Skip to main content

Advertisement

SpringerLink
Log in

The GPSM2/LGN GoLoco motifs are essential for hearing

  • Published:
Mammalian Genome Aims and scope Submit manuscript

Abstract

The planar cell polarity (PCP) pathway is responsible for polarizing and orienting cochlear hair cells during development through movement of a primary cilium, the kinocilium. GPSM2/LGN, a mitotic spindle-orienting protein associated with deafness in humans, is a PCP effector involved in kinocilium migration. Here, we link human and mouse truncating mutations in the GPSM2/LGN gene, both leading to hearing loss. The human variant, p.(Trp326*), was identified by targeted genomic enrichment of genes associated with deafness, followed by massively parallel sequencing. Lgn ΔC mice, with a targeted deletion truncating the C-terminal GoLoco motifs, are profoundly deaf and show misorientation of the hair bundle and severe malformations in stereocilia shape that deteriorates over time. Full-length protein levels are greatly reduced in mutant mice, with upregulated mRNA levels. The truncated Lgn ΔC allele is translated in vitro, suggesting that mutant mice may have partially functioning Lgn. Gαi and aPKC, known to function in the same pathway as Lgn, are dependent on Lgn for proper localization. The polarization of core PCP proteins is not affected in Lgn mutants; however, Lgn and Gαi are misoriented in a PCP mutant, supporting the role of Lgn as a PCP effector. The kinocilium, previously shown to be dependent on Lgn for robust localization, is essential for proper localization of Lgn, as well as Gαi and aPKC, suggesting that cilium function plays a role in positioning of apical proteins. Taken together, our data provide a mechanism for the loss of hearing found in human patients with GPSM2/LGN variants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Almomani R, Sun Y et al (2013) GPSM2 and Chudley-McCullough syndrome: a Dutch founder variant brought to North America. Am J Med Genet Part A 161A(5):973–976

    Article  PubMed  Google Scholar 

  • Bhonker Y, Ushakov K et al (2014) Human gene discovery for understanding development of the inner ear and hearing loss. In: Romand R, Varela-Neito I (eds) Development of auditory and vestibular systems. Academic Press, New York, pp 107–128

    Google Scholar 

  • Brownstein Z, Friedman LM et al (2011) Targeted genomic capture and massively parallel sequencing to identify genes for hereditary hearing loss in Middle Eastern families. Genome Biol 12(9):R89

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Brownstein Z, Bhonker Y et al (2012) High-throughput sequencing to decipher the genetic heterogeneity of deafness. Genome Biol 13(5):245

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Brownstein Z, Abu-Rayyan A et al (2014) Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing. Eur J Hum Genet 22(6):768–775

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Chan HY, Sivakamasundari V et al (2011) Comparison of IRES and F2A-based locus-specific multicistronic expression in stable mouse lines. PLoS ONE 6(12):e28885

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Curtin JA, Quint E et al (2003) Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse. Curr Biol 13(13):1129–1133

    Article  PubMed  CAS  Google Scholar 

  • Diaz-Horta O, Sirmaci A et al (2012) GPSM2 mutations in Chudley-McCullough syndrome. Am J Med Genet A 158A(11):2972–2973

    Article  PubMed  Google Scholar 

  • Doherty D, Chudley AE et al (2012) GPSM2 mutations cause the brain malformations and hearing loss in Chudley-McCullough syndrome. Am J Hum Genet 90(6):1088–1093

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Du Q, Macara IG (2004) Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119(4):503–516

    Article  PubMed  CAS  Google Scholar 

  • Ezan J, Lasvaux L et al (2013) Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton. Nat Cell Biol 15(9):1107–1115

    Article  PubMed  CAS  Google Scholar 

  • Gegg M, Böttcher A et al (2014) Flattop regulates basal body docking and positioning in mono- and multiciliated cells. eLife 3:e03842

    Article  PubMed Central  Google Scholar 

  • Gilissen C, Hoischen A et al (2012) Disease gene identification strategies for exome sequencing. Eur J Hum Genet 20(5):490–497

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Goetz SC, Anderson KV (2010) The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet 11(5):331–344

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Grimsley-Myers CM, Sipe CW et al (2009) The small GTPase Rac1 regulates auditory hair cell morphogenesis. J Neurosci 29(50):15859–15869

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41(1999):95–98

    CAS  Google Scholar 

  • Horn HF, Brownstein Z et al (2013) The LINC complex is essential for hearing. J Clin Invest 123(2):740–750

    PubMed  CAS  PubMed Central  Google Scholar 

  • Hunter-Duvar IM (1978) A technique for preparation of cochlear specimens for assessment with the scanning electron microscope. Acta Oto-Laryngol Suppl 351:3–23

    Article  CAS  Google Scholar 

  • Jacobo A, Hudspeth AJ (2014) Reaction–diffusion model of hair-bundle morphogenesis. Proc Natl Acad Sci USA 111(43):15444–15449

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Johnston CA, Hirono K, Prehoda KE, Doe CQ (2009) Identification of an Aurora-A/PinsLINKER/Dlg spindle orientation pathway using induced cell polarity in S2 cells. Cell 138(6):1150–1163

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Jones C, Chen P (2007) Planar cell polarity signaling in vertebrates. BioEssays 29(2):120–132

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Jones C, Roper VC et al (2007) Ciliary proteins link basal body polarization to planar cell polarity regulation. Nat Genet 40(1):69–77

    Article  PubMed  Google Scholar 

  • Kaushik R, Yu F et al (2003) Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression. Mol Biol Cell 14(8):3144–3155

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Kibar Z, Vogan KJ et al (2001) Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail. Nat Genet 28(3):251–255

    Article  PubMed  CAS  Google Scholar 

  • Kirjavainen A, Laos M et al (2015) The Rho GTPase Cdc42 regulates hair cell planar polarity and cellular patterning in the developing cochlea. Biol Open 4(4):516–526

    Article  PubMed  PubMed Central  Google Scholar 

  • Konno D, Shioi G et al (2008) Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat Cell Biol 10(1):93–101

    Article  PubMed  CAS  Google Scholar 

  • Kuzmiak HA, Maquat LE (2006) Applying nonsense-mediated mRNA decay research to the clinic: progress and challenges. Trends Mol Med 12(7):306–316

    Article  PubMed  CAS  Google Scholar 

  • Lepelletier L, de Monvel JB et al (2013) Auditory hair cell centrioles undergo confined brownian motion throughout the developmental migration of the kinocilium. Biophys J 105(1):48–58

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408

    Article  PubMed  CAS  Google Scholar 

  • Mapelli M, Gonzalez C (2012) On the inscrutable role of Inscuteable: structural basis and functional implications for the competitive binding of NuMA and Inscuteable to LGN. Open Biol 2(8):120102

    Article  PubMed  PubMed Central  Google Scholar 

  • Mauser JF, Prehoda KE (2012) Inscuteable regulates the Pins-Mud spindle orientation pathway. PLoS One 7(1):e29611

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Mochizuki N, Cho G et al (1996) Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G alpha i2. Gene 181(1):39–43

    Article  PubMed  CAS  Google Scholar 

  • Montcouquiol M, Kelley MW (2003) Planar and vertical signals control cellular differentiation and patterning in the mammalian cochlea. J Neurosci 23(28):9469–9478

    PubMed  CAS  Google Scholar 

  • Montcouquiol M, Rachel RA et al (2003) Identification of Vangl2 and Scrb1 as planar polarity genes in mammals. Nature 423(6936):173–177

    Article  PubMed  CAS  Google Scholar 

  • Montcouquiol M, Sans N et al (2006) Asymmetric localization of Vangl2 and Fz3 indicate novel mechanisms for planar cell polarity in mammals. J Neurosci 26(19):5265–5275

    Article  PubMed  CAS  Google Scholar 

  • Pan Z, Shang Y et al (2013) Structural and biochemical characterization of the interaction between LGN and Frmpd1. J Mol Biol 425(6):1039–1049

    Article  PubMed  CAS  Google Scholar 

  • Pettersen EF, Goddard TD et al (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612

    Article  PubMed  CAS  Google Scholar 

  • Richardson GP, de Monvel JB et al (2011) How the genetics of deafness illuminates auditory physiology. Ann Rev Physiol 73:311–334

    Article  CAS  Google Scholar 

  • Schaefer M, Shevchenko A et al (2000) A protein complex containing Inscuteable and the Gα-binding protein Pins orients asymmetric cell divisions in Drosophila. Curr Biol 10(7):353–362

    Article  PubMed  CAS  Google Scholar 

  • Schwander M, Kachar B et al (2010) The cell biology of hearing. J Cell Biol 190(1):9–20

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Shahin H, Walsh T et al (2010) Five novel loci for inherited hearing loss mapped by SNP-based homozygosity profiles in Palestinian families. Eur J Hum Genet 18(4):407–413

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Shearer AE, Smith RJH (2015) Massively Parallel Sequencing for genetic diagnosis of hearing loss: the new standard of care. Otolaryngol Head Neck Surg 153(2):175–182

    Article  PubMed  Google Scholar 

  • Sim JCH, White SM et al (2015) ARID1B-mediated disorders: mutations and possible mechanisms. Intractable Rare Dis Res 4(1):17–23

    Article  PubMed  PubMed Central  Google Scholar 

  • Sipe CW, Lu X (2011) Kif3a regulates planar polarization of auditory hair cells through both ciliary and non-ciliary mechanisms. Dev Camb Engl 138(16):3441–3449

    CAS  Google Scholar 

  • Sipe CW, Liu L et al (2013) Lis1 mediates planar polarity of auditory hair cells through regulation of microtubule organization. Development 140(8):1785–1795

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Stevermann L, Liakopoulos D (2012) Molecular mechanisms in spindle positioning: structures and new concepts. Curr Opin Cell Biol 24(6):816–824

    Article  PubMed  CAS  Google Scholar 

  • Tall GG, Gilman AG (2005) Resistance to inhibitors of cholinesterase 8A catalyzes release of Gαi-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Gαi-GDP complexes. Proc Natl Acad Sci USA 102(46):16584–16589

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Tarchini B, Jolicoeur C, Cayouette M (2013) A molecular blueprint at the apical surface establishes planar asymmetry in cochlear hair cells. Dev Cell 27(1):88–102

    Article  PubMed  CAS  Google Scholar 

  • The UniProt Consortium (2014) UniProt: a hub for protein information. Nucleic Acids Res 43:D204–D212

    Article  PubMed Central  Google Scholar 

  • Walsh T, Shahin H et al (2010) Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am J Hum Genet 87(1):90–94

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Wang J, Mark S et al (2005) Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway. Nat Genet 37(9):980–985

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Waterhouse AM, Procter JB et al (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9):1189–1191

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Webb SW, Grillet N et al (2011) Regulation of PCDH15 function in mechanosensory hair cells by alternative splicing of the cytoplasmic domain. Development 138(8):1607–1617

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Williams SE, Beronja S et al (2011) Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 470(7334):353–358

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yariz KO, Walsh T et al (2012) A truncating mutation in GPSM2 is associated with recessive non-syndromic hearing loss. Clin Genet 81(3):289–293

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Yuzawa S, Kamakura S et al (2011) Structural basis for interaction between the conserved cell polarity proteins Inscuteable and Leu-Gly-Asn repeat-enriched protein (LGN). Proc Natl Acad Sci USA 108(48):19210–19215

    Article  PubMed  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by the Human Frontier Science Program RGP0012/2012 (K. B. A., P. C., F. M., D. S.); National Institutes of Health (NIDCD) R01DC011835 (K. B. A., M. K.); I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation, Centers of Excellence in Gene Regulation in Complex Human Disease, Grant No. 41/11 (K. B. A.) and in Integrated Structural Cell Biology, Grant No. 1775/12 (E. T, M. L.); and a TEVA Pharmaceutical Industries Ltd. as part of the Israeli National Network of Excellence in Neuroscience (NNE) (Y. B.). This work was performed in partial fulfillment of the requirements for a Ph.D. degree by Yoni Bhonker at the Sackler Faculty of Medicine, Tel Aviv University, Israel. We wish to thank Megy Cemel and Michal Meir for their valuable contributions.

Author information

Authors and Affiliations

  1. Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, 6997801, Israel

    Yoni Bhonker, Kathy Ushakov, Shaked Shivatzki, Ofer Yizhar-Barnea, Tal Elkan-Miller & Karen B. Avraham

  2. Department of Biological Sciences, Bethlehem University, Bethlehem, Palestine

    Amal Abu-Rayyan & Moien Kanaan

  3. Department of Biochemistry and Molecular Biology, Weiss Faculty of Life Sciences, Tel Aviv University, 69978, Tel Aviv, Israel

    Liat Amir-Zilberstein & David Sprinzak

  4. Department of Cell Biology, Emory University, Atlanta, GA, 30322, USA

    Sun Myoung Kim & Ping Chen

  5. Department of Biology, Technion-Israel Institute of Technology, 32000, Haifa, Israel

    Einav Tayeb-Fligelman & Meytal Landau

  6. Laboratory of Cell Asymmetry, Center for Developmental Biology, Riken, Kobe, 650-0047, Japan

    Fumio Matsuzaki

Authors
  1. Yoni Bhonker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amal Abu-Rayyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kathy Ushakov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Liat Amir-Zilberstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shaked Shivatzki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ofer Yizhar-Barnea
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tal Elkan-Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Einav Tayeb-Fligelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sun Myoung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Meytal Landau
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Moien Kanaan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ping Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Fumio Matsuzaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. David Sprinzak
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Karen B. Avraham
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karen B. Avraham.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. S1

Neuroimaging of mouse brains. No qualitative gross morphological changes were observed by serial MRI scanning of coronal sections of brains from 5-month old, a Lgn ΔC/+ and b Lgn ΔC/ΔC mice. Supplementary material 1 (TIFF 2598 kb)

Supplementary Fig. S2

SEM images of P30 hair cells. a, b OHCs and c IHC of Lgn ΔC/+ mice. d, e OHCs of Lgn ΔC/ΔC mice. The presence of medial and lateral hair bundles atop a single cell, first observed in P5 cochleae, is maintained at this age (d, arrow). f Stereocilia of an IHC showing the same multi-row uniform thickness as observed at P5. Supplementary material 2 (TIFF 1736 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bhonker, Y., Abu-Rayyan, A., Ushakov, K. et al. The GPSM2/LGN GoLoco motifs are essential for hearing. Mamm Genome 27, 29–46 (2016). https://doi.org/10.1007/s00335-015-9614-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00335-015-9614-7

Keywords

Search

Navigation