Imaging molecules in their native environment: cryo-electron tomography of PCDH15 complexes in mouse stereocilia
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.
Johannes Elferich is currently a postdoctoral fellow at the UMass Chen Medical School in the laboratory of Nikolaus Grigorieff. He obtained his PhD in 2015 in the laboratory of Ujwal Shinde at Oregon Health and Science University working on pH-mediated activation of the mammalian serine protease furin. He then joined Eric Gouaux at Oregon Health and Science University to study mechano-sensory transduction during hearing. Working together with Jingpeng Ge and Sarah Clark, he solved the structures of the PCDH15/LHFPL5 complex that forms the lower end of the mammalian tip-link and of the motor protein prestin that underlies acoustic amplification in the inner ear of mammals. They also used cryo-electron tomography to study the localization, stoichiometry and structural flexibility of the tip-link in stereocilia extracted from the inner ear of mice, furthering our understanding of hearing by taking direct images of single molecules of the underlying protein machinery. In 2021 he joined Nikolaus Grigorieff to work on developing methods and software for high-throughput molecule detection in cryo-electron micrographs to enable visual proteomics.
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