In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment
In vivo and in vitro effects of two novel gamma-actin (ACTG1) mutations that cause DFNA20/26 hearing impairment
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DOI：
10.1093/hmg/ddp249
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发表时间：
2009-08-15
影响因子：
3.5
通讯作者：
Angel Moreno-Pelayo, Miguel
中科院分区：
文献类型：
--
作者：
Morin, Matias;Bryan, Keith E.;Angel Moreno-Pelayo, Miguel
在此，我们通过结合酵母和哺乳动物细胞中的生化和细胞生物学分析，报告了两种新型耳聋相关伽马肌动蛋白突变体 K118N 和 E241K 在生物复杂性不断增加的一系列不同情况下的功能评估。我们的体内实验表明，虽然 K118N 对酵母行为的影响非常轻微，但 E241K 突变引起的表型非常严重，其特征是以甘油作为碳源生长的能力高度受损、异常的多液泡模式以及粗 F-肌动蛋白束在细胞中随机沉积。后一个特征与 E241K 突变体在体外形成束的高度不寻常的自发倾向一致，尽管这种成束倾向被原肌球蛋白中和，并且 E241K 丝束在丝切蛋白存在下对切断高度敏感。在瞬时转染的 NIH3T3 细胞中，两种突变肌动蛋白通常都整合到细胞骨架结构中，尽管也观察到细胞质聚集体，表明体内突变引起的异常因素。有趣的是，基因枪介导的这些突变体在耳蜗毛细胞中的表达不会导致细胞骨架结构或静纤毛形态的总体改变。我们的结果提供了与耳聋相关的 γ-肌动蛋白突变体的生物学后果的更完整的图景，并支持这样的假设：DFNA20/26 听力损失的语后和进行性性质是毛细胞细胞骨架随着时间的推移逐渐恶化的结果。
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.