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

Shanthini Mahendrasingam,Robert Fettiplace,Kumar N Alagramam,David N Furness,Ellen Cross,Manuel S Malmierca

doi:10.1371/journal.pone.0185285

Shanthini Mahendrasingam, Robert Fettiplace + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0185285

Copy DOI

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.

Highlights

Mechanoelectrical transduction (MET) channels in vertebrate hair cells are gated by a fine extracellular filament, the tip link, that is located in the sensory stereociliary bundle
Pcdh15 mutants heterozygous (Pcdh15+/av3J) and homozygous (Pcdh15av3J/av3J) mice were bred at Case Western Reserve University (CWRU) and their use was approved under protocol number 2010–0074 by the Institutional Animal Care and Use Committee (IACUC)
A detailed account of hair bundle morphogenesis is not intended here, but in order to understand the structural context of the labelling patterns, we provide Scanning electron microscopy (SEM) images of different ages

Summary

Introduction

Mechanoelectrical transduction (MET) channels in vertebrate hair cells are gated by a fine extracellular filament, the tip link, that is located in the sensory stereociliary bundle (see review [1]). Anomalous currents occur following BAPTA treatment in wild-type mice, which is known to destroy the tip links [9] and can be evoked by stimulating the hair cell apex during development up to postnatal day (P) 2–3 in apical inner or outer hair cells [10]. This current diminishes in immature hair cells as the normal MET current develops and is independent of the putative subunit proteins of the MET, transmembrane-like channel protein isoforms 1 (TMC1) or 2 (TMC2), as it occurs in Tmc1/Tmc knockouts [10,11]. Recent evidence suggests that the anomalous reverse-polarity currents flow through PIEZO2, mechanically sensitive channels on the hair cell apical surface [12]

Methods

Results

Discussion

Conclusion