Electron cryo-tomography of vestibular hair-cell stereocilia

Abstract

18High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our 19 understanding of auditory and vestibular function. To provide three-dimensional views of the structure of 20 stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia 21 on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. 22Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal 23 was not perfectly ordered. This sample-preparation and imaging procedure will allow for examination of 24 structural features of stereocilia in a near-native state. 25 2 Keywords 26 Hair cells; stereocilia; cryo-electron microscopy; actin 27

A cryo-tomography-based volumetric model of the actin core of mouse vestibular hair cell stereocilia lacking plastin 1

“…As we noted in our preliminary study (Metlagel et al, 2019), the actin-actin spacing measured here (∼13 nm) is considerably larger than the ∼8 nm we measured previously for Pls1 -/- mutants (Krey et al, 2016). One of the major advantages of the electron cryo-tomography approach we used here is that we prepare samples by rapid freezing, with no fixation, dehydration, and staining steps that could distort stereocilia dimensions.…”

“…Our quantitative results presented here stem from one tomogram each of the shaft and taper region and the conclusions derived from these data were confirmed by the other two tomograms. All Pls1 -/- stereocilia reconstructed and examined displayed a higher degree of order for the actin core when compared to wild-type samples (Krey et al, 2016, Metlagel et al, 2019), with Fourier analysis yielding an estimated 4.3 nm resolution for the Pls1 -/- density maps examined. This limited resolution prevented us from docking atomic models into the density maps, but allowed us to build simplified volumetric ball-and-stick models into the density map, and to determine filament and cross-link numbers, dimensions, curvature, distances, and spacings.…”

“…We focused here on the 3D structure of Pls1 -/- mutant mouse utricle stereocilia; a preliminary comparison of wild-type and knockout tomographic data sets revealed the higher order of the actin core in Pls1 -/- knockout stereocilia (Metlagel et al, 2019), which improved our model of the 3D organization of the actin core. The resolution in our tomograms was limited to 4.3 nm, however, which did not allow us to directly reveal the molecular identity of features visible in the density maps.…”

“…Blotting of stereocilia onto microscope grids, vitrification, cryo-tomographic data collection and 3D reconstruction have all been described in detail previously (Metlagel et al, 2019). In short, the sensory epithelium was blotted onto the lacey carbon support film of an EM grid, transferring intact stereocilia to the grid.…”

“…Electron cryo-tomography (cryo-ET) has emerged as a powerful method for examining macromolecular structures in cells in general (Baker et al, 2017, Oikonomou and Jensen, 2017, Hutchings and Zanetti, 2018) and the cytoskeleton in particular (Jasnin et al, 2013, Turgay et al, 2017, McIntosh et al, 2018, Sun et al, 2018). We have previously reported cryo-ET studies at ∼3-4 nm resolution of frozen-hydrated individual stereocilia, which were isolated by blotting them away from the sensory epithelia onto a poly-lysine-coated grid (Metlagel et al, 2019).…”