Auditory hair cell function requires proper assembly and regulation of the nonmuscle gamma isoactin-rich cytoskeleton, and six point mutations in this isoactin cause a type of delayed onset autosomal dominant nonsyndromic progressive hearing loss, DFNA20/26. The molecular basis underlying this actin-dependent hearing loss is unknown. To address this problem, the mutations have been introduced into yeast actin, and their effects on actin function were assessed in vivo and in vitro. Because we previously showed that polymerization was unaffected in five of the six mutants, we have focused on proteins that regulate actin, in particular cofilin, which severs F-actin and sequesters actin monomers. The mutations do not affect the interaction of cofilin with G-actin. However, T89I and V370A mutant F-actins are much more susceptible to cofilin disassembly than WT filaments in vitro. Conversely, P332A filaments demonstrate enhanced resistance. Wild type actin solutions containing T89I, K118M, or P332A mutant actins at mole fractions similar to those found in the hair cell respond in vitro toward cofilin in a manner proportional to the level of the mutant present. Finally, depression of cofilin action in vivo by elimination of the cofilin-activating protein, Aip1p, rescues the inability to grow on glycerol caused by K118M, T278I, P332A, and V370A. These results suggest that a filament instability caused by these mutations can be balanced by decreasing a system in vivo that promotes increased filament turnover. Such mutant-dependent filament destabilization could easily result in hair cell malfunction leading to the late-onset hearing loss observed in these patients.Our ability to perceive sounds hinges on the proper functioning of a highly specialized group of cells, called hair cells, which are housed in the cochlea of the inner ear. Their actin cytoskeleton plays a vital role in the ability of these cells to transduce mechanical stimuli into electrical responses (1). The columnar auditory hair cell consists of three actin-rich components (2). First are the three graded rows of finger-like projections from the apical surface of the cell referred to as stereocilia. These protrusions, comprised predominantly of bundles of actin filaments, are surrounded by the cellular membrane (3, 4). Soundgenerated pressure waves in the cochlear fluid displace the basilar membrane, which in turn forces the stereocilia into the overlaying tectorial membrane, resulting in their mechanical deformation. Deflection of the stereocilia results in the opening of mechanoelectrical transduction ion channels residing on the stereocilia and ultimately triggers the initiation of a nerve impulse, which is relayed by the auditory nerve to the brain for interpretation (5).Normal development, functioning, and maintenance of hair cell structures depend on tight regulation of the hair cell cytoskeleton as it controls the height and width of the stereociliary bundles. Each stereocilium inserts into the second actinrich structure, the cuticular plate, a web-...