Abstract
Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.
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McMichael CM, Bednarek SY (2013) Cytoskeletal and membrane dynamics during higher plant cytokinesis. New Phytol 197:1039–1057
Lee YR, Liu B (2013) The rise and fall of the phragmoplast microtubule array. Curr Opin Plant Biol 16:757–763
Nishihama R, Soyano T, Ishikawa M, Araki S, Tanaka H, Asada T, Irie K, Ito M, Terada M, Banno H, Yamazaki Y, Machida Y (2002) Expansion of the cell plate in plant cytokinesis requires a kinesin-like protein/MAPKKK complex. Cell 109:87–99
Krupnova T, Sasabe M, Ghebreghiorghis L, Gruber CW, Hamada T, Dehmel V, Strompen G, Stierhof YD, Lukowitz W, Kemmerling B, Machida Y, Hashimoto T, Mayer U, Jurgens G (2009) Microtubule-associated kinase-like protein RUNKEL needed [corrected] for cell plate expansion in Arabidopsis cytokinesis. Curr Biol 19:518–523
Oh SA, Johnson A, Smertenko A, Rahman D, Park SK, Hussey PJ, Twell D (2005) A divergent cellular role for the FUSED kinase family in the plant-specific cytokinetic phragmoplast. Curr Biol 15:2107–2111
Ho CM, Hotta T, Kong Z, Zeng CJ, Sun J, Lee YR, Liu B (2011) Augmin plays a critical role in organizing the spindle and phragmoplast microtubule arrays in Arabidopsis. Plant Cell 23:2606–2618
Muller S, Fuchs E, Ovecka M, Wysocka-Diller J, Benfey PN, Hauser MT (2002) Two new loci, PLEIADE and HYADE, implicate organ-specific regulation of cytokinesis in Arabidopsis. Plant Physiol 130:312–324
Muller S, Smertenko A, Wagner V, Heinrich M, Hussey PJ, Hauser MT (2004) The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function. Curr Biol 14:412–417
Twell D, Park SK, Hawkins TJ, Schubert D, Schmidt R, Smertenko A, Hussey PJ (2002) MOR1/GEM1 has an essential role in the plant-specific cytokinetic phragmoplast. Nat Cell Biol 4:711–714
Eleftheriou EP, Baskin TI, Hepler PK (2005) Aberrant cell plate formation in the Arabidopsis thaliana microtubule organization 1 mutant. Plant Cell Physiol 46:671–675
Kang BH (2003) Members of the Arabidopsis dynamin-like gene family, ADL1, are essential for plant cytokinesis and polarized cell growth. Plant Cell 15:899–913
Backues SK, Korasick DA, Heese A, Bednarek SY (2010) The Arabidopsis dynamin-related protein2 family is essential for gametophyte development. Plant Cell 22:3218–3231
Fendrych M, Synek L, Pecenkova T, Toupalova H, Cole R, Drdova E, Nebesarova J, Sedinova M, Hala M, Fowler JE, Zarsky V (2010) The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell 22:3053–3065
Lukowitz W, Mayer U, Jurgens G (1996) Cytokinesis in the Arabidopsis embryo involves the syntaxin-related KNOLLE gene product. Cell 84:61–71
Thiele K, Wanner G, Kindzierski V, Juergens G, Mayer U, Pachl F, Assaad FF (2009) The timely deposition of callose is essential for cytokinesis in Arabidopsis. Plant J 58:13–26
Walker KL, Muller S, Moss D, Ehrhardt DW, Smith LG (2007) Arabidopsis TANGLED identifies the division plane throughout mitosis and cytokinesis. Curr Biol 17:1827–1836
Xu XM, Zhao Q, Rodrigo-Peiris T, Brkljacic J, He CS, Muller S, Meier I (2008) RanGAP1 is a continuous marker of the Arabidopsis cell division plane. Proc Natl Acad Sci U S A 105:18637–18642
Moschou PN, Smertenko AP, Minina EA, Fukada K, Savenkov EI, Robert S, Hussey PJ, Bozhkov PV (2013) The caspase-related protease separase (extra spindle poles) regulates cell polarity and cytokinesis in Arabidopsis. Plant Cell 25:2171–2186
Acknowledgements
The authors thank Eleftherios Eleftheriou, Aristotle University of Thessaloniki, and Tobias Baskin, University of Amherst for micrographs of cytokinesis in mor1-1.
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Smertenko, A., Moschou, P., Zhang, L., Fahy, D., Bozhkov, P. (2016). Characterization of Cytokinetic Mutants Using Small Fluorescent Probes. In: Caillaud, MC. (eds) Plant Cell Division. Methods in Molecular Biology, vol 1370. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3142-2_15
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DOI: https://doi.org/10.1007/978-1-4939-3142-2_15
Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3142-2
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