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Chloroplast-actin (cp-actin) filaments play a pivotal role in chloroplast photorelocation movement. This protocol describes observation of cp-actin filaments in intact palisade cells of Arabidopsis leaves (Kong et al., 2013). The live cell imaging of cp-actin filaments is taken on moving chloroplasts, so that this protocol is useful for analysis of cp-actin dynamics that are induced by blue light.

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Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis
拟南芥叶片中叶绿体-肌动蛋白微丝动态变化的观测

植物科学 > 植物细胞生物学 > 细胞结构
作者: Sam-Geun Kong
Sam-Geun KongAffiliation: Department of Biology, Kyushu University, Fukuoka, Japan
For correspondence: kong.samgeun@gmail.com
Bio-protocol author page: a1082
 and Masamitsu Wada
Masamitsu Wada Affiliation: Department of Biology, Kyushu University, Fukuoka, Japan
Bio-protocol author page: a1083
Vol 3, Iss 24, 12/20/2013, 2911 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1008

[Abstract] Chloroplast-actin (cp-actin) filaments play a pivotal role in chloroplast photorelocation movement. This protocol describes observation of cp-actin filaments in intact palisade cells of Arabidopsis leaves (Kong et al., 2013). The live cell imaging of cp-actin filaments is taken on moving chloroplasts, so that this protocol is useful for analysis of cp-actin dynamics that are induced by blue light.
Keywords: Actin(肌动蛋白), Arabidopsis(拟南芥), Blue light(蓝色的光), Chloroplast(叶绿体), Organelle(细胞器)

[Abstract]

Materials and Reagents

  1. Plant materials
    1. Arabidopsis thaliana transgenic plants expressing GFP-TALIN in wild-type or mutant plants (Kong et al., 2013). Actin probe lines such as LIFEACT-YFP and GFP-fABD2, THRUMIN1-GFP transgenic lines are also useful.
    2. Young and fully expanded rosette leaves of 3-4-week-old plants grown under 100 μmol/m2/s white light (16 h)/dark (8 h) cycles at 23 °C.
      Note: Plants grown on soil are better than those in the plate of MS medium. The reason is that the leaves of the plants grown in the plate are more susceptible to wither during sample handling than those grown on soil.
  2. Red cellophane film (TokyoButaiShowmei, catalog number: No. 20 )
  3. (Optional) 2,3-butanedione monoxime (BDM) (Sigma-Aldrich, catalog number: B0753 ) as an inhibitor of myosin ATPase that inhibits actin dynamics
  4. Evacuation solution containing Silwet L-77 (Bristol-Myers Squibb Company, catalog number: BMS-SL7755 ) (see Recipes)

Equipment

  1. Syringe (10 ml)
  2. Confocal microscope (Leica Microsystems, model: SP5 equipped with a 63x/1.20 W objective lens and multi-line 100 mW argon laser)
  3. Plant growth room or chamber
  4. Red safe-light (red LED or fluorescent lamp filtered with red films)
  5. Forceps
  6. Razor blade
  7. Scissors
  8. A custom-made cuvette system and ring holder: this system is composed of two steel rings mating each other with complementary threads, two round cover glasses (22 mm in diameter, No. 1 and No. 5), and a silicon ring (100 μm in thickness) (Wada and Kong, 2011; see Video 1)

Software

  1. Open source software ImageJ (http://rsbweb.nih.gov/ij/)

Procedure

  1. Preparation of leaf specimen (see Video 1)

    Video 1. Preparation of leaf specimen in the cuvette system

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

    1. Incubate plants under weak light conditions (30 μmol m-2 s-1 white light) for a couple of hours.
      Notes:
      1. Under weak light conditions, chloroplasts accumulate along the cell walls that are perpendicular to the incident light (accumulation response). However, they move away from the area under strong light conditions and accumulate to the anticlinal walls that are parallel to the incident light (avoidance response).
      2. This step is useful to observe chloroplasts during avoidance response. In addition, this step can be skipped or modified depending on your experimental purposes.
    2. Detach a rosette leaf and eliminate airspaces in the leaf by evacuation in a syringe filled with an evacuation solution (0.01% Silwet L-77).
    3. Cut the leaf into small pieces (ca. 4 mm x 4 mm) with a razor blade.
    4. (Optional) Inhibitor treatment such as 50 mM 2,3-butanedione monoxime (BDM).
      1. Dilute interesting inhibitors to appropriate concentrations in the evacuation solution. As a mock treatment, carrier solution (DMSO) without the inhibitor is added to the evacuation solution at the same dilution rate.
      2. Eliminate air spaces in the leaf by gentle evacuation using a syringe that is filled with the solution.
      3. Incubate the leaf samples for the appropriate times (0.5 ~ 1 h) under dim red light.
    5. Place the excised and cut leaf (treated with inhibitor solution, if necessary) adaxial side up on a round coverslip (No. 5)
    6. Cover the leaf specimen carefully with a round coverslip (No. 1) and set it in a cuvette system.
      Note: Remove carefully any bubbles in the space between leaf sample and coverslips not only the air in the air space of a leaf.

  2. Confocal microscopy
    1. Find an appropriate chloroplast(s) by observing the specimen under dim red light and adjust focus at the vicinity between the plasma membrane and chloroplast(s) in palisade cells using rapid scans at weak laser power.
      Note: Eye observation under dim red light conditions is recommended to reduce any light effect on cp-actin filament dynamics. The dim red light condition is made using a piece of red cellophane film that is put in the light path of halogen lamp. If necessary to adjust focus, set output laser at the lowest power (1-5% of 488 nm) as much as chlorophyll fluorescence is detectable.
    2. Incubate the specimen in darkness for several minutes on the microscope stage.
      Note: Disappearance (depolymerization) of cp-actin filaments is induced by several scans of optical blue laser. However, dark incubation induces polymerization of cp-actin filaments following the blue laser scanning.
    3. Set optical parameters to optimize signal to noise ratio using SP5 software (live data mode).
      1. Excitation lasers: the multi-line argon laser (100 mW of laser power) is set at 20% output power, and further at 10% of 488 nm for GFP excitation and 5% of 458 nm for the induction of chloroplast avoidance response.
        Note: Laser powers are empirically adjustable to obtain high signal to noise images depending on experimental conditions and individual systems' performances that may vary.
      2. Emission bands for the specific fluorescent signals: 500 to 550 nm for GFP and 640 to 740 nm for chlorophyll.
      3. Capture the images (three to four images in 0.5-μm steps, if necessary) with a lens (63x/1.20 W) at an appropriate resolution (for example, 256 x 256, or 512 x 256) with 4x digital zooming for 20 or 30 time-lapse cycles with at least 20-sec time-interval (see below). Scope is in XYZT mode.
        Note: Scanning speed of confocal microscopy is limited and cp-actin filaments are dynamically regulated. Hence, temporal and spatial resolutions can be controlled at some ranges by changing scan speed and y-axes resolution. Bidirectional mode can also be used to shorten acquisition time.
      4. To induce chloroplast avoidance response, scan an appropriate rectangular or circular regions using region of interest (ROI) with the stimulating laser (458 nm, 2.8 μW) during intervals for the appropriate times.
        Note: To induce avoidance response, blue light is given directly on a half side of chloroplast. The width of ROI is usually set around 10-20 μm to observe all processes (depolymerization, polymerization, asymmetric distribution) of cp-actin filament dynamics on moving chloroplasts (see Video 2).

        Video 2. Cp-actin filament dynamics during the blue light-induced chloroplast avoidance response. Time-lapse images (maximized with four images in a 1.5-μm depth) were collected at approximately 33-sec intervals and played back at 5 frames per second (fps). The total elapsed time is 11:33 (mm:ss). The images are false-colored to indicate GFP (green) and chlorophyll (red) fluorescence. The region indicated with the blue circle (15 μm in diameter) was irradiated using 458-nm laser scans during the intervals between the image acquisitions to induce the avoidance response. Scale bar = 10 μm.

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


      5. (Optional) The sequential images of cp-actin filament disappearance could be captured at the higher optical and temporal resolutions (for example, a resolution of 128 x 68 with 6x digital zooming).
    4. The other microscopic conditions are adjusted to optimize image quality.
      Note: The optical set should be variable depending on experimental conditions. The imaging quality is mostly dependent on the fluorescent intensity that is variable on the expression level of actin probes and the property of fluorescent proteins. Hence, the best optical set should be empirically determined on a case-by-case basis.

  3. Quantitative Analysis
    1. Obtain enough time-lapse images from independent experiments for quantitative analysis.
    2. Analyze the time-lapse images using the ImageJ software program (http://rsbweb.nih.gov/ij/), which is populated with the appropriate plugins such as kymograph, intensity and length of actin filaments (Kong et al., 2013).

Recipes

  1. Evacuation solution
    0.01% (v/v) Silwet in deionized water

Acknowledgments

This protocol was based on the procedure described by Kong et al. (2013). This work was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants 25440140 to S.-G.K. and 25120721 and 25251033 to M.W.).

References

  1. Kong, S. G., Arai, Y., Suetsugu, N., Yanagida, T. and Wada, M. (2013). Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis. Plant Cell 25(2): 572-590.
  2. Wada, M. and Kong, S. G. (2011). Analysis of chloroplast movement and relocation in Arabidopsis. Methods Mol Biol 774: 87-102.

材料和试剂

  1. 植物材料
    1. 在野生型或突变植物中表达GFP-TALIN的拟南芥转基因植物(Kong等人,2013)。 肌动蛋白探针品系如LIFEACT-YFP和GFP-fABD2,THRUMIN1-GFP转基因品系也是有用的。
    2. 在23℃下在100μmol/m 2/s白光(16h)/黑暗(8h)循环下生长的3-4周龄植物的年轻和完全膨胀的莲座叶。
      注意:在土壤中生长的植物比在MS培养基的平板中生长的植物更好。 原因是植物生长在植物板上的叶子在样品处理过程中比在土壤上生长的植物更容易枯萎。
  2. 红色玻璃纸膜(TokyoButaiShowmei,目录号:20)
  3. (任选的)作为抑制肌动蛋白动力学的肌球蛋白ATP酶抑制剂的2,3-丁二酮单肟(BDM)(Sigma-Aldrich,目录号:B0753)
  4. 含有Silwet L-77(Bristol-Myers Squibb公司,目录号:BMS-SL7755)的疏散溶液(参见Recipes)

设备

  1. 注射器(10ml)
  2. 共聚焦显微镜(Leica Microsystems,型号:SP5,配有63x/1.20W物镜和多线100mW氩激光器)
  3. 植物生长室或房间
  4. 红色安全灯(红色LED或荧光灯过滤红色膜)
  5. 镊子
  6. 剃刀刀片
  7. 剪刀
  8. 定制的比色杯系统和环架:该系统由两个互补配合的钢环组成,两个圆形盖玻片(直径22mm,1号和5号)和硅环(100个) μm)(Wada和Kong,2011;见视频1)

软件

  1. 开放源代码软件ImageJ( http://rsbweb.nih.gov/ij/

程序

  1. 叶标本的制备(见视频1)

    视频1.在比色杯系统中制备叶片样本
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    获取Adobe Flash Player

    <! - [if!IE]> - >
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    1. 在弱光条件下(30μmol/cm 2 -1 白光)孵育植物几个小时。
      注意:
      1. 在弱光条件下,叶绿体沿着垂直于入射光的细胞壁积累(积累反应)。 然而,它们在强光照条件下远离该区域并且累积到与入射光平行的背斜壁(回避反应)。
      2. 此步骤对于在回避反应期间观察叶绿体是有用的。 此外,根据您的实验目的,此步骤可以跳过或修改。
    2. 通过在填充有排出溶液(0.01%Silwet L-77)的注射器中抽真空来分离玫瑰叶并消除叶中的空气。
    3. 用刀片将叶子切成小块(约4 mm x 4 mm)。
    4. (任选的)抑制剂处理,例如50mM 2,3-丁二酮单肟(BDM)
      1. 在疏散溶液中稀释感兴趣的抑制剂至合适的浓度。 作为模拟处理,将不含抑制剂的载体溶液(DMSO)以相同的稀释率加入到排出溶液中。
      2. 通过使用填充有溶液的注射器温和抽真空消除叶中的空隙
      3. 在暗红色光下孵育叶片样品适当的时间(0.5〜1小时)
    5. 将切下并切割的叶(用抑制剂溶液处理,如有必要)正面朝上放在圆盖玻片(5号)上
    6. 用圆盖玻片(1号)小心地盖住叶子样本,并将其放置在比色杯系统中。
      注意:小心移除叶片样品和盖玻片之间的空间中的任何气泡,不仅是叶片空气空间中的空气。

  2. 共聚焦显微镜
    1. 通过在暗红色光下观察样本来找到合适的叶绿体,并使用在弱激光功率下的快速扫描在栅栏细胞中的质膜和叶绿体之间的附近调整焦点。
      注意:在暗红色光条件下的眼睛观察被推荐以减少对cp-肌动蛋白丝动力学的任何光影响。 暗淡的红光条件是使用放在光路中的一片红色玻璃纸薄膜制成的 卤素灯。如果需要调整焦距,将输出激光器设置在最低功率(488 nm的1-5%),尽可能检测到叶绿素荧光。
    2. 在黑暗中在显微镜载物台上孵育样品几分钟。
      注意:cp-肌动蛋白丝的消失(解聚)由光学蓝色激光的几次扫描诱导。然而,黑暗孵育诱导cp-肌动蛋白丝的聚合在蓝色激光扫描后。
    3. 使用SP5软件(实时数据模式)设置光学参数以优化信噪比。
      1. 激发激光器:多线氩激光器(100mW的激光功率)被设置为20%输出功率,并且进一步在GFP激发的488nm的10%和用于诱导叶绿体回避反应的458nm的5%。
        注意:根据实验条件和各个系统的性能,激光功率可根据经验进行调整,以获得高信噪比的图像。
      2. 特定荧光信号的发射带:GFP为500至550nm,叶绿素为640至740nm。
      3. 使用具有4x数字变焦的适当分辨率(例如256 x 256或512 x 256)的镜头(63x/1.20 W)捕获图像(以0.5-μm步长从三到四个图像 持续20或30个延时周期,至少20秒的时间间隔(见下文)。范围处于XYZT模式。
        注意:共聚焦显微镜的扫描速度是有限的,并且cp-肌动蛋白丝是动态调节的。因此,可以通过改变扫描速度和y轴分辨率来在一些范围内控制时间和空间分辨率。双向模式也可以用来缩短采集时间。
      4. 为了诱导叶绿体回避反应,在适当时间的间隔期间使用刺激激光(458nm,2.8μW)使用感兴趣区域(ROI)扫描适当的矩形或圆形区域。
        注意:为了诱导回避反应,蓝光直接在叶绿体的一侧。 ROI的宽度通常设置在10-20μm左右,以观察移动叶绿体上cp-肌动蛋白丝动力学的所有过程(解聚,聚合,不对称分布)。

        视频2.在蓝光诱导叶绿体回避反应期间的Cp-肌动蛋白细丝动力学。 以约33秒的间隔收集延时图像(以1.5-μm深度的四个图像最大化),并以5帧/秒(fps)播放。总耗用时间为11:33(mm:ss)。图像是假色的,以指示GFP(绿色)和叶绿素(红色)荧光。在图像采集之间的间隔期间,使用458nm激光扫描照射用蓝色圆(直径15μm)表示的区域,以诱导回避反应。比例尺=10μm。
                                           <! - [if!IE]> - > <! - <![endif] - >                                             

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        获取Adobe Flash Player

        <! - [if!IE]> - >
        <! - <![endif] - >

      5. (可选)可以在较高的光学和时间分辨率(例如,具有6x数字变焦的128×68的分辨率)捕获cp-肌动蛋白丝消失的连续图像。
    4. 调整其他微观条件以优化图像质量。
      注意:光学组应根据实验条件而变化。 成像质量主要取决于在肌动蛋白探针的表达水平和荧光蛋白的性质上可变的荧光强度。 因此,最好的光学集合应该根据具体情况确定。

  3. 定量分析
    1. 从定量分析的独立实验获得足够的时间推移图像。
    2. 使用ImageJ软件程序分析延时图像( http://rsbweb.nih.gov/ij/),其填充有适当的插件,例如,肌动描记器,肌动蛋白丝的强度和长度(Kong等人,2013)。

食谱

  1. 疏散解决方案
    0.01%(v/v)Silwet在去离子水中

致谢

该方案基于Kong等人(2013)描述的方法。 这项工作得到了日本教育,文化,体育,科学和技术部的科学研究的Grants-in-Aid(批准号25440140至S.-G.K.和25120721和25251033至M.W.)的部分支持。

参考文献

  1. Kong,S.G.,Arai,Y.,Suetsugu,N.,Yanagida,T.and Wada,M。(2013)。 叶绿体 - 肌动蛋白丝的快速切断和运动性是拟南芥中叶绿体避免反应所必需的 植物细胞 25(2):572-590
  2. Wada,M。和Kong,S.G。(2011)。 分析拟南芥中的叶绿体运动和重新定位。 < em> Methods Mol Biol 774:87-102。
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How to cite this protocol: Kong, S. and Wada, M. (2013). Observation of Chloroplast-actin Filaments in Leaves of Arabidopsis. Bio-protocol 3(24): e1008. DOI: 10.21769/BioProtoc.1008; Full Text



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