In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment

Abstract

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Plant Vegetative and Animal Cytoplasmic Actins Share Functional Competence for Spatial Development with Protists

“…Moreover, because the human cytoplasmic actins are functional in Arabidopsis, missense mutations in these human actins that are associated with a large number of diseases, including blindness, deafness, myopathies, immune deficiency, and aberrant kidney function, could be easily tested in this plant model. For example, the influence of dominant missense mutations in b-actin on the structure of lamellapodia in lymphoblasts (Procaccio et al, 2006) and in g-actin on the maintenance of stereocilia in the ear (Morín et al, 2009) might be studied in Arabidopsis root hairs. By contrast, examining actin functions using transgenic mouse models would be orders of magnitude more expensive and time consuming.…”

Importance of a Lys113–Glu195 Intermonomer Ionic Bond in F-actin Stabilization and Regulation by Yeast Formins Bni1p and Bnr1p

“…12). Two mutations at residue 118 in ␥-nonmuscle actin lead to early onset deafness (38,39). Mutations in ␣-smooth muscle actin at positions 115, 116, and 256 lead to the development of aortic aneurysms (40).…”

Insights into the effects of disease‐causing mutations in human actins

“…We also obtained a range of vacuolar phenotypes including hypovesiculation, hypervesiculation and in some cases a mixture of both in an allele-specific fashion. These studies were also extended to mammalian cultured cells that had been transfected with constructs that produced either GFP-labeled WT or mutant b-cytoplasmic actins [Morin et al, 2009].…”

“…Again, a general feature of these studies is that the mutations produced a spectrum of effects, generally in a dominant negative fashion, but the severity of the effects and the morphology of the irregularities produced did not necessarily correlate with the severity of associated human disease Morin et al, 2009;.…”

“…We also obtained a range of vacuolar phenotypes including hypovesiculation, hypervesiculation and in some cases a mixture of both in an allele-specific fashion. These studies were also extended to mammalian cultured cells that had been transfected with constructs that produced either GFP-labeled WT or mutant b-cytoplasmic actins [Morin et al, 2009].In some of these cases, the mutation led to abnormal aggregation or bundling that seemed to match the bundling tendencies of the yeast mutants in vitro. However, in none of these cases did the exogenous mutant actin appear to produce substantial effects on overall cell organization or morphology.…”