Advertisement

Functional Analysis of LINC Complexes in the Skin

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1840)

Abstract

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro.

Key words

Nesprin SUN Mouse embryonic fibroblasts Primary keratinocytes Primary fibroblasts Directed cell migration 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

This work was supported by Cologne Cluster of Excellence on Cellular Stress Responses in Aging-Associated Diseases (CECAD), the International Graduate School in Development Health and Disease (IGSDHD), and the BBSRC (Biotechnology and Biological Sciences Research Council).

References

  1. 1.
    Crisp M, Liu Q, Roux K et al (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 172:41–53CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Xiong H, Rivero F, Euteneuer U et al (2008) Dictyostelium Sun-1 connects the centrosome to chromatin and ensures genome stability. Traffic 9:708–724CrossRefPubMedGoogle Scholar
  3. 3.
    Haque F, Mazzeo D, Patel JT et al (2010) Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes. J Biol Chem 285:3487–3498CrossRefPubMedGoogle Scholar
  4. 4.
    Li P, Noegel AA (2015) Inner nuclear envelope protein SUN1 plays a prominent role in mammalian mRNA export. Nucleic Acids Res 43:9874–9888PubMedPubMedCentralGoogle Scholar
  5. 5.
    Luxton GW, Starr DA (2014) KASHing up with the nucleus: novel functional roles of KASH proteins at the cytoplasmic surface of the nucleus. Curr Opin Cell Biol 28:69–75CrossRefPubMedGoogle Scholar
  6. 6.
    Worman HJ (2012) Nuclear lamins and laminopathies. J Pathol 226:316–325CrossRefPubMedGoogle Scholar
  7. 7.
    Sosa BA, Kutay U, Schwartz TU (2013) Structural insights into LINC complexes. Curr Opin Struct Biol 23:285–291CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Rothballer A, Kutay U (2013) The diverse functional LINCs of the nuclear envelope to the cytoskeleton and chromatin. Chromosoma 122:415–429CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lattanzi G, Marmiroli S, Facchini A et al (2012) Nuclear damages and oxidative stress: new perspectives for laminopathies. Eur J Histochem e45:56Google Scholar
  10. 10.
    Rajgor D, Mellad JA, Autore F et al (2012) Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds. PLoS One 7:e40098CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Zhen YY, Libotte T, Munck M et al (2002) NUANCE, a giant protein connecting the nucleus and actin cytoskeleton. J Cell Sci 115:3207–3222PubMedGoogle Scholar
  12. 12.
    Padmakumar VC, Abraham S, Braune S et al (2004) Enaptin, a giant actin-binding protein, is an element of the nuclear membrane and the actin cytoskeleton. Exp Cell Res 295:330–339CrossRefPubMedGoogle Scholar
  13. 13.
    Wilhelmsen K, Ketema M, Truong H et al (2006) KASH-domain proteins in nuclear migration, anchorage and other processes. J Cell Sci 119:5021–5029CrossRefPubMedGoogle Scholar
  14. 14.
    Lüke Y, Zaim H, Karakesisoglou I et al (2007) Nesprin-2 Giant (NUANCE) maintains nuclear envelope architecture and composition in skin. J Cell Sci 121:1887–1898CrossRefGoogle Scholar
  15. 15.
    Rashmi RN, Eckes B, Glöckner G et al (2012) The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing. Nucleus 3:172–186CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Bhattacharya K, Swaminathan K, Peche VS et al (2016) Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology. Sci Rep 6:25411CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zhang ZG, Bothe I, Hirche F et al (2006) Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of alpha2beta1 integrin. J Cell Sci 119:1886–1895CrossRefPubMedGoogle Scholar
  18. 18.
    Carthew J, Karakesisoglou I (2016) Detection of diverse and high molecular weight Nesprin-1 and Nesprin-2 isoforms using Western blotting. Methods Mol Biol 1411:221–232CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiosciencesUniversity of DurhamDurhamUK
  2. 2.Institute of Biochemistry I, Medical FacultyUniversity Hospital CologneKölnGermany
  3. 3.Center for Molecular Medicine Cologne (CMMC) and Cologne Cluster of Excellence on Cellular Stress Responses in Aging-Associated Diseases (CECAD)University of CologneKölnGermany

Personalised recommendations

Cite protocol

Buy options