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Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei

使用结构照明显微镜（SIM）和光敏定位显微镜(PALM）分析RNA聚合酶II分子在流式分选的拟南芥细胞核中的丰度及分布

Klaus Weisshart Klaus Weisshart
JF Jörg Fuchs
VS Veit Schubert

发布: 2016年02月05日第6卷第3期 DOI: 10.21769/BioProtoc.1725 浏览次数: 13652

评审: Samik BhattacharyaAnonymous reviewer(s)

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Abstract

RNA polymerase II (RNAPII) is the enzyme transcribing most of the eukaryotic protein-coding genes. Analysing the distribution and quantification of RNAPII can help understanding its function in interphase nuclei. Although several investigations in mammals indicate the organization of RNAPII in so-called ‘transcription factories’ (Jackson et al., 1993; Rieder et al., 2012; Papantonis and Cook, 2013), their existence is still controversially discussed (Zhao et al., 2014).

Recently, based on super-resolution microscopy the presence of transcription factories was also suggested in plants. Applying structured illumination microscopy (SIM) and photoactivated localization microscopy (PALM) the distribution and number of RNAPII molecules in Arabidopsis nuclei were analysed and a positive correlation between RNAPII abundance and endopolyploidy was found (Schubert, 2014; Schubert and Weisshart, 2015).

Here, we present a protocol describing the isolation of Arabidopsis thaliana interphase nuclei via flow-sorting according to their endopolyploidy level, followed by a double immunostaining using antibodies specific for different RNAPII modifications and the subsequent evaluation by spatial SIM and PALM to achieve results regarding the abundance, distribution and co-localization of single inactive and active RNAPII molecules.

Keywords: Super-resolution microscopy (超高分辨率显微镜) search Cell nucleus (细胞核) search Transcription (转录) search RNA polymerase (RNA聚合酶) search Single molecule localization (单分子定位) search


Part I. Flow sorting of Arabidopsis interphase nuclei

Materials and Reagents

  1. 22x22 mm high precision coverslips (170±5 µm, no. 1.5H) (Marienfeld-Superior) Rubber cement (Marabu Australia, catalog number: 290110000 )
  2. 5 ml Polystyrene Round-Bottom Tubes with 35 µm cell strainer cap (BD Biosciences, catalog number: 352235 ) or alternatively, 5 ml Polystyrene Round-Bottom Tube (BD Biosciences, catalog number: 352063 ) in combination with disposable filters CellTrics, 30 or 50 µm (Sysmex-Partec, catalog numbers: 04-0042-2316 or 04-0042-2317 )
    Note: Currently, it is "Corning, Falcon®, catalog number: 352235” and "Corning, Falcon®, catalog number: 352063”.
  3. 0.22 µm filter
  4. A. thaliana rosette leaves
  5. Calibration beads [SpheroTM Rainbow Calibration Particles (8 peaks)] (BD Biosciences, catalog number: 559123 )
  6. Accudrop Beads (BD FACSTM Accudrop Beads) (BD Biosciences, catalog number: 345249 )
  7. Formaldehyde solution 37% (Carl Roth GmbH + Co, catalog number: 7398.1 )
  8. 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Molecular ProbesTM, catalog number: D-1306 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D-1306”.
  9. Milli-Q water
  10. NaCl
  11. Na2HPO4.2H2O
  12. NaH2PO4
  13. Tris
  14. Na2EDTA
  15. Spermin
  16. KCl
  17. Triton X-100
  18. NaOH
  19. β-mercaptoethanol
  20. MgCl2
  21. Sucrose
  22. Tween 20
  23. HCl
  24. BD FACSFlow sheath fluid (BD Biosciences, catalog number: 342003 ) or alternatively, 1 x PBS (see Recipes)
  25. Formaldehyde fixative (see Recipes)
  26. 500 ml Tris-Buffer (see Recipes)
  27. 200 ml lysis buffer LB01 (Dolezel et al., 1989) (see Recipes)
  28. 100 µg/ml DAPI stock solution (see Recipes)
  29. 25 ml sucrose buffer (see Recipes)

Equipment

  1. FACSAria IIu (BD Biosciences)

Software

  1. FACSDiva software

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Weisshart, K., Fuchs, J. and Schubert, V. (2016). Structured Illumination Microscopy (SIM) and Photoactivated Localization Microscopy (PALM) to Analyze the Abundance and Distribution of RNA Polymerase II Molecules on Flow-sorted Arabidopsis Nuclei. Bio-protocol 6(3): e1725. DOI: 10.21769/BioProtoc.1725.

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分类

植物科学 > 植物细胞生物学 > 细胞成像

植物科学 > 植物细胞生物学 > 细胞器分离

细胞生物学 > 细胞成像 > 结构照明显微镜（SIM）

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