Advertisement

Detection of Endogenous Nuclear Proteins in Plant Cells: Localizing Nuclear Matrix Constituent Proteins (NMCPs), the Plant Analogs of Lamins

  • Malgorzata Ciska
  • Susana Moreno Díaz de la EspinaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1560)

Abstract

At present, two complementary approaches are used for in situ protein visualization in plant nuclei. Imaging of transformed fluorescent proteins is the election tool for the analysis of protein movement and interaction. However, this methodology presents several drawbacks for the identification/localization of endogenous nuclear factors, such as over-expression or mislocalization of transformed proteins. In contrast, immunocytochemistry with specific antibodies represents a powerful tool for the localization of endogenous nuclear proteins at their “native” nuclear sub-compartments. In plant cells, the cell wall hampers antibody accessibility during immunocytochemical analysis thereby reducing the effectivity of the technique, particularly in the case of lowly expressed proteins. To overcome this problem in nuclear protein immunodetection, we developed a method based on the in vitro incubation of isolated nuclei with specific antibodies followed by imaging by confocal fluorescence or electron microscopy. Here we describe the application of this methodology to the localization of Nuclear Matrix Constituent Proteins (NMCP), the plant analogs of lamins, of the monocot Allium cepa, using antibodies raised against highly conserved regions of the proteins.

Key words

Immunofluorescence confocal microscopy Immunoelectron microscopy Nuclear isolation Pre-embedding labeling NMCP proteins Plant cells Allium cepa 

Notes

Acknowledgments

We would like to thank Mrs M. Carnota and past members of the lab for their contribution to the development of the methods and protocols presented here. We acknowledge the support from the Spanish Ministry of Science and Innovation [BFU2010-15,900] and the CSIC [PIE 201020E019].

References

  1. 1.
    Ciska M, Moreno Diaz de la Espina S (2014) The intriguing plant nuclear lamina. Front Plant Sci 5:166. doi: 10.3389/fpls.2014.00166 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Dittmer TA, Stacey NJ, Sugimoto-Shirasu K et al (2007) LITTLE NUCLEI genes affecting nuclear morphology in Arabidopsis thaliana. Plant Cell 19(9):2793–2803. doi: 10.1105/tpc.107.053231 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Sakamoto Y, Takagi S (2013) LITTLE NUCLEI 1 and 4 regulate nuclear morphology in Arabidopsis thaliana. Plant Cell Physiol 54(4):622–633. doi: 10.1093/pcp/pct031 CrossRefPubMedGoogle Scholar
  4. 4.
    Masuda K, Xu ZJ, Takahashi S et al (1997) Peripheral framework of carrot cell nucleus contains a novel protein predicted to exhibit a long alpha-helical domain. Exp Cell Res 232(1):173–181. doi: 10.1006/excr.1997.3531 CrossRefPubMedGoogle Scholar
  5. 5.
    Masuda K, Haruyama S, Fujino K (1999) Assembly and disassembly of the peripheral architecture of the plant cell nucleus during mitosis. Planta 210(1):165–167CrossRefPubMedGoogle Scholar
  6. 6.
    Ciska M, Masuda K, Moreno Diaz de la Espina S (2013) Lamin-like analogues in plants: the characterization of NMCP1 in Allium cepa. J Exp Bot 64(6):1553–1564. doi: 10.1093/jxb/ert020 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kimura Y, Kuroda C, Masuda K (2010) Differential nuclear envelope assembly at the end of mitosis in suspension-cultured Apium graveolens cells. Chromosoma 119(2):195–204. doi: 10.1007/s00412-009-0248-y CrossRefPubMedGoogle Scholar
  8. 8.
    Kimura Y, Fujino K, Ogawa K et al (2014) Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain. Front Plant Sci 5:62. doi: 10.3389/fpls.2014.00062 PubMedPubMedCentralGoogle Scholar
  9. 9.
    Graumann K (2014) Evidence for LINC1-SUN associations at the plant nuclear periphery. PLoS One 9(3):e93406. doi: 10.1371/journal.pone.0093406 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hanson MR, Kohler RH (2001) GFP imaging: methodology and application to investigate cellular compartmentation in plants. J Exp Bot 52(356):529–539CrossRefPubMedGoogle Scholar
  11. 11.
    Brown RC, Lemmon BE (2011) Dividing without centrioles: innovative plant microtubule organizing centres organize mitotic spindles in bryophytes, the earliest extant lineages of land plants. AoB Plants 2011:plr028. doi: 10.1093/aobpla/plr028 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Acevedo R, Cuadrado A, De la Torre C et al (2002) Behaviour of ribosomal genes and nucleolar domains during activation in sugarcane (Saccharum officinarum L.) root primordia: from the unsoaked quiescent state to the steady state of proliferation. Eur J Histochem 46(2):143–158CrossRefPubMedGoogle Scholar
  13. 13.
    Acevedo R, Samaniego R, Moreno Diaz de la Espina S (2002) Coiled bodies in nuclei from plant cells evolving from dormancy to proliferation. Chromosoma 110(8):559–569CrossRefPubMedGoogle Scholar
  14. 14.
    Samaniego R, Jeong SY, de la Torre C et al (2006) CK2 phosphorylation weakens 90 kDa MFP1 association to the nuclear matrix in Allium cepa. J Exp Bot 57(1):113–124. doi: 10.1093/jxb/erj010 CrossRefPubMedGoogle Scholar
  15. 15.
    Cruz JR, Moreno Diaz de la Espina S (2009) Subnuclear compartmentalization and function of actin and nuclear myosin I in plants. Chromosoma 118(2):193–207. doi: 10.1007/s00412-008-0188-y CrossRefPubMedGoogle Scholar
  16. 16.
    Perez-Munive C, Moreno Diaz de la Espina S (2011) Nuclear spectrin-like proteins are structural actin-binding proteins in plants. Biol Cell 103(3):145–157. doi: 10.1042/BC20100083 CrossRefPubMedGoogle Scholar
  17. 17.
    Stirling JW, Graff PS (1995) Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J Histochem Cytochem 43(2):115–123CrossRefPubMedGoogle Scholar
  18. 18.
    Beisker W, Dolbeare F, Gray JW (1987) An improved immunocytochemical procedure for high-sensitivity detection of incorporated bromodeoxyuridine. Cytometry 8(2):235–239. doi: 10.1002/cyto.990080218 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Malgorzata Ciska
    • 1
  • Susana Moreno Díaz de la Espina
    • 1
    Email author
  1. 1.Department of Cell and Molecular Biology, Biological Research CenterCSICMadridSpain

Personalised recommendations