Allele-specific Effects of Human Deafness γ-Actin Mutations (DFNA20/26) on the Actin/Cofilin Interaction

Abstract

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-...

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

“…Interestingly, Lys-113 is part of a helix, Lys-113-Thr-125, that contains two other TAAD mutations we have previously characterized, N115T and R116Q, in addition to Lys-118, the site of two deafness-causing mutations (14,45). If our hypothesis is correct, the mutations become part of a pathogenic module that, as a unit, can affect F-actin conformation.…”

Allele-specific Effects of Thoracic Aortic Aneurysm and Dissection α-Smooth Muscle Actin Mutations on Actin Function

“…Cofilin also sequesters monomers, preferably when bound to ADP, as is the case when the monomer dissociates from the filament (44 -46). We examined the susceptibility of the wild type and mutant actin to increasing amounts of yeast cofilin (20). Yeast cofilin tends to decorate rather than sever actin filaments at stoichiometric levels, resulting in an increase in light scattering as seen with wild type actin in Fig.…”

“…Accelerating voltage of the transmission electron microscope was 100 kV. Image J was used to process the images and measure the individual filament lengths for N115T, R116Q, and wild type actins (20).…”

“…All actins were stored at 4°C and used within 4 days of purification. Yeast cofilin was purified from Escherichia coli carrying a recombinant construct for the protein according to Lappalainen et al (20,28), and the concentration of the purified cofilin was determined by absorption at 280 nm with an extinction coefficient of 14,650 M Ϫ1 cm…”

“…We have observed resistance to cofilin previously in the deafness mutant P332A but to a lesser extent. At an actin/cofilin ratio of 1:2.5, approximately half of the original P332A actin filaments persisted (P332A at 55% versus N115T at 80%) (20).…”