Human Molecular Genetics Advance Access originally published online on May 28, 2009
Human Molecular Genetics 2009 18(16):3075-3089; doi:10.1093/hmg/ddp249
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In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment
1 Unidad de Genética Molecular, Hospital Ramón y Cajal, 28034 Madrid, Spain 2 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain 3 Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA 4 School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
* To whom correspondence should be addressed at: Unidad de Genética Molecular, Hospital Ramón y Cajal, Carretera de Colmenar Km 9, 28034 Madrid, Spain. Tel: +34 913368541; Fax: +34 913369016; Email: mmoreno.hrc{at}salud.madrid.org or mopelayo{at}hotmail.com
Received March 18, 2009; Accepted May 21, 2009
Here we report the functional assessment of two novel deafness-associated 
-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 -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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.