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Measuring the Interactions between Peroxisomes and Chloroplasts by in situ Laser Analysis
原位激光分析法测定过氧化物酶体和叶绿体之间的相互作用   

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Abstract

Quantitative analysis has been necessary for deeply understanding characteristic of organelles function. This is the detailed protocol for the quantification of the physical interaction between peroxisomes and chloroplasts taken by laser scanning microscopy described by Oikawa et al. (2015). To clarify the morphological interactions between both organelles, we measured the contact length between two organelles (interaction length) in the fluorescent microscope image by using image analysis software ImageJ. The result clearly revealed that the contact length in light condition is much longer than that in dark condition. In addition, the force of the morphological interaction was quantified utilizing intersection technology of femtosecond laser and atomic force microscope (AFM). When an intense femtosecond laser is focused near the interface of two organelles, the adhesion is broken by a force due to the laser. The adhesion strength in light and dark conditions was estimated from the force calibrated by AFM. The detailed procedure is described in Bio-protocol as another protocol entitled “Quantification of the adhesion strength between peroxisomes and chloroplasts by femtosecond laser technology” (Hosokawa et al., 2016). These methods can be applied to other physical interaction between different types of organelles such as nuclei, mitochondria, Golgi, and chloroplasts.

Keywords: Peroxisome(过氧化物酶体), Chloroplast(叶绿体), Organelle interaction(细胞器的相互作用), Measurement of interaction(测量的作用)

Materials and Reagents

  1. Glass slide (super frost) (Matsunami Glass)
    Note: Any types of glass slide suitable for fluorescence observation can be used. We attached black tape on the glass slide to envelop the sample with cover slip (see Figure 1D).
  2. Cover slip (24 x 60 No.1, Thickness 0.12-0.17 mm) (Matsunami Glass)
  3. 10 ml disposable syringe (Terumo Medical Corporation)
  4. Arabidopsis thaliana (ecotype Columbia) expressing peroxisome-targeted GFP (GFP-PTS1) (Mano et al., 2002)
    Note: 1-4 is shown in Figure 1A.
  5. 1/3 x Murashige and Skoog salts (MS) medium (Wako Pure Chemical Industries, catalog number: 392-00591 ) containing vitamins, 1% sucrose, and MES buffer (pH 5.7) (Wako Pure Chemical Industries, catalog number: 341-01622 )
  6. Agar powder for plant growth (Funakoshi, catalog number: BA-10 )
  7. Distilled water
  8. KOH
  9. Plant culture medium (see Recipes)

Equipment

  1. Scissors and tweezers (shown in Figure 1A)
  2. 40x dry system objective lens (ZEISS, EC Plan-Neofluar®)
  3. Growth chamber for growing plants (100 µmol m-2 sec-1 white Light for 16 h and dark for 8 h, 22 °C)
  4. Black box for dark condition
  5. Confocal laser scanning microscope (ZEISS, model: LSM510 META )

Software

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

Procedure

  1. Preparation of leaf section
    1. Cut the rosette leaves of 3-week-old Arabidopsis plants in 1 cm diameter and placed in a syringe filled with water (Figure 1B).
    2. Plug the top of syringe by finger and pull plunger to deaerate the leaves (Figure 1B).
    3. Mount one piece of the deaerated-leaf section (Figure 1C) on slide glass with water and cover with cover glass (Figure 1D).
    4. Incubate the pretreated-leaf sample in the dark or light for 2 h in growth chamber.


    Figure 1. Preparation of leaf section for CLSM observation. A. All instruments used for preparation of leaf section. B. Deaeration of the leaves by pulling plunger while plugging the top of syringe. C. Leaf section used for observation. Before (left) and after (right) deaeration. D. Deaerated-leaf section mounted on the slide glass.

  2. Taking organelles images by CLSM
    1. The images of organelles in leaf palisade mesophyll cells are focused and obtained through a 40x dry system objective lens using LSM 510 (Figure 4).
    2. To observe GFP fluorescence, a 488 nm Ar/Kr laser is used and the fluorescence is detected through an emission filter BP505-550. To detect the autofluorescence from chlorophyll, a 543 nm He/Ne laser is used and the signal is acquired through emission filters LP580.
    3. Obtain both fluorescence simultaneously and save the data as a single image of tif. file.
      Note: The observation of organelles should be finished within 30 min after moving the sample from growth chamber, since the change of light condition affects the organelle motility and interaction.

  3. Quantification of the organelle interaction
    1. Launch an Image J software and open the obtained images.
    2. Set the scale of s: Analyze > Set Scale
    3. Set the measurements: Analyze > Set Measurements > check the column of Shape descriptors (Figure 2).


      Figure 2. Analyze menu of ImageJ used to measure the length of segments lines. Choose Shape descriptors in Set Measurements window. Results of 10 interaction lengths are shown as an example of calculation after selecting Measure mode below Analyze menu.

    4. Using line tool, draw a line connecting two intersection points between two organelles, or using segment tool, draw the curve line (red line in the right panel of Figure 3 and lower panel of Figure 4) for measuring.
      Note: The image is magnified by zoom (in) function of ImageJ when drawing a line. Image > Zoom > In{+}.


      Figure 3. Segmented Line tool of ImageJ used to measure the interaction length (red line in the right panel) between a peroxisome and a chloroplast

    5. Measure the length of lines: Analyze > Measure
      Note: An average of at least 50 interactions between peroxisomes and chloroplasts should be measured to calculate the average length in independent three experiments (Figure 4, Table 1). Here, we show results of ten samples for measurements of interaction lengths between peroxisomes and chloroplasts.


      Figure 4. Measurements of the 10 selected-interaction lengths of membrane contact area between peroxisomes (green) and chloroplasts (magenta) in the dark (left panel) and light (right panel) at lower panels.
      Spherical peroxisomes in the dark change their shape to elongation in light, suggesting that their physiological interaction is enhanced in light. Upper panels are original images. Bars = 10 µm

      Table 1. Results of measurements of the 10 selected-contact lengths between peroxisomes and chloroplasts in dark and light.
      The contact length in light condition was longer than that in dark condition. This fact suggests that the peroxisomes assist photorespiration by tightly contacting with the chloroplasts in photosynthesis.

Recipes

  1. Plant culture medium
    Dissolve 10 g sucrose, 1.53 g MS salt, and 0.5 g MES in 1 L pure water, and then adjust the pH to 5.7 with 1 M KOH
    Add 8 g agar in the medium, and then autoclave

Acknowledgments

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) [KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas to M. N. (No. 22120007) and Y. H. (No. 22120010)].

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. Hosokawa, Y., Iino, T., Oikawa, K., Mano, S., Yamada, K. and Nishimura, M., Quantification of the adhesion strength between peroxisomes and chloroplasts by femtosecond laser technology. Bio-protocol 6(11): e1834.
  3. Oikawa, K., Matsunaga, S., Man, S., Kondo, M., Yamada, K., Hayashi, M., Kagawa, T., Kadota, A., Sakamoto, W., Higashi, H., Watanabe, M., Mitsui, T., Shigemasa, A., Iino, T., Hosokawa, Y. and Nishimura, M. (2015). Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis. Nat Plants 1: 15035.
  4. Shibata, M., Oikawa, K., Mano, S. and Nishimura, M. (2014). Measurement of the number of peroxisomes. Bio-protocol 4(21): e1284.

简介

定量分析对于深入了解细胞器功能的特征是必要的。这是通过由Oikawa等人(2015)描述的激光扫描显微术获取的用于定量过氧化物酶体和叶绿体之间的物理相互作用的详细方案。为了澄清两种细胞器之间的形态学相互作用,我们使用图像分析软件Image J测量荧光显微镜图像中两个细胞器之间的接触长度(相互作用长度)。结果清楚地表明,光照条件下的接触长度比在黑暗的条件。此外,利用飞秒激光和原子力显微镜(AFM)的交叉技术来量化形态相互作用的力。当强激光飞秒激光聚焦在两个细胞器的界面附近时,粘附力由于激光的力而断裂。在光和暗条件下的粘合强度由通过AFM校准的力估计。详细过程在Bio-protocol中描述为另一个名为"Quantification of the adhesion strength between peroxisomes and chloroplasts by femtosecond laser technology"的另一个协议(Hosokawa等人,2016)。这些方法可以应用于不同类型的细胞器如细胞核,线粒体,高尔基体和叶绿体之间的其他物理相互作用。

关键字:过氧化物酶体, 叶绿体, 细胞器的相互作用, 测量的作用

材料和试剂

  1. 玻璃幻灯片(超级霜)(Matsunami玻璃)
    注意:可以使用适合于荧光观察的任何类型的载玻片。我们在载玻片上贴上黑色胶带,用盖玻片包住样品(见图1D)。
  2. 盖滑(24×60 No.1,厚度0.12-0.17mm)(Matsunami玻璃)
  3. 10ml一次性注射器(Terumo Medical Corporation)
  4. 表达过氧化物酶体靶向GFP(GFP-PTS1)的拟南芥(Columbia生态型)(Mano等人,2002)
    注意:1-4如图1A所示。
  5. 含有维生素,1%蔗糖和MES缓冲液(pH5.7)(Wako Pure Chemical Industries,目录号:341-5)的1/3 x Murashige和Skoog盐(MS)培养基(Wako Pure Chemical Industries,目录号:392-00591) 01622)
  6. 用于植物生长的琼脂粉(Funakoshi,目录号:BA-10)
  7. 蒸馏水
  8. KOH
  9. 植物培养基(见配方)

设备

  1. 剪刀和镊子(如图1A所示)
  2. 40x干系统物镜(ZEISS,EC Plan-Neofluar
  3. 用于生长植物的生长室(100μmol.m-2 s -1白光照明16h和黑暗8h,22℃)
  4. 暗盒黑盒
  5. 共聚焦激光扫描显微镜(ZEISS,型号:LSM510 META)

软件

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

程序

  1. 叶区的准备
    1. 切割3周龄拟南芥植物的玫瑰花叶,直径1厘米,并置于充满水的注射器(图1B)。
    2. 用手指插入注射器的顶部,并拉动柱塞使叶子脱气(图1B)。
    3. 将一片脱气叶片部分(图1C)装在载玻片上,并用盖玻片覆盖(图1D)。
    4. 在黑暗或光照下在生长室中孵育预处理的叶样品2小时


    图1. CLSM观测的叶片部分的制备。 A。所有仪器用于制备叶片。 B.通过在插入注射器的顶部的同时拉动柱塞来使叶子脱气。 C.用于观察的叶片。前(左)和后(右)脱气。 D.安装在载玻片上的除叶片部分。

  2. 通过CLSM采取细胞器图像
    1. 叶栅叶肉细胞中的细胞器的图像被聚焦 并通过使用LSM 510的40x干系统物镜获得 (图4)。
    2. 为了观察GFP荧光,使用488nm Ar/Kr激光, 通过发射滤光片BP505-550检测荧光。检测 ?使用来自叶绿素的自发荧光,543nm He/Ne激光器 ?通过发射滤波器LP580获取信号。
    3. 同时获得两个荧光并将数据保存为tif的单个图像。文件。
      注意:在从生长室移动样品后30分钟内,细胞器的观察应该完成,因为光条件的改变影响细胞器的运动和相互作用。

  3. 细胞器相互作用的量化
    1. 启动Image J软件并打开获取的图像。
    2. 设置s的比例:Analyze>设置缩放
    3. 设置测量:分析>设置测量>检查Shape描述符的列(图2)。


      图2.图像J的分析菜单用于测量线段的长度。在"设置测量"窗口中选择"形状描述符"。结果:10 相互作用长度作为计算的实例示出 选择分析菜单下的测量模式。

    4. 使用线工具,绘制连接两个细胞器之间的两个交叉点的线,或使用线段工具,绘制曲线(图3的右图中的红线和图4的下图)用于测量。 注意:绘制线条时,图像通过放大(放大)功能放大。图像>缩放>在{+}。


      数字 ?3.图像J的分段线工具用于测量过氧化物酶体和叶绿体之间的相互作用长度(右图中的红线)

    5. 测量线的长度:分析>测量
      注意:应当测量过氧化物酶体和叶绿体之间的至少50个相互作用的平均值,以计算独立的三次实验中的平均长度(图4,表1)。在这里,我们显示十个样品的结果,用于测量过氧化物酶体和叶绿体之间的相互作用长度

      图4.黑暗(左图)和浅色(右图)的过氧化物酶体(绿色)和叶绿体(洋红色)之间的膜接触面积的10个选择的相互作用长度的测量。它们的形状对光的伸长,提示它们的光的生理相互作用增强。上图是原始图像。条=10μm

      表1.在黑暗和光照下过氧化物酶体和叶绿体之间的10个选择接触长度的测量结果。在光照条件下的接触长度比在黑暗条件下的接触长度长。这一事实表明,过氧化物酶体通过在光合作用中与叶绿体紧密接触来协助光呼吸。

食谱

  1. 植物培养基
    在1L纯水中溶解10g蔗糖,1.53g MS盐和0.5g MES,然后用1M KOH将pH调节至5.7 / 在培养基中加入8g琼脂,然后高压灭菌

致谢

这项工作得到教育,文化,体育,科学和技术部(MEXT)[KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas to M.N。(No. 22120007)和Y. H.(No. 22120010)]的支持。

参考文献

  1. Mano,S.,Nakamori,C.,Hayashi,M.,Kato,A.,Kondo,M。和Nishimura,M。(2002)。 通过用GFP可视化的拟南芥中过氧化物酶体的分布和表征:动态形态和肌动蛋白依赖性运动。/a> Plant Cell Physiol 43(3):331-341。
  2. Hosokawa,Y.,Iino,T.,Oikawa,K.,Mano,S.,Yamada,K.and Nishimura,M。,< a class ="ke-insertfile"href ="http:通过飞秒激光技术定量测定过氧化物酶体和叶绿体之间的粘附强度。生物方案 6(11):e1834 br />
  3. Oikawa,K.,Matsunaga,S.,Man,S.,Kondo,M.,Yamada,K.,Hayashi,M.,Kagawa,T.,Kadota,A.,Sakamoto,W.,Higashi, Watanabe,M.,Mitsui,T.,Shigemasa,A.,Iino,T.,Hosokawa,Y.and Nishimura,M.(2015)。 原位激光分析所阐明的过氧化物酶体和叶绿体之间的物理相互作用。 em> Nat Plants 1:15035.
  4. Shibata,M.,Oikawa,K.,Mano,S.and Nishimura,M.(2014)。 过氧化物酶体的数量测量 生物协议 4(21) :e1284。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Oikawa, K., Mano, S., Yamada, K., Hosokawa, Y. and Nishimura, M. (2016). Measuring the Interactions between Peroxisomes and Chloroplasts by in situ Laser Analysis. Bio-protocol 6(8): e1790. DOI: 10.21769/BioProtoc.1790.
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