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

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.

Redefining the Structure of Tip Links in Hair Cells

“…We used the base dimers anticipating that Cdh23 exists as a base dimer in hair cells via the PICA-like domain. Though such a PICA-like domain has been bioinformatically verified for Cdh23, recent cryo-electron micrographs clearly demonstrated bifurcated C-termini for Cdh23 in tip links …”

“…The in situ electron micrograph of stereocilia clearly depicted the thread-like arrangement of tip links. , Subsequently, myriad studies have been employed to identify the conformational and structural origin of the thread-like tip links . It was soon revealed that the constituent cadherins, Pcdh15 and Cdh23, may exist as lateral dimers and form helically shaped tip links.…”

“…However, no single studies have reported the stable and predominant existence of the Cdh23 cis dimer. Instead, cryo-electron micrographs of tip links on stereocilia indicated random bifurcated regions between two Cdh23 molecules engaged in tip links, and more frequently at the C-terminus . The trans dimer of Cdh23, on the contrary, was established long ago along with its molecular details. − Overall, a continuous delay in capturing and deciphering the lateral dimer of Cdh23 has raised serious concerns about its existence.…”

“…The lower part of the helical coil is formed by Pcdh15. In situ imaging of stereocilia using cryo-electron microscopy and antibody-modified Au nanoparticle staining clearly depicted the intertwined lateral dimers of Pcdh15 . The residues at the EC3 domain and the membrane-proximal PICA domain that drive lateral interactions in Pcdh15 were identified from X-ray crystallography of varying EC domains. ,, Finally, a series of mutations, including Leu138Asp (L138D), Leu138Ala (L138A), Arg198Ala (R198A), Asp255Lys (D255K),Val250Asp (V250D), Lys345Ala (K345A), and Lys345Asp (K345D), were made to pinpoint the residues through the disruption of the lateral interactions .…”

Redefining the structure of tip-links in hair-cells

“…The in-situ electron micrograph of stereocilia clearly depicted the thread-like arrangement of tip-links 12 , 25 . Subsequently, myriads of studies have been employed to identify the conformational and structural origin of the thread-like tip-links 26 .…”