Abstract
Nucleolar isolation allows exhaustive characterization of the nucleolar content. Centrifugation-based protocols are not adapted to isolation of nucleoli directly from a plant tissue because of copurification of cellular debris. We describe here a method that allows the purification of nucleoli using fluorescent-activated cell sorting from Arabidopsis thaliana leaves. This approach requires the expression of a specific nucleolar protein such as fibrillarin fused to green fluorescent protein in planta.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Galbraith DW, Anderson MT, Herzenberg LA (1999) Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. Methods Cell Biol 58:315–341
Troussellier M, Courties C, Vaquer A (1993) Recent applications of flow cytometry in aquatic microbial ecology. Biol Cell 78(1-2):111–121
Galbraith DW, Harkins KR, Jefferson RA (1988) Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts. Cytometry 9(1):75–83
Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF (2011) Cytometric assessment of mitochondria using fluorescent probes. Cytometry A 79(6):405–425
Bargmann BO, Birnbaum KD. (2010) Fluorescence activated cell sorting of plant protoplasts. J Vis Exp (36)
Galbraith DW, Janda J, Lambert GM (2011) Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type. Methods Mol Biol 699:407–429
You MK, Lim SH, Kim MJ, Jeong YS, Lee MG, Ha SH (2014) Improvement of the fluorescence intensity during a flow cytometric analysis for rice protoplasts by localization of a green fluorescent protein into chloroplasts. Int J Mol Sci 16(1):788–804
Zhang C, Barthelson RA, Lambert GM, Galbraith DW (2008) Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol 147(1):30–40
Vrána J, Simková H, Kubaláková M, Cíhalíková J, Doležel J (2012) Flow cytometric chromosome sorting in plants: the next generation. Methods 57(3):331–337
Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585
Pederson T (2010) “Compact” nuclear domains: reconsidering the nucleolus. Nucleus 1:444–445
Boulon S, Westman BJ, Hutten S, Boisvert FM, Lamond AI (2010) The nucleolus under stress. Mol Cell 40(2):216–227
Hacot S, Coute Y, Belin S, Albaret MA, Mertani HC, Sanchez JC, Rosa-Calatrava M, Diaz JJ (2010) Isolation of nucleoli. Curr Protoc Cell Biol. Chapter 3: Unit 3.36
Pendle AF, Clark GP, Boon R, Lewandowska D, Lam YW, Andersen J, Mann M, Lamond AI, Brown JW, Shaw PJ (2005) Proteomic analysis of the Arabidopsis nucleolus suggests novel nucleolar functions. Mol Biol Cell 16(1):260–269
Pontvianne F, Blevins T, Chandrasekhara C, Mozgová I, Hassel C, Pontes OM, Tucker S, Mokros P, Muchová V, Fajkus J, Pikaard CS (2013) Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states. Genes Dev 27(14):1545–1550
Durut N, Abou-Ellail M, Pontvianne F, Das S, Kojima H, Ukai S, de Bures A, Comella P, Nidelet S, Rialle S, Merret R, Echeverria M, Bouvet P, Nakamura K, Sáez-Vásquez J (2014) A duplicated NUCLEOLIN gene with antagonistic activity is required for chromatin organization of silent 45S rDNA in Arabidopsis. Plant Cell 26(3):1330–1344
Barneche F, Steinmetz F, Echeverría M (2000) Fibrillarin genes encode both a conserved nucleolar protein and a novel small nucleolar RNA involved in ribosomal RNA methylation in Arabidopsis thaliana. J Biol Chem 275(35):27212–27220
Sáez-Vasquez J, Caparros-Ruiz D, Barneche F, Echeverría M (2004) A plant snoRNP complex containing snoRNAs, fibrillarin, and nucleolin-like proteins is competent for both rRNA gene binding and pre-rRNA processing in vitro. Mol Cell Biol 24(16):7284–7297
Acknowledgments
This work was supported by the CNRS, the UPVD foundation BQR 2014 FANoS and the ANR NucleoReg ANR-15-CE12-0013-01 to FP, and ANR SubCellif to JSV. The cell sorter is an instrument of the Montpellier Rio Imaging platform. The authors thank Richard Cooke for English corrections.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Pontvianne, F., Boyer-Clavel, M., Sáez-Vásquez, J. (2016). Fluorescence-Activated Nucleolus Sorting in Arabidopsis. In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3792-9_15
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3790-5
Online ISBN: 978-1-4939-3792-9
eBook Packages: Springer Protocols