Myosin-dependent short actin filaments contribute to peripheral widening in developing stereocilia.

Stereocilia, the actin-based mechanosensory protrusions of inner ear sensory hair cells, require precise dimensional control for proper mechanotransduction, yet the mechanisms governing actin assembly during development remain unclear. Their size and shape are determined by a stable core of long, parallel, unbranched filamentous (F-) actin. We find that during stereocilia widening, which is a key process for function and stability, newly expressed actin first integrates at the tip, then along the periphery of the core.

Select autosomal dominant DFNA11 deafness variants activate Myo7A targeting in epithelial cells.

Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Variants in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements that control Myo7A within cells are not well understood.

The complete absence of cytoplasmic γ-actin results in no discernible phenotype in mice or primary fibroblasts.

Mice and primary fibroblasts derived from mouse embryos completely lacking cytoplasmic β-actin, because the Actb gene was engineered to instead express γ-actin protein, have previously been found to be virtually devoid of phenotype. Here, we report the characterization of mice and mouse embryonic fibroblasts homozygous for an Actg1 allele edited to translate β-actin instead of γ-actin (Actg1-coding beta; Actg1), which resulted in mice and fibroblasts that are devoid of γ-actin. We demonstrate that these Actg1 mice present with no measurable phenotype in survival, body mass, activity, muscle contractility, or auditory function.

F-actin in the cuticular plate and junctions of auditory hair cells is regulated by ADF and cofilin to allow for normal stereocilia bundle patterning and maintenance.

Auditory hair cells, which convert sound-induced vibrations in the inner ear into neural signals, depend on multiple actin populations for normal function. Stereocilia are mechanosensory protrusions formed around a core of linear, crosslinked F-actin. They are anchored in the cuticular plate, which predominantly consists of randomly oriented actin filaments.

The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies.

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial.

Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening).

Nucleotide- and Protein-Dependent Functions of .

Cytoplasmic β- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences  and , respectively, are more divergent but still 89% similar.  mice are embryonic lethal and  cells fail to proliferate, but editing the  gene to express γ-actin () resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for .

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin).

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin.

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips.

Cell Biology: Function Guides Form of Auditory Sensory Cells.

Mechanosensory bundles on auditory sensory cells are composed of stereocilia that grow in rows of decreasing height. This pattern depends on the specification of the eventual tallest row, then the assignment of distinct molecular identities to the shorter rows. Mechanotransduction refines and maintains row identity, thus instructing the form of the bundle.

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2.

GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing.

Mutations in human GRXCR2, which encodes a protein of undetermined function, cause hearing loss by unknown mechanisms. We found that mouse GRXCR2 localizes to the base of the stereocilia, which are actin-based mechanosensing organelles in cochlear hair cells that convert sound-induced vibrations into electrical signals. The stereocilia base also contains taperin, another protein of unknown function required for human hearing.

PNPase knockout results in mtDNA loss and an altered metabolic gene expression program.

Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Interestingly, PKO established in mouse embryonic fibroblasts (MEFs) resulted in the loss of mitochondrial DNA (mtDNA).

Essential nucleotide- and protein-dependent functions of /β-actin.

The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between and null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout.

The stable actin core of mechanosensory stereocilia features continuous turnover of actin cross-linkers.

Stereocilia are mechanosensitive protrusions on the surfaces of sensory hair cells in the inner ear that detect sound, gravity, and head movement. Their cores are composed of parallel actin filaments that are cross-linked and stabilized by several actin-binding proteins, including fascin-2, plastin-1, espin, and XIRP2. The actin filaments are the most stable known, with actin turnover primarily occurring at the stereocilia tips.

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms.

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic β - or γ -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific β - and γ -actin KO mice. We found β - and γ -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics.

Relative importance of β- and γ-actin in primary mouse embryonic fibroblasts.

The highly homologous β (β) and γ (γ) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine β- and γ-isoform--specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle α-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary β- or β/γ-actin double-knockout fibroblasts.

Stereocilia morphogenesis and maintenance through regulation of actin stability.

Stereocilia are actin-based protrusions on auditory and vestibular sensory cells that are required for hearing and balance. They convert physical force from sound, head movement or gravity into an electrical signal, a process that is called mechanoelectrical transduction. This function depends on the ability of sensory cells to grow stereocilia of defined lengths.

High Frequency Hearing Loss and Hyperactivity in DUX4 Transgenic Mice.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by mutations leading to ectopic expression of the transcription factor DUX4, and encompasses both muscle-related and non-muscle phenotypes. Mouse models bearing this gene represent valuable tools to investigate which pathologies are due to DUX4 expression, and how DUX4 leads to these pathologies. The iDUX4(2.

Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament-severing proteins.

Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of crosslinked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants.

β-Actin and fascin-2 cooperate to maintain stereocilia length.

Stereocilia are actin-based protrusions on auditory sensory hair cells that are deflected by sound waves to initiate the conversion of mechanical energy to neuronal signals. Stereocilia maintenance is essential because auditory hair cells are not renewed in mammals. This process requires both β-actin and γ-actin as knock-out mice lacking either isoform develop distinct stereocilia pathology during aging.