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Centrifugal Displacement of Nuclei in Adherent Cells to Study LINC Complex-Dependent Mechanisms of Homeostatic Nuclear Positioning

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The LINC Complex

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

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Abstract

The positioning of the nucleus is critical for key cellular processes including division, migration, and differentiation. Traditional approaches to understanding the functions and mechanisms of nuclear positioning have relied upon cellular systems in which nuclei move in response to stimuli or developmental programs and use molecular or pharmacological perturbations of nuclear and cytoskeletal elements. Here, we describe a complimentary approach to perturbing nuclear position in adherent cells using centrifugal force and how this may be used to understand LINC complex mechanisms of homeostatic nuclear positioning.

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References

  1. Gundersen GG, Worman HJ (2013) Nuclear positioning. Cell 152(6):1376–1389

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  2. Chang W, Worman HJ, Gundersen GG (2015) Accessorizing and anchoring the LINC complex for multifunctionality. J Cell Biol 208(1):11–22

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Crisp M et al (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 172(1):41–53

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Haque F et al (2006) SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton. Mol Cell Biol 26(10):3738–3751

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Adames NR, Cooper JA (2000) Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J Cell Biol 149(4):863–874

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Borrego-Pinto J et al (2012) Samp1 is a component of TAN lines and is required for nuclear movement. J Cell Sci 125(Pt 5):1099–1105

    Article  PubMed  CAS  Google Scholar 

  7. Chang W et al (2013) Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts. Mol Biol Cell 24(24):3869–3880

    Article  PubMed  PubMed Central  Google Scholar 

  8. Folker ES et al (2011) Lamin a variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement. Proc Natl Acad Sci 108(1):131–136

    Article  PubMed  Google Scholar 

  9. Kutscheidt S et al (2014) FHOD1 interaction with nesprin-2G mediates TAN line formation and nuclear movement. Nat Cell Biol 16(7):708–715

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Luxton GW et al (2010) Linear arrays of nuclear envelope proteins harness retrograde actin flow for nuclear movement. Science 329(5994):956–959

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Fridolfsson HN et al (2010) UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration. Dev Biol 338(2):237–250

    Article  PubMed  CAS  Google Scholar 

  12. Fridolfsson HN, Starr DA (2010) Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei. J Cell Biol 191(1):115–128

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Lee KK et al (2002) Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans. Mol Biol Cell 13(3):892–901

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  14. Meyerzon M et al (2009) UNC-83 is a nuclear-specific cargo adaptor for kinesin-1-mediated nuclear migration. Development 136(16):2725–2733

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Starr DA, Han M (2002) Role of ANC-1 in tethering nuclei to the actin cytoskeleton. Science 298(5592):406–409

    Article  CAS  PubMed  Google Scholar 

  16. Roux KJ et al (2009) Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization. Proc Natl Acad Sci 106(7):2194–2199

    Article  PubMed  PubMed Central  Google Scholar 

  17. Hu DJ et al (2013) Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells. Cell 154(6):1300–1313

    Article  PubMed  CAS  Google Scholar 

  18. Jayo A et al (2016) Fascin regulates nuclear movement and deformation in migrating cells. Dev Cell 38(4):371–383

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Neelam S et al (2015) Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell. Proc Natl Acad Sci 112(18):5720–5725

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. Tkachenko E et al (2013) The nucleus of endothelial cell as a sensor of blood flow direction. Biol Open 2(10):1007–1012

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Veomett G et al (1974) Reconstruction of mammalian cells from nuclear and cytoplasmic components separated by treatment with cytochalasin B. Proc Natl Acad Sci U S A 71(5):1999–2002

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  22. Prescott DM, Kirkpatrick JB (1973) Mass enucleation of cultured animal cells. Methods Cell Biol 7:189–202

    Article  CAS  PubMed  Google Scholar 

  23. Poste G (1973) Anucleate mammalian cells: applications in cell biology and virology. Methods Cell Biol 7:211–249

    Article  CAS  PubMed  Google Scholar 

  24. Daga RR, Chang F (2005) Dynamic positioning of the fission yeast cell division plane. Proc Natl Acad Sci U S A 102(23):8228–8232

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Rodionov V et al (2001) Digital fluorescence microscopy of cell cytoplasts with and without the centrosome. Methods Cell Biol 67:43–51

    Article  CAS  PubMed  Google Scholar 

  26. Zhu R, Antoku S, Gundersen GG (2017) Centrifugal displacement of nuclei reveals multiple LINC complex mechanisms for homeostatic nuclear positioning. Curr Biol 27(20):3097–3110

    Google Scholar 

  27. Gomes ER, Jani S, Gundersen GG (2005) Nuclear movement regulated by Cdc42, MRCK, myosin, and actin flow establishes MTOC polarization in migrating cells. Cell 121(3):451–463

    Article  CAS  PubMed  Google Scholar 

  28. Wright WE (1973) The production of mass populations of anucleate cytoplasms. Methods Cell Biol 7:203–210

    Article  PubMed  CAS  Google Scholar 

  29. Chang W, Antoku S, Gundersen GG (2016) Wound-healing assays to study mechanisms of nuclear movement in fibroblasts and myoblasts. Methods Mol Biol 1411:255–267

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Gregg G. Gundersen .

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Zhu, R., Gundersen, G.G. (2018). Centrifugal Displacement of Nuclei in Adherent Cells to Study LINC Complex-Dependent Mechanisms of Homeostatic Nuclear Positioning. In: Gundersen, G., Worman, H. (eds) The LINC Complex. Methods in Molecular Biology, vol 1840. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8691-0_9

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  • DOI: https://doi.org/10.1007/978-1-4939-8691-0_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8690-3

  • Online ISBN: 978-1-4939-8691-0

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