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
发布: 2013年11月05日第3卷第21期 DOI: 10.21769/BioProtoc.964 浏览次数: 9226
评审: Tie Liu
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参见作者原研究论文
The authors used this protocol in:
Mar 2013
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
- Rice protoplasts
- Plasmid pUN1301-EGFP-ABD2-EGFP (Yang et al., 2011)
- D-Mannitol (Sigma-Aldrich, catalog number: M1902 )
- 2-(N-Morpholino) ethanesulfonic acid (MES) (Merck KGaA, catalog number: 475893 )
- Cellulase “Onozuka” RS (Yakult Pharmaceutical Industry, catalog number: 9012-54-8 )
- Macerozyme R-10 (Yakult Pharmaceutical Industry, catalog number: 8032-75-1 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A1933 )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C5670 )
- β-mercaptoethanol (Merck KGaA, catalog number: 444203 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
- Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M2393 )
- PEG4000 (Merck KGaA, catalog number: 817006 )
- Murashige and Skoog basal salt mixture powder (MS) (Sigma-Aldrich, catalog number: M5524 )
- Digestion buffer (see Recipes)
- W5 buffer (see Recipes)
- Mmg buffer (see Recipes)
- Polyethylene glycol solution (see Recipes)
Equipment
- 35 μm nylon mesh
- 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)
- Shaker
- Centrifuge
- Concave slide (diameter 15 mm) (Beyotime, catalog number: FSL011 )
- Olympus BX61 inverted microscope equipped with a 100x 1.4 NA UPLSAPO (Universal Plan Super Apochromat Objective) objective
- 488 nm laser
- 525/50 nm band-pass filter
Software
- Andor IQ2 software (available at http://www.andor.com)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物细胞生物学 > 细胞成像
细胞生物学 > 细胞成像 > 共聚焦显微镜
植物科学 > 植物细胞生物学 > 细胞结构
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