Integration of Tmc1/2 into the mechanotransduction complex in zebrafish hair cells is regulated by Transmembrane O-methyltransferase (Tomt)

Abstract

Transmembrane O-methyltransferase (TOMT/LRTOMT) is responsible for non-syndromic deafness DFNB63. However, the specific defects that lead to hearing loss have not been described. Using a zebrafish model of DFNB63, we show that the auditory and vestibular phenotypes are due to a lack of mechanotransduction (MET) in Tomt-deficient hair cells. GFP-tagged Tomt is enriched in the Golgi of hair cells, suggesting that Tomt might regulate the trafficking of other MET components to the hair bundle. We found that Tmc1/2 proteins are specifically excluded from the hair bundle in tomt mutants, whereas other MET complex proteins can still localize to the bundle. Furthermore, mouse TOMT and TMC1 can directly interact in HEK 293 cells, and this interaction is modulated by His183 in TOMT. Thus, we propose a model of MET complex assembly where Tomt and the Tmcs interact within the secretory pathway to traffic Tmc proteins to the hair bundle.DOI: http://dx.doi.org/10.7554/eLife.28474.001

Putative pore-forming subunits of the mechano-electrical transduction channel, Tmc1/2b, require Tmie to localize to the site of mechanotransduction in zebrafish sensory hair cells

“…First, we characterized Tmie’s interactions with other MET proteins in vivo by expressing transgenic Tmie-GFP in mutant pcdh15a , lhfpl5a , and tomt larvae (Fig 2). Because a triple knock-out of zebrafish tmc has not been reported, we used tomt mutants as a proxy for tmc -deficient fish based on recent studies of defective bundle localization of the Tmcs in tomt -deficient fish and mice [11, 35]. As in wild type bundles (Fig 2A), Tmie-GFP is detectable in the stereocilia in each of these MET mutants (Fig 2B and 2D), even if hair bundles are splayed (Fig 2B and 2C, arrowheads).…”

“…Thin bundles have been observed in other zebrafish MET mutants, such as those carrying mutations in ap1b1 and tomt . Both genes have been previously implicated in protein trafficking in hair cells, with tomt having a specific role in targeting Tmc1/2 proteins to the hair bundle [11, 33].…”

“…To determine the interdependence of trafficking of Tmie and the other MET components, we examined the localization of Tmie-GFP in mutants of essential MET genes: the tip-link protein pcdh15a ; the accessory protein lhfpl5a ; and in tomt mutants (Fig 3). In both zebrafish and mice, the secretory pathway protein Tomt is required for proper localization of the Tmc1/2 [11, 35]; the tomt mutant likely simulates the condition of a triple knockout of all three zebrafish tmc genes ( tmc1/2a/2b ). Despite the absence of Pcdh15a, Lhfpl5a, and Tmcs, we found that Tmie-GFP still traffics to the bundles of hair cells.…”

“…All animal research was in compliance with guidelines from the Institutional Animal Care and Use Committee at Oregon Health and Science University. In this study, the following zebrafish mutant lines were used: tmie ru1000 [21], tmie t26171 , pcdh15a psi7 [9], lhfpl5a tm290d [44], tmc2b sa8817 [11]. All zebrafish lines in this study were maintained in a Tübingen or Top long fin wild type background.…”

TRPV5, TRPV6, TRPM6, and TRPM7 do not contribute to hair-cell mechanotransduction

“…An alternative view is that PCDH15 or the TRPMs bind membrane proteins promiscuously, which may call into question interactions described based on similar immunoprecipitation experiments (Ramakrishnan et al, 2009;Ramakrishnan et al, 2012;Maeda et al, 2014;Beurg et al, 2015;Cunningham et al, 2017;Erickson et al, 2017). While our experiments were well controlled, including the use of alternative cadherins and TRP channels, reliance simply on cell-culture immunoprecipitation experiments is fraught.…”