Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15

Abstract

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15.

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice

“…We have presented the following findings ( Fig. 10): 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies previous findings in neonatal hair cells (7,9); 2) LHFPL5 persists in the hair bundle of hair cells after P7, which clarifies the unexplained absence of LHFPL5 after P7 previously reported (7,10) and supports the notion that LHFPL5 is a permanent component in the MT complex; 3) TMC1 remains at the tip of the shorter rows of stereocilia in adult OHCs but is uniformly distributed in both the tallest row and the shorter rows of stereocilia in adult IHCs; and 4) LHFPL5 remains at the tip of the shorter rows of stereocilia in adult OHCs but is uniformly distributed in the shorter rows of stereocilia in adult IHCs. The unexpected uniform distribution of TMC1 and LHFPL5 in the hair bundle of adult IHCs raises intriguing questions regarding the 2 proteins' turnover rates, regulations, additional functions, and functional interactions.…”

“…Similar to what was reported by Xiong et al (10) again found that LHFPL5 labeling declined substantially by P6, although the labeling remained detectable at the tip of very few shorter rows of stereocilia at P21. We speculate that the marked reduction in LHFPL5 detection in this study is also a consequence of the dynamic change of antibodyepitope accessibility.…”

“…Kurima et al (9) comprehensively examined TMC1 localization in outer hair cells (OHCs) up to postnatal day (P) 15 and in inner hair cells (IHCs) up to P10 in the mouse but did not investigate TMC1 localization in adult hair cells. By using immunofluorescence staining, Xiong et al (7) characterized LHFPL5 localization in P1 and P5 mouse hair cells but, unexpectedly, did not detect LHFPL5 in hair bundles after P7 despite its abundant mRNA expression in hair cells; similar results were also reported by Mahendrasingam et al (10), who, by using the relatively more sensitive immunogold staining method, found that LHFPL5 labeling declined substantially by P6, although it could still be detected at very few tips of shorter rows of stereocilia at P21.…”

“…As a candidate channel or component of the MT complex, TMC1 and LHFPL5 are expected to localize, exclusively or nonexclusively, at the lower end of the tip link in the hair bundle of hair cells, the presumed location of the MT complex; thus, characterization of TMC1 and LHFPL5 localization is fundamentally critical for understanding their functions. However, the localization of these proteins has been examined in detail in only 1 or 2 studies each (7,9,10), and TMC1 and LHFPL5 localization, particularly in adult hair cells, remains incompletely elucidated. Kurima et al (9) comprehensively examined TMC1 localization in outer hair cells (OHCs) up to postnatal day (P) 15 and in inner hair cells (IHCs) up to P10 in the mouse but did not investigate TMC1 localization in adult hair cells.…”

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

“…For example, PCDH15 largely requires LHFPL5 for trafficking to the stereocilia [17, 18, 38], and depends on Cadherin 23 to maintain its localization at the site of MET [17, 39]. LHFPL5 also requires PCDH15 to maintain localization at the stereocilia tips [18, 38]. Thus, Tmie appears to be the exception to the rule of co-dependent transport to the hair bundle.…”