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Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy

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The LINC Complex

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

Abstract

Linkers of nucleoskeleton and cytoskeleton (LINC) complexes are conserved nuclear envelope (NE) spanning molecular bridges which mechanically integrate the nucleus with the cytoskeleton and mediate force transmission into the nucleoplasm. Despite their critical roles in fundamental cellular processes such as meiotic chromosome and nuclear positioning, the mechanism of LINC complex assembly in cells remains unclear. To begin to address this deficit, we recently developed z-scan fluorescence fluctuation spectroscopy (FFS) and brightness analysis as a method for quantifying the oligomeric states of fluorescent protein-tagged NE proteins including nesprins and SUN proteins. Since the homo-oligomerization of SUN2 is critical for its ability to interact with nesprins within the perinuclear space, the knowledge obtained through quantitative brightness experiments reveals important insights into the in vivo mechanisms of LINC complex assembly. Here we describe the procedure we use to determine the brightness of proteins in the NE of living cells. In addition to the measurement procedure, we discuss the instrumentation requirements and present the results of applying this procedure to measure the brightness of nesprin-2 and SUN2.

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Abbreviations

b :

Normalized brightness

KASH:

Klarsicht, ANC-1, SYNE homology

LINC:

Linker of nucleoskeleton and cytoskeleton

N :

Number concentration

NE:

Nuclear envelope

SUN:

Sad1/UNC-84

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Acknowledgments

This work was financially supported in part by the NIH (J.D.M. (GM064589), and C.A.S. (AR007612)), and the Dystonia Medical Research Foundation (G.W.G.L. and J.D.M.).

Author information

Authors and Affiliations

  1. School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, USA

    Jared Hennen, Isaac Angert, Kwang-Ho Hur & Joachim D. Mueller

  2. Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA

    G. W. Gant Luxton

Authors
  1. Jared Hennen
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  2. Isaac Angert
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  3. Kwang-Ho Hur
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  4. G. W. Gant Luxton
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  5. Joachim D. Mueller
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Corresponding authors

Correspondence to G. W. Gant Luxton or Joachim D. Mueller .

Editor information

Editors and Affiliations

  1. Department of Pathology and Cell Biology, Columbia University, New York, New York, USA

    Gregg G. Gundersen

  2. Department of Medicine and Department of Pathology and Cell Biology, Columbia University, New York, New York, USA

    Howard J. Worman

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Hennen, J., Angert, I., Hur, KH., Gant Luxton, G.W., Mueller, J.D. (2018). Investigating LINC Complex Protein Homo-oligomerization in the Nuclear Envelopes of Living Cells Using Fluorescence Fluctuation Spectroscopy. 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_11

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  • DOI: https://doi.org/10.1007/978-1-4939-8691-0_11

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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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