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

Abstract

Electron cryo-tomography allows for high-resolution imaging of stereocilia in their native state. Because their actin filaments have a higher degree of order, we imaged stereocilia from mice lacking the actin crosslinker plastin 1 (PLS1). We found that while stereocilia actin filaments run 13 nm apart in parallel for long distances, there were gaps of significant size that were stochastically distributed throughout the actin core. Actin crosslinkers were distributed through the stereocilium, but did not occupy all possible binding sites. At stereocilia tips, protein density extended beyond actin filaments, especially on the side of the tip where a tip link should anchor. Along the shaft, repeating density was observed that corresponds to actin-to-membrane connectors. In the taper region, most actin filaments terminated near the plasma membrane. The remaining filaments twisted together to make a tighter bundle than was present in the shaft region; the spacing between them decreased from 13 nm to 9 nm. Our models illustrate detailed features of distinct structural domains that are present within the stereocilium.

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

“…More recently, cryo-electron microscopy imaging has been used to image stereocilia in their native state, avoiding these artifacts (Metlagel et al, 2019;Song et al, 2020). However, due to rapid damage of the specimen by high-energy electrons, the contrast of this imaging modality is low, making it challenging to visually identify relatively small features, such as the tip link.…”

“…Therefore, labeling approaches are required to unambiguously identify components of the MT machinery. In this study, we combine a stereocilia preparation technique for cryo-EM imaging (Metlagel et al, 2019;Song et al, 2020) with a highly specific, immune-affinity labeling approach using anti-PCDH15 antibody-coupled gold nanoparticles (AuNP) (Azubel et al, 2019) to elucidate the molecular structures and conformational states of tip links under near native conditions.…”

Allosteric Regulation of Human Plastins

“…Specifically, the loop 184-189 and the first five residues of the helix 190-206 of CH1 are sufficiently close to make contacts at both sides (subdomains 1 and 3) of the hydrophobic cleft of the "i" actin subunit but also with the 44-48 segment of the D-loop (subdomain 2) of the longitudinally adjacent subunit "i+2". Also, in subdomain 1 of the "i+2" subunit, the tip of [79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94][95] helix is in proximity to residues Gln118, Asn213, and His225 of plastin's CH1. With the exception of Asn213, all these PLS3 residues are different from those determined in a previous model based on a ~ 30-Å resolution reconstruction of ABD1-decorated actin filaments 65 .…”

Correction of Missing-Wedge Artifacts in Filamentous Tomograms by Template-Based Constrained Deconvolution

“…The success of this approach, however, was partial, since the missing wedge artifacts made it necessary to restrict the z coordinate in order to obtain reasonable traces. Given the more complex organization of the taper region [9], we were concerned that this approach may not work in the taper region of the bundle.…”