Identification of deafness genes has revealed many essential proteins of the mechanotransduction complex of hair cells, including TMC1, PCDH15, TMIE, CIB2 and LHFPL5. Other proteins, not encoded by known deafness genes, might also play an important role. We used data from a comprehensive transcriptome study (SHIELD database) to identify mouse genes preferentially expressed in cochlear and vestibular hair cells. We identified Lhfpl4, a close homolog of Lhfpl5, which has a similarly selective expression in hair cells with increased expression during the first postnatal week. Both LHFPL4 and LHFPL5 are small proteins (220-250 aa) with four predicted transmembrane domains and intracellular N- and C-termini. LHFPL5 binds to PCDH15 as a tetrameric membrane complex. However, little is known about LHFPL4. Could LHFPL4 have an essential role in hair cells, similar to that of LHFPL5?

We first optimized an in vitro electroporation method to deliver plasmids up to 18 kb to mouse cochlear hair cells. In P2-P5 mice we electroporated plasmids encoding LHFPL4 and LHFPL5. To label LHFPLs, we used a novel tandem GCN epitope and a cognate engineered antibody with picomolar affinity and high specificity. Placement of these epitopes in extracellular domains, with normal function confirmed by rescue of knockout mice, allowed both live-cell fluorescence imaging and immunogold electron microscopic localization of LHFPLs. We confirmed localization of LHFPL5 near the tips of the stereocilia, especially the shorter rows. Anti-GCN labeling of native LHFPL4 also showed localization near the tips of the stereocilia. Immunogold SEM further localized LHFPL4 near the tips of all stereocilia.

We used co-immunoprecipitation to assess interaction with other transduction proteins. Both LHFPL4 and LHFPL5 bound to both PCDH15 and TMC1. Based on the localization of LHFPL4 near the tips of stereocilia and its binding to TMC1, we suggest that LHFPL4 plays a role in the mechanotransduction complex.

Predicted structure of LHFPL5. A protein essential for transduction, LHFPL5 is predicted to have four transmembrane domains and an extracellular beta sheet. LHFPL5 binds to PCDH15 and may be a part of the mature mechanotransduction complex. LHFPL4 also binds PCDH15 and is essential in the absence of LHFPL5.

PAPERS

Ivanchenko MV, Indzhykulian AA, Corey DP (2021) Electron Microscopy Techniques for Investigating Structure and Composition of Hair-Cell Stereociliary Bundles. Front Cell Dev Biol. 9:744248.