基于Tape-Sandwich方法(Wu等人,2009),稍作修改。
    1. 收集3周龄拟南芥植物的第六叶和第七叶。四叶对于这个实验是足够的。
    2. 通过剥离使用两种类型的胶带,时间去除叶表皮 磁带和修补磁带。将时间胶带和修补胶带放在上面 侧和叶的下侧。轻轻取出 修补胶带剥离叶片的表皮。有关详情,请参阅Wu 等 (2009)。
      注意:一旦找到兼容的磁带集, 您可以轻松,干净地剥离表皮。但是,有 即使在同一产品中也会带来不便。
    3. 把带叶的时间胶带放入带有5ml酶溶液的烧杯中(见下面的食谱)。
    4. 将杯子包裹在铝箔中,避免轻微损伤和温和 在室温下摇动(〜40rpm)30分钟。 在此步骤中,单元格 壁用酶溶液消化,并分离原生质体 从叶子
    5. 通过倾析将含原生质体的溶液轻轻转移到试管中
    6. 在室温下使用摇摆转子在100×g下旋转1分钟。
    7. 弃去上清液,轻轻加入2ml洗涤溶液(见下面的配方)。
    8. 在室温下使用摇摆转子在100×g下旋转1分钟。
    9. 重复步骤A7-8。
    10. 弃去上清液,轻轻加入1 ml洗涤液。

  2. 通过显微镜获取原生质体图像
    1. 使用巴斯德移液管小心地将原生质体溶液放在MAS涂层的载玻片上
    2. 在幻灯片上轻轻盖上盖子。
    3. 登上准备的标本在显微镜和焦点。 寻找直径40-55μm的原生质体。
    4. 从上到下(或中间)位置获取原生质体的Z系列图像。
      我们使用LSM510的条件如下:氩激光488nm,发射 过滤器BP505-550,40x干物镜(Plan-Neofluar 40x/0.75),1×变焦, 像素尺寸为0.22μm。 每个焦平面之间的步长为0.887μm。 要快速获取图像,请使用"投资回报率"最小化扫描区域
    5. 执行Z投影,通过LSM510 META的操作软件在一个图像中编译获取的连续图像 注意:原生质体的观察应在一小时内完成   分离后,因为分离的原生质体迅速变得 不健康,容易爆裂。 Z投影也可以在ImageJ中完成: 图像>堆栈> Z项目>最大强度投影。

  3. 过氧化物酶体计数
    1. 启动ImageJ软件并打开Z投影图像。
    2. 使用分析&测量菜单计算每个原生质体的直径。
      注意:在计算前需要执行"设置刻度"。 点击 "直线"工具并跟踪打开图像的比例尺。 走 分析>设置缩放,在框中输入距离"已知距离", 选中"全局",然后单击"确定"关闭窗口
    3. Go插件>分析>单元计数器并手动计数过氧化物酶体(图1)。


      图1.使用ImageJ的过氧化物酶计数

      注意:对于统计分析,我们计数15-20原生质体。参见表  1和2.有必要确认的直径的平均值 控制和目标之间的原生质体几乎相等。在 表1的情况,具有小直径的原生质体(样品1) 由于直径太小而从分析中消除。小心 区分细胞器和噪声,因为信号来自 底部低,其中一些被观察为连接 信号。为了最小化人的主观性,建议 多个人根据相同的数据独立地计数过氧化物酶体 盲测。

      表1.原生质体中过氧化物酶体的直径和数量

      直径
      号码
      示例1 * 1
      29.8
      31
      样品2
      36.1
      29
      样品3
      36.6
      25
      样品4
      37.7
      47
      样品5
      40.4
      42
      样品6
      42.0
      40
      样品7
      43.5
      59
      样品8
      44.0
      42
      样品9
      45.0
      58
      样品10
      45.5
      49
      样品11
      45.9
      54
      样品12
      45.9
      81
      样品13
      46.0
      45
      样品14
      47.0
      52
      样品15
      47.3
      33
      样品16
      47.6
      66
      样品17
      47.9
      52
      * 1 具有小直径的原生质体,例如样品1,由于直径太小而从分析中消除。

      表2.原生质体中过氧化物酶体的直径和数量的平均值

      直径(D)
      号码
      n
      35 43.6
      48.4
      16
      SD
      5.1
      14.2

      SE
      1.3 
      3.6


食谱

  1. 酶溶液
    1%(v/w)纤维素酶Onozuka R-10 0.25%(v/w)巨噬酶R-10 0.4M甘露醇 20mM MES(pH 5.7)
  2. 洗液
    0.4M甘露醇 20mM MES(pH 5.7)

致谢

这是用于测量由Shibata等人(2013)描述的过氧化物酶体的数量的详细方案。

参考文献

  1. Mano,S.,Nakamori,C.,Hayashi,M.,Kato,A.,Kondo,M。和Nishimura,M。(2002)。 阿拉伯语 中的过氧化物酶体的分布和表征通过用GFP可视化:动态形态和肌动蛋白依赖性运动。植物细胞生理学43(3):331-341。
  2. Shibata,M.,Oikawa,K.,Yoshimoto,K.,Kondo,M.,Mano,S.,Yamada,K.,Hayashi,M.,Sakamoto,W.,Ohsumi,Y.and Nishimura, 2013)。 高度氧化的过氧化物酶体通过拟南芥中的自噬而选择性降解。 > 植物细胞 25(12):4967-4983。
  3. Wu,F.H.,Shen,S.C.,Lee,L.Y.,Lee,S.H.,Chan,M.T.and Lin,C.S。(2009)。 磁带 - 拟南芥三明治 - 更简单的拟南芥 >原生质体分离方法。 植物方法 5:16.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Shibata, M., Oikawa, K., Mano, S. and Nishimura, M. (2014). Measurement of the Number of Peroxisomes. Bio-protocol 4(21): e1284. DOI: 10.21769/BioProtoc.1284.
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