β-Actin and Fascin-2 Cooperate to Maintain Stereocilia Length

Abstract

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 knockout mice lacking either isoform develop distinct stereocilia pathology during aging. In addition, stereocilia integrity may hinge on immobilizing actin, which outside of a small region at stereocilia tips turns over with a very slow, months-long half-life. Here, we establish that β-actin and the actin crosslinking protein fascin-2 cooperate to maintain stereocilia length and auditory function. We observed that mice expressing mutant fascin-2 (p.R109H) or mice lacking β-actin share a common phenotype including progressive, high-frequency hearing loss together with shortening of a defined subset of stereocilia in the hair cell bundle. Fascin-2 binds β-actin and γ-actin filaments with similar affinity in vitro and fascin-2 does not depend on β-actin for localization in vivo. Nevertheless, double mutant mice lacking β-actin and expressing fascin-2 p.R109H have a more severe phenotype suggesting that each protein has a different function in a common stereocilia maintenance pathway. Since the fascin-2 p.R109H mutant binds but fails to efficiently crosslink actin filaments, we propose that fascin-2 crosslinks function to slow actin depolymerization at stereocilia tips to maintain stereocilia length.

Stereocilia morphogenesis and maintenance through regulation of actin stability

“…4C). The morphology of the mechanotransducing stereocilia is also particularly sensitive to perturbation of the actin cytoskeleton as loss or mutation of several cytoskeletal proteins, including the long isoform of myosin XVa, fascin-2, β-actin, Esp8L2, actin interacting protein 1 (AIP1) and ADF result in shortening of these stereocilia while not impacting the length of the non-mechanotransducing row [2,4,5,69,27]. …”

MyTH4-FERM myosins in the assembly and maintenance of actin-based protrusions

“…However a number of factors controlling elongation have been identified, including EPS8 [37,91] and espin-1 [139]. Conversely, the bundling proteins fascin-2 [140–142] and fimbrin [143] are dispensible for assembly but required for maintaining normal stereocilia width and height, and TRIOBP, taperin and Fam65b are required for bundling actin filaments specifically in the taper at the stereocilia base where the bundles terminate in the cuticular plate [144,145 • ]. Additionally, the actin capping protein twinfilin-2 [146] as well as gelsolin [147,148], an actin severing and capping protein, serve to restrict the length of stereocilia by inhibiting actin polymerization at the tips.…”

Creatine kinase B is necessary to limit myoblast fusion during myogenesis

“…To generate an NH 2-terminal FLAG-tagged Fascin-1 construct, a cDNA fragment encoding the mouse full-length Fscn1 together with FLAG was cloned into pENTR/D-TOPO (Invitrogen), recombined into pDEST8, and expressed in Sf9 cells as previously described (54). For all experiments, fusion protein expression was verified by immunoblotting.…”