Abstract
As components of diverse tissues and organs, metazoan cells have to display a wide variety of specialized functions. Implementation of such functions invariably entails the establishment of tissue-specific cellular architecture (Bone and Starr, J Cell Sci 129:1951–1961, 2016). In animal cells, the nucleus is typically the largest organelle and in many respects acts as a landmark for multiple subcellular structures. For instance, in epithelial cells, the nucleus is frequently positioned close to the basal membrane via association with the cytoskeleton. Clearly such associations must be mediated by protein components of the outer nuclear membrane. One such protein is Nesprin-4, a member of the KASH domain family that is expressed in a variety of epithelial cells, including sensory outer hair cells of the inner ear. In this chapter, I describe a proximity-based biotinylation technique, BioID, that can be applied to Nesprin-4 to map its interactions at the nuclear periphery.
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References
Bone CR, Starr DA (2016) Nuclear migration events throughout development. J Cell Sci 129:1951–1961
Gundersen GG, Worman HJ (2013) Nuclear positioning. Cell 152:1376–1389
Sosa BA, Rothballer A, Kutay U et al (2012) LINC complexes form by binding of three KASH peptides to domain interfaces of trimeric SUN proteins. Cell 149:1035–1047
Zhou Z, Du X, Cai Z et al (2012) Structure of Sad1-UNC84 homology (SUN) domain defines features of molecular bridge in nuclear envelope. J Biol Chem 287:5317–5326
Crisp M, Liu Q, Roux K et al (2006) Coupling of the nucleus and cytoplasm: role of the LINC complex. J Cell Biol 172:41–53
Haque F, Lloyd DJ, Smallwood DT 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 26:3738–3371
Hasan S, Guttinger S, Muhlhausser P et al (2006) Nuclear envelope localization of human UNC84A does not require nuclear lamins. FEBS Lett 580:1263–1268
Padmakumar VC, Libotte T, Lu W et al (2005) The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope. J Cell Sci 118:3419–3430
Burke B, Roux KJ (2009) Nuclei take a position: managing nuclear location. Dev Cell 17:587–597
Hudspeth AJ (2014) Integrating the active process of hair cells with cochlear function. Nat Rev Neurosci 15:600–614
Horn HF, Brownstein Z, Lenz DR et al (2013) The LINC complex is essential for hearing. J Clin Invest 123:740–750
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 106:2194–2199
Roux KJ, Kim DI, Raida M et al (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196:801–810
Roux KJ, Kim DI, Burke B (2013) BioID: a screen for protein-protein interactions. Curr Protoc Protein Sci 74:Unit 19.23
Kim DI, Birendra KC, Zhu W et al (2014) Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A 111:E2453–E2461
Rivolta MN, Holley MC (2002) Cell lines in inner ear research. J Neurobiol 53:306–318
Ball RK, Friis RR, Schoenenberger CA et al (1988) Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. EMBO J 7:2089–2095
Sosa BA, Kutay U, Schwartz TU (2013) Structural insights into LINC complexes. Curr Opin Struct Biol 23:285–291
Dreesen O, Chojnowski A, Ong PF et al (2013) Lamin B1 fluctuations have differential effects on cellular proliferation and senescence. J Cell Biol 200:605–617
Chojnowski A, Ong PF, Wong ES et al (2015) Progerin reduces LAP2alpha-telomere association in Hutchinson-Gilford progeria. Elife 4:e07759
Thompson A, Schafer J, Kuhn K et al (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75:1895–1904
Ash JF, Louvard D, Singer SJ (1977) Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc Natl Acad Sci U S A 74:5584–5588
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Burke, B. (2018). Interactions of Nesprin-4-Containing LINC Complexes in Outer Hair Cells Explored by BioID. 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_5
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DOI: https://doi.org/10.1007/978-1-4939-8691-0_5
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