Abstract
Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.
Highlights
Many molecular components that are necessary for the development and maintenance of hearing have been discovered by identifying the genes that underlie hearing impairment in humans and mice [1,2,3,4]
Our ex vivo studies reveal that fluorescently tagged ELMOD3 localized with the actin-based microvilli of LLC-PK1-CL4 epithelial cells, in the stereocilia of sensory hair cells of mouse organ of Corti explants, and to a lesser extent to the actin cytoskeleton of MDCK cells, whereas the deafness-associated allele (p.Leu265Ser) was deficient in each case
While ELMOD3 antibody reactivity was detected in outer hair cells stereocilia at P02, more pronounced accumulation of ELMOD3 immunoreactivity was detected in rat cochlear inner hair cell stereocilia only by P12, which is when hair bundles are in the late phase of maturation
Summary
Many molecular components that are necessary for the development and maintenance of hearing have been discovered by identifying the genes that underlie hearing impairment in humans and mice [1,2,3,4]. One of the key structures in the mechanotransduction process is the hair cell stereocilium. At the tapered end of the stereocilium, the actin filaments form a rootlet that has been proposed to anchor the structure in the actin-rich meshwork of the cuticular plate [5]. Among the identified hearing loss-associated genes, nineteen encode proteins that interact with actin [6,7]. Numerous studies have demonstrated that actin cytoskeleton-associated proteins are involved in the development, maintenance and stabilization of the stereocilia (for review, see [6])
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