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The GPSM2/LGN GoLoco motifs are essential for hearing

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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.

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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.

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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

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  1. Yoni Bhonker
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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)

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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

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