Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography.

Johannes Elferich,Aya Matsui,April Goehring,Sarah Clark,Eric Gouaux,Jingpeng Ge

doi:10.7554/elife.74512

Abstract

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the 'tip link.' Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location, and conformational states of intact tip links, on stereocilia, remains unknown. Here, we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.

Highlights

Vertebrates sense sound, head movement and gravity using specialized sensory cells, called hair cells (McPherson, 2018)
Immunostaining of mouse cochlea using these anti protocadherin 15 (PCDH15) pAbs produces robust staining of stereocilia in hair cells derived from wild-type (WT) mice that is entirely absent from the stereocilia of PCDH15 knock-out mice, demonstrating the specificity and utility of the anti-PCDH15 pAbs
We observed similar staining of stereocilia derived from mouse utricles, both in the presence of calcium and after chelation of calcium using BAPTA, indicating that the antibody binding was independent of calcium and, independent of PCDH15-cadherin 23 (CDH23) binding (Figure 1B)

Summary

Introduction

Vertebrates sense sound, head movement and gravity using specialized sensory cells, called hair cells (McPherson, 2018). These images reveal a 120-170 nm long, helically coiled, filament with a diameter of 5 nm and a repeat of 40 nm (Kachar et al, 2000) These links appear to bifurcate at the upper and lower insertion sites into 2-3 individual strands, an observation that is difficult to reconcile in light of high resolution structural data showing that PCDH15 and CDH23 are parallel dimers and that both proteins harbor membrane-proximal ‘dimerization domains’ (De-la-Torre et al., 2018; Dionne et al, 2018; Ge et al, 2018). To elucidate the molecular structures and conformational states of tip links under near native conditions

Results

Discussion

Methods