Simplified Volumetric Models as an Effective Strategy for Segmenting Actin Networks in Cryo-Electron Tomograms.

Efficient methods for the extraction of features of interest remain one of the biggest challenges for the interpretation of cryo-electron tomograms. Various automated approaches have been proposed, many of which work well for high-contrast datasets where the features of interest can be easily detected and are clearly separated from one another. Our inner ear stereocilia cryo-electron tomographic datasets are characterized by a dense array of hexagonally packed actin filaments that are frequently cross-connected.

Pericytes control vascular stability and auditory spiral ganglion neuron survival.

The inner ear has a rich population of pericytes, a multi-functional mural cell essential for sensory hair cell heath and normal hearing. However, the mechanics of how pericytes contribute to the homeostasis of the auditory vascular-neuronal complex in the spiral ganglion are not yet known. In this study, using an inducible and conditional pericyte depletion mouse (PDGFRB-CreER; ROSA26iDTR) model, we demonstrate, for the first time, that pericyte depletion causes loss of vascular volume and spiral ganglion neurons (SGNs) and adversely affects hearing sensitivity.

VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.

Millions of people are affected by hearing loss. Hearing loss is frequently caused by noise or aging and often associated with loss of pericytes. Pericytes populate the small vessels in the adult cochlea.

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

Cryo-electron tomography maps often exhibit considerable noise and anisotropic resolution, due to the low-dose requirements and the missing wedge in Fourier space. These spurious features are visually unappealing and, more importantly, prevent an automated segmentation of geometric shapes, requiring a subjective and labor-intensive manual tracing. We developed a novel computational strategy for objectively denoising and correcting missing-wedge artifacts in homogeneous specimen areas of tomograms, where it is assumed that a template repeats itself across the volume under consideration, as happens in the case of filaments.

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

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.

3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.

Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall.

Electron cryo-tomography of vestibular hair-cell stereocilia.

High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered.

Regulation of Oxygenic Photosynthesis during Trophic Transitions in the Green Alga .

Light and nutrients are critical regulators of photosynthesis and metabolism in plants and algae. Many algae have the metabolic flexibility to grow photoautotrophically, heterotrophically, or mixotrophically. Here, we describe reversible Glc-dependent repression/activation of oxygenic photosynthesis in the unicellular green alga We observed rapid and reversible changes in photosynthesis, in the photosynthetic apparatus, in thylakoid ultrastructure, and in energy stores including lipids and starch.

A Pattern Recognition Tool for Medium-resolution Cryo-EM Density Maps and Low-resolution Cryo-ET Density maps.

Cryo-electron microscopy (Cryo-EM) and cryo-electron tomography (cryo-ET) produce 3-D density maps of biological molecules at a range of resolution levels. Pattern recognition tools are important in distinguishing biological components from volumetric maps with the available resolutions. One of the most distinct characters in density maps at medium (5-10 Å) resolution is the visibility of protein secondary structures.

Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia.

Cryo-electron tomography (cryo-ET) is a powerful method of visualizing the three-dimensional organization of supramolecular complexes, such as the cytoskeleton, in their native cell and tissue contexts. Due to its minimal electron dose and reconstruction artifacts arising from the missing wedge during data collection, cryo-ET typically results in noisy density maps that display anisotropic XY versus Z resolution. Molecular crowding further exacerbates the challenge of automatically detecting supramolecular complexes, such as the actin bundle in hair cell stereocilia.