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Visualization and Quantification of Actin Dynamics in Rice Protoplasts
水稻原生质体肌动蛋白动力变化的观察与定量分析

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Abstract

Direct visualization of the organization and dynamics of the actin cytoskeleton in rice cells is essential to understand its roles in regulating rice growth and development. Visualization of actin dynamics in protoplasts transformed with actin probe is a relatively quick and simple strategy, compared to the strategy of generating stable transgenic rice plants that harbor actin probe, which is time-consuming. Here is a protocol described in details regarding transforming rice protoplasts as well as visualizing and quantifying actin dynamics in rice protoplasts, which is based on the method previously reported (Shi et al., 2013).

Keywords: Rice protoplast(水稻原生质体), Actin dynamics(肌动蛋白), Filament severing frequency(纤维切断频率), Actin depolymerization rate(肌动蛋白解聚率), Filament elongation rate(丝伸长率)

Materials and Reagents

  1. Rice protoplasts
  2. Plasmid pUN1301-EGFP-ABD2-EGFP (Yang et al., 2011)
  3. D-Mannitol (Sigma-Aldrich, catalog number: M1902 )
  4. 2-(N-Morpholino) ethanesulfonic acid (MES) (Merck KGaA, catalog number: 475893 )
  5. Cellulase “Onozuka” RS (Yakult Pharmaceutical Industry, catalog number: 9012-54-8 )
  6. Macerozyme R-10 (Yakult Pharmaceutical Industry, catalog number: 8032-75-1 )
  7. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
  8. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
  9. β-mercaptoethanol (Merck KGaA, catalog number: 444203 )
  10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  11. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  12. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M2393 )
  13. PEG4000 (Merck KGaA, catalog number: 817006 )
  14. Murashige and Skoog basal salt mixture powder (MS) (Sigma-Aldrich, catalog number: M5524 )
  15. Digestion buffer (see Recipes)
  16. W5 buffer (see Recipes)
  17. Mmg buffer (see Recipes)
  18. Polyethylene glycol solution (see Recipes)

Equipment

  1. 35 μm nylon mesh
  2. Spinning disk confocal microscope equipped with a Yokogawa CSU-X1 spinning disk head using a 512 x 512 Andor iXON electron-multiplying CCD camera (Andor)
  3. Shaker
  4. Centrifuge
  5. Concave slide (diameter 15 mm) (Beyotime, catalog number: FSL011 )
  6. Olympus BX61 inverted microscope equipped with a 100x 1.4 NA UPLSAPO (Universal Plan Super Apochromat Objective) objective
  7. 488 nm laser
  8. 525/50 nm band-pass filter

Software

  1. Andor IQ2 software (available at http://www.andor.com)

Procedure

  1. Sterilize rice seeds with 2.5% NaClO and 0.01% Tween-20 for 15 min and wash the seeds with sterilized water for five times, then sterilize the seeds with 2.5% NaClO for another 15 min and wash the seeds with sterilized water for five times. Finally put these seeds on sterilized filter paper and dry them.
  2. Germinate and grow sterilized rice seeds above on half-strength MS medium at 28 °C for 14 d in darkness.
  3. Cut the leaf sheaths of the etiolated seedlings into 0.5-1.0 mm length and immediately immerge into digestion buffer to digest the tissues in darkness at 28 °C for 4 h with gentle shaking (40-80 rpm). All steps below are operated at room temperature unless otherwise noted.
  4. Add one volume of pre-cooling W5 buffer into above mixture and collect the protoplasts on ice by sifting out the undigested tissues with 35 μm nylon mesh.
  5. Collect the protoplasts by a 5 min spinning of 300 x g at 4 °C and re-suspend the protoplasts in fresh cool W5 buffer.
  6. Harvest the pellet and dilute the protoplasts into 2 x 106 cells/ml with Mmg buffer.
  7. For transformation, 20 μg of plasmid pUN1301-EGFP-ABD2-EGFP was added into 100 μl protoplasts, followed by the addition of an equal volume of polyethylene glycol solution. The mixture was incubated at room temperature for 20 min.
  8. Add 10 volume of W5 buffer and harvest the protoplasts with centrifugation at 300 x g for 5 min.
  9. Re-suspend the pellet with fresh W5 buffer and incubate the protoplasts in darkness for 20 h.
  10. Mount the protoplasts into a concave slide (diameter 15 mm) with a pipet and cover them with a coverslip. The dynamics of actin filaments were observed under an Olympus BX61 inverted microscope equipped with a 100x 1.4 NA UPLSAPO objective. Figure 1 shows the organization of actin filaments in a typical rice protoplast.


    Figure 1. Organization of actin filaments in a typical rice protoplast. This protoplast is transformed with plasmid pUN1301-EGFP-ABD2-EGFP. Scale bar = 10 μm.

  11. The images were collected using a spinning disk confocal microscope equipped with a Yokogawa CSU-X1 spinning disk head using a 512 x 512 Andor iXON electron-multiplying CCD camera. GFP was excited using a 488 nm laser, and fluorescence emission was collected using a 525/50 nm band-pass filter. Time-lapse Z-series images (with a step of 0.5 μm) were collected with a time interval of 100 msec using Andor IQ2, and the time interval for the whole Z-series collection was 5 sec. The Z-stack image is made into a movie to shown the actin filaments in a typical rice protoplast (see Video 1).

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    Video 1. A video of the Z-stack image displays the actin filaments in a typical rice protoplast harboring pUN1301-EGFP-ABD2-EGFP. Images were taken every 0.5 μm and were compressed into a 2.8 s-video. Scale bar = 10 μm.

  12. The severing frequency of filament is quantified as the number of breaks, per unit filament length, per unit time (breaks/μm/s) and the depolymerization rate is calculated by the length of shortening filament within the given time interval (μm/s). Table 1 makes an example for one of our results.

    Table 1. The severing frequency and depolymerization rate of filaments in rice protoplasts
    Severing frequency (breaks/μm/s)
    0.0165 ± 0.0080  (n = 15)
    Depolymerization rate (μm/s) 0.0023 ± 6.58e-4  (n = 20)
    Quantification of parameters associated with single actin dynamics in rice protoplasts harboring plasmid pUN1301-EGFP-ABD2-EGFP. More than 15 protoplasts were used to analyze. Values given are means± SE.

Recipes

  1. Digestion buffer
    0.6 M mannitol
    10 mM MES pH 5.7
    1.5% cellulose RS
    0.75% macerozyme R10
    0.1% BSA
    1 mM CaCl2
    5 mM β-mercaptoethanol
  2. W5 buffer
    154 mM NaCl
    125 mM CaCl2
    5 mM KCl
    2 mM MES
  3. Mmg buffer
    0.6 M mannitol
    15 mM MgCl2
    4 mM MES
  4. Polyethylene glycol solution
    0.6 M mannitol
    100 mM CaCl2
    40% v/v PEG 4,000

Acknowledgments

This protocol was adapted from our previously published work (Shi et al., 2013). We thank Meng Shi and Shaojie Cui for helpful suggestions on rice protoplast preparation and transformation. The research in the Huang lab was supported by grants from Ministry of Science of Technology (2013CB945100) and National Natural Science Foundation of China (31125004).

References

  1. Shi, M., Xie, Y., Zheng, Y., Wang, J., Su, Y., Yang, Q. and Huang, S. (2013). Oryza sativa actin-interacting protein 1 is required for rice growth by promoting actin turnover. Plant J 73(5): 747-760.    
  2. Yang, W., Ren, S., Zhang, X., Gao, M., Ye, S., Qi, Y., Zheng, Y., Wang, J., Zeng, L., Li, Q., Huang, S. and He, Z. (2011). BENT UPPERMOST INTERNODE1 encodes the class II formin FH5 crucial for actin organization and rice development. Plant Cell 23(2): 661-680.  


简介

直接可视化的肌动蛋白细胞骨架在水稻细胞的组织和动态是必不可少的理解其在调节水稻生长和发展中的作用。 与产生含有肌动蛋白探针的稳定转基因水稻植物的策略相比,用肌动蛋白探针转化的原生质体中肌动蛋白动力学的可视化是相对快速和简单的策略,这是耗时的。 这里是详细描述关于转化水稻原生质体以及可视化和量化水稻原生质体中的肌动蛋白动力学的方案,其基于先前报道的方法(Shi等人,2013)。

关键字:水稻原生质体, 肌动蛋白, 纤维切断频率, 肌动蛋白解聚率, 丝伸长率

材料和试剂

  1. 水稻原生质体
  2. 质粒pUN1301-EGFP-ABD2-EGFP(Yang等人,2011)
  3. D-甘露醇(Sigma-Aldrich,目录号:M1902)
  4. 2-(N-吗啉代)乙磺酸(MES)(Merck KGaA,目录号:475893)
  5. 纤维素酶"Onozuka"RS(Yakult Pharmaceutical Industry,目录号:9012-54-8)
  6. Macerozyme R-10(Yakult Pharmaceutical Industry,目录号:8032-75-1)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A1933)
  8. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C5670)
  9. β-巯基乙醇(Merck KGaA,目录号:444203)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  11. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  12. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M2393)
  13. PEG4000(Merck KGaA,目录号:817006)
  14. Murashige和Skoog基础盐混合物粉末(MS)(Sigma-Aldrich,目录号:M5524)
  15. 消化缓冲液(参见配方)
  16. W5缓冲区(见配方)
  17. Mmg缓冲区(参见配方)
  18. 聚乙二醇溶液(见配方)

设备

  1. 35μm尼龙网
  2. 旋转盘共聚焦显微镜,配备有使用512×512 Andor iXON电子倍增的 Yokogawa CSU-X1旋转盘头 CCD相机( Andor
  3. 振动器
  4. 离心机
  5. 凹形滑动(直径15mm)(Beyotime,目录号:FSL011)
  6. 配备100x 1.4 NA UPLSAPO(通用计划超级消色差物镜)物镜的Olympus BX61倒置显微镜
  7. 488 nm激光
  8. 525/50 nm带通滤波器

软件

  1. 安道IQ2软件(可从 http://www.andor.com 获取)

程序

  1. 用2.5%NaClO和0.01%Tween-20消毒水稻种子15分钟,用灭菌水洗涤种子5次,然后用2.5%NaClO将种子灭菌15分钟,用灭菌水冲洗种子5次。 最后将这些种子放在灭菌的滤纸上并干燥
  2. 在28℃下在半强度MS培养基上在黑暗中发芽和生长灭菌的水稻种子14天。
  3. 切割叶鞘的叶鞘为0.5-1.0毫米长度,立即浸入消化缓冲液中消化组织在黑暗中28℃下轻轻摇动(40-80 rpm)4小时。 除非另有说明,以下所有步骤均在室温下操作。
  4. 向上述混合物中加入一体积的预冷却W5缓冲液,并通过用35μm尼龙网筛选未消化的组织在冰上收集原生质体。
  5. 通过在4℃下300分钟旋转5分钟来收集原生质体,并将原生质体重悬在新鲜的W5缓冲液中。
  6. 收获沉淀,并用Mmg缓冲液将原生质体稀释成2×10 6个细胞/ml。
  7. 为了转化,将20μg质粒pUN1301-EGFP-ABD2-EGFP加入到100μl原生质体中,然后加入等体积的聚乙二醇溶液。 将混合物在室温下温育20分钟
  8. 加入10体积的W5缓冲液,并在300×g离心5分钟,收获原生质体。
  9. 用新鲜的W5缓冲液重新悬浮沉淀,并在黑暗中孵育原生质体20小时
  10. 用移液管将原生质体装入凹形载玻片(直径15mm)中,并用盖玻片覆盖。 在装备有100×1.4NA的Olympus BX61倒置显微镜下观察肌动蛋白丝的动力学 UPLSAPO目标。图1显示了典型的稻原生质体中肌动蛋白丝的组织

    图1.典型的水稻原生质体中肌动蛋白丝的组织。 用质粒pUN1301-EGFP-ABD2-EGFP转化该原生质体。比例尺=10μm。

  11. 使用配备有使用512×512 Andor iXON电子倍增CCD照相机的Yokogawa CSU-X1旋转盘头的旋转盘共聚焦显微镜收集图像。使用488nm激光激发GFP,并使用525/50nm带通滤光片收集荧光发射。使用Andor IQ2以100msec的时间间隔收集延时Z系列图像(具有0.5μm的台阶),并且整个Z系列收集的时间间隔为5秒。将Z-堆叠图像制成电影以在典型的稻原生质体中显示肌动蛋白丝(见视频1)。

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

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    获取Adobe Flash Player

    <! - [if!IE]> - >
    <! - <![endif] - >
    视频1.Z-堆叠图像的视频在含有pUN1301-EGFP-ABD2-EGFP的典型稻原生质体中显示肌动蛋白丝。每0.5μm取图像,并压缩成2.8s- 视频。 比例尺=10μm。

  12. 丝的切断频率定量为每单位时间的断裂次数(单位长度/断裂/μm/s),解聚速率通过在给定时间间隔(μm/s)内的起酥油长度计算, 。 表1列出了我们的一个结果示例。

    表1.水稻原生质体中丝的切断频率和解聚速率
    切断频率(断裂/μm/s)
    0.0165±0.0080  ( n = 15)
    解聚速率(μm/s) 0.0023±6.58e-4  ( n = 20)
    在携带质粒pUN1301-EGFP-ABD2-EGFP的稻原生质体中与单肌动蛋白动力学相关的参数的定量。 使用超过15个原生质体进行分析。 给出的值是平均值±SE。

食谱

  1. 消化缓冲区
    0.6 M甘露糖 10 mM MES pH 5.7
    1.5%纤维素RS
    0.75%macerozyme R10
    0.1%BSA
    1mM CaCl 2
    5mMβ-巯基乙醇
  2. W5缓冲区
    154 mM NaCl 125mM CaCl 2。 5 mM KCl
    2 mM MES
  3. Mmg缓冲区
    0.6 M甘露糖 15mM MgCl 2·h/v 4 mM MES
  4. 聚乙二醇溶液
    0.6 M甘露糖 100mM CaCl 2
    40%v/v PEG 4,000

致谢

该协议改编自我们以前公开的工作(Shi等人,2013年)。 我们感谢Meng Shi和Shaojie Cui对水稻原生质体制备和转化提出了有益的建议。 黄实验室的研究由科技部(2013CB945100)和国家自然科学基金(31125004)资助。

参考文献

  1. Shi,M.,Xie,Y.,Zheng,Y.,Wang,J.,Su,Y.,Yang,Q.and Huang, 水稻肌动蛋白相互作用蛋白1是水稻生长所必需的 肌动蛋白周转。植物J 73(5):747-760。    
  2. Yang,W.,Ren,S.,Zhang,X.,Gao,M.,Ye,S.,Qi,Y.,Zheng,Y.,Wang,J.,Zeng, Huang,S。和He,Z。(2011)。 BENT UPPERMOST INTERNODE1 编码II类formin FH5对肌动蛋白组织至关重要, 水稻发育。 植物细胞 23(2):661-680。  


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引用:Xie, Y. and Huang, S. (2013). Visualization and Quantification of Actin Dynamics in Rice Protoplasts. Bio-protocol 3(21): e964. DOI: 10.21769/BioProtoc.964.
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