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Detection of SUN1 Splicing Variants at the mRNA and Protein Levels in Cancer

  • Ayaka Matsumoto
  • Nariaki Matsuura
  • Miki Hieda
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1840)

Abstract

The linker of nucleoskeleton and cytoskeleton (LINC) complex, containing the proteins SUN and nesprin, is the fundamental structural unit of the nuclear envelope. The neoplastic-based regulation of the LINC complex in cancer tissues has become increasingly recognized in recent years, including the altered expression, somatic mutation, and methylation of genes. However, precisely how mutations and deregulated expression of the LINC complex contribute to the pathogenic mechanisms of tumorigenesis remain to be elucidated, mainly because of several technical difficulties. First, both the SUN and SYNE (encoding nesprin) genes give rise to a vast number of splicing variants. Second, immunoprecipitation experiments of endogenous SUN and nesprin proteins are difficult owing to the lack of suitable reagents as well as the limited solubility of these proteins in mild extraction conditions. Here, we describe three protocols to investigate these aspects: (1) immunohistochemistry to determine the expression levels and localization of the LINC complex in cancer tissue, (2) detection of SUN1 splicing variants at the mRNA level, and (3) detection of SUN1 splicing variants and binding partners at the protein level.

Key words

SUN1 SUN2 Nesprin Immunohistochemistry Splicing variants 

Notes

Acknowledgment

We thank Ms Yu Nishioka and Ms Junko Imada for technical assistance. This work is supported by the Education and Research Grant Program of Ehime Prefectural University of Health Science to MH.

References

  1. 1.
    Fischer AH, Bardarov S Jr, Jiang Z (2004) Molecular aspects of diagnostic nucleolar and nuclear envelope changes in prostate cancer. J Cell Biochem 91:170–184CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Zink D, Fischer AH, Nickerson JA (2004) Nuclear structure in cancer cells. Nat Rev Cancer 4:677–687CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    de Las Heras JI, Schirmer EC (2014) The nuclear envelope and cancer: a diagnostic perspective and historical overview. Adv Exp Med Biol 773:5–26CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Jevtić P, Levy DL (2014) Mechanisms of nuclear size regulation in model systems and cancer. Adv Exp Med Biol 773:537–569CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Fischer AH (2014) The diagnostic pathology of the nuclear envelope in human cancers. Adv Exp Med Biol 773:49–75CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Bell ES, Lammerding J (2016) Causes and consequences of nuclear envelope alterations in tumour progression. Eur J Cell Biol 95:449–464CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Crisp M, Liu Q, Roux K, Rattner JB et al (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 172:41–53CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Hodzic DM, Yeater DB, Bengtsson L et al (2004) Sun2 is a novel mammalian inner nuclear membrane protein. J Biol Chem 279:25805–25812CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Shao X, Tarnasky HA, Lee JP et al (1999) Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme. Dev Biol 211:109–123CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Frohnert C, Schweizer S, Hoyer-Fender S (2011) SPAG4L/SPAG4L-2 are testis-specific SUN domain proteins restricted to the apical nuclear envelope of round spermatids facing the acrosome. Mol Hum Reprod 17:207–218CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Jiang XZ, Yang MG, Huang LH et al (2011) SPAG4L, a novel nuclear envelope protein involved in the meiotic stage of spermatogenesis. DNA Cell Biol 30:875–882CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Rajgor D, Shanahan CM (2013) Nesprins: from the nuclear envelope and beyond. Expert Rev Mol Med 15:e5CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Roux KJ, Crisp ML, Liu Q et al (2009) Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization. Proc Natl Acad Sci U S A 106:2194–2199CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Gundersen GG, Worman HJ (2013) Nuclear positioning. Cell 152:1376–1389CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Zhang X, Lei K, Yuan X et al (2017) SUN1/2 and Syne/Nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice. Neuron 2009(64):173–187Google Scholar
  16. 16.
    Lei K, Zhu X, Xu R et al (2012) Inner nuclear envelope proteins SUN1 and SUN2 play a prominent role in the DNA damage response. Curr Biol 22:1609–1615CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Lawrence KS, Tapley EC, Cruz VE et al (2016) LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining. J Cell Biol 215:801–821CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Luxton GW, Gomes ER, Folker ES et al (2010) Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement. Science 329:956–959CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Luxton GW, Gomes ER, Folker ES et al (2011) TAN lines: a novel nuclear envelope structure involved in nuclear positioning. Nucleus 2:173–181CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Chang W, Antoku S, Östlund C et al (2015) Linker of nucleoskeleton and cytoskeleton (LINC) complex-mediated actin-dependent nuclear positioning orients centrosomes in migrating myoblasts. Nucleus 6:77–88CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Nishioka Y, Imaizumi H, Imada J et al (2016) SUN1 splice variants, SUN1_888, SUN1_785, and predominant SUN1_916, variably function in directional cell migration. Nucleus 7:572–584CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Hiraoka Y, Dernburg AF (2009) The SUN rises on meiotic chromosome dynamics. Dev Cell 17:598–605CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Adam SA (2017) The nucleoskeleton. Cold Spring Harb Perspect Biol 9. pii a023556Google Scholar
  24. 24.
    Hsieh TH, Chien CL, Lee YH et al (2014) Downregulation of SUN2, a novel tumor suppressor, mediates miR-221/222-induced malignancy in central nervous system embryonal tumors. Carcinogenesis 35:2164–2174CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Lv XB, Liu L, Cheng C et al (2015) SUN2 exerts tumor suppressor functions by suppressing the Warburg effect in lung cancer. Sci Rep 5:17940CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Matsumoto A, Hieda M, Yokoyama Y et al (2015) Global loss of a nuclear lamina component, lamin A/C, and LINC complex components SUN1, SUN2, and nesprin-2 in breast cancer. Cancer Med 4:1547–1557CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Matsumoto A, Sakamoto C, Matsumori H et al (2016) Loss of the integral nuclear envelope protein SUN1 induces alteration of nucleoli. Nucleus 7:68–83CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Kennedy C, Sebire K, de Kretser DM et al (2004) Human sperm associated antigen 4 (SPAG4) is a potential cancer marker. Cell Tissue Res 315:279–283CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Shoji K, Murayama T, Mimura I et al (2013) Sperm-associated antigen 4, a novel hypoxia-inducible factor 1 target, regulates cytokinesis, and its expression correlates with the prognosis of renal cell carcinoma. Am J Pathol 182:2191–2203CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Knaup KX, Monti J, Hackenbeck T et al (2014) Hypoxia regulates the sperm associated antigen 4 (SPAG4) via HIF, which is expressed in renal clear cell carcinoma and promotes migration and invasion in vitro. Mol Carcinog 53:970–978PubMedPubMedCentralGoogle Scholar
  31. 31.
    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:e40098CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Doherty JA, Rossing MA, Cushing-Haugen KL et al (2010) SR1/SYNE1 polymorphism and invasive epithelial ovarian cancer risk: an Ovarian Cancer Association Consortium study. Cancer Epidemiol Biomark Prev 19:245–250CrossRefGoogle Scholar
  33. 33.
    Sjöblom T, Jones S, Wood LD et al (2006) The consensus coding sequences of human breast and colorectal cancers. Science 314:268–274CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Tessema M, Willink R, Do K et al (2008) Promoter methylation of genes in and around the candidate lung cancer susceptibility locus 6q23-25. Cancer Res 68:1707–1714CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Schuebel KE, Chen W, Cope L et al (2007) Comparing the DNA hypermethylome with gene mutations in human colorectal cancer. PLoS Genet 3:1709–1723CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Marmé A, Zimmermann HP, Moldenhauer G et al (2008) Loss of Drop1 expression already at early tumor stages in a wide range of human carcinomas. Int J Cancer 123:2048–2056CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    Schoppmann SF, Vinatzer U, Popitsch N et al (2013) Novel clinically relevant genes in gastrointestinal stromal tumors identified by exome sequencing. Clin Cancer Res 19:5329–5339CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Warren DT, Tajsic T, Mellad JA et al (2010) Novel nuclear nesprin-2 variants tether active extracellular signal-regulated MAPK1 and MAPK2 at promyelocytic leukemia protein nuclear bodies and act to regulate smooth muscle cell proliferation. J Biol Chem 285:1311–1320CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Vinayagam A, Stelzl U, Foulle R et al (2011) A directed protein interaction network for investigating intracellular signal transduction. Sci Signal 4(189):rs8CrossRefPubMedCentralPubMedGoogle Scholar
  40. 40.
    Autore F, Shanahan CM, Zhang Q (2016) Identification and validation of putative nesprin variants. Methods Mol Biol 1411:211–220CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Holt I, Duong NT, Zhang Q et al (2016) Specific localization of nesprin-1-α2, the short isoform of nesprin-1 with a KASH domain, in developing, fetal and regenerating muscle, using a new monoclonal antibody. BMC Cell Biol 17:26CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Göb E, Meyer-Natus E, Benavente R et al (2011) Expression of individual mammalian Sun1 isoforms depends on the cell type. Commun Integr Biol 4:440–442CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Razafsky D, Wirtz D, Hodzic D (2014) Nuclear envelope in nuclear positioning and cell migration. Adv Exp Med Biol 773:471–490CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Ayaka Matsumoto
    • 1
  • Nariaki Matsuura
    • 1
    • 2
  • Miki Hieda
    • 1
    • 3
  1. 1.Graduate School of Medicine and Health ScienceOsaka UniversityOsakaJapan
  2. 2.Osaka International Cancer InstituteOsakaJapan
  3. 3.Graduate School of Health SciencesEhime Prefectural University of Health SciencesIyoJapan

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