Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia.
Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia.
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DOI：
10.1038/nature10745
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发表时间：
2012-01-15
期刊：
影响因子：
64.8
通讯作者：
Lechene, Claude P.
中科院分区：
文献类型：
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作者：
Zhang, Duan-Sun;Piazza, Valeria;Perrin, Benjamin J.;Rzadzinska, Agnieszka K.;Poczatek, J. Collin;Wang, Mei;Prosser, Haydn M.;Ervasti, James M.;Corey, David P.;Lechene, Claude P.
内耳的毛细胞在动物的一生中通常不会更换，并且必须不断更新其各种细胞器的组成部分。其中包括静纤毛，每一个都有一个由数百根肌动蛋白丝组成的核心，这些肌动蛋白丝从它们的顶端表面产生，并在它们的顶端承载机械转导装置。此前曾通过用β-肌动蛋白-GFP融合物转染培养的新生大鼠毛细胞来研究静纤毛中的肌动蛋白更新，发现肌动蛋白在2-3天内从上而下被替换的证据。肌动蛋白结合蛋白espin的过度表达导致静纤毛在12-24小时内伸长，也表明静纤毛长度的快速调节。类似地，在鸡和哺乳动物的毛细胞中，机械感觉“尖端链接”在卵裂后5-10小时内被替换。相反，鸡体内静纤毛的周转要慢得多。这可能是因为静纤毛只有某些成分能快速转换，快速转换只发生在新生动物中，只发生在培养中，或者只发生在对断裂或肌动蛋白过度表达等挑战的反应中。在这里，我们定量蛋白质周转喂养动物与a15 N-标记的前体氨基酸，并使用多同位素成像质谱测量新蛋白质的外观。令人惊讶的是，在成年青蛙和小鼠以及新生小鼠中，在体内和体外，静纤毛非常稳定，每天以<10%的速度掺入新合成的蛋白质。只有静纤毛尖端有快速周转，没有观察到铣削。其他方法证实了这一点：在表达β-actin-GFP的毛细胞中，我们漂白了毛束上的基准线，但它们在6天内没有移动。当我们用他莫昔芬诱导的重组停止β-或γ-肌动蛋白的表达时，除了在尖端，两种肌动蛋白亚型都没有离开静纤毛。因此，静纤毛的快速周转只发生在尖端，而不是通过一个快速铣削过程。
Hair cells of the inner ear are not normally replaced during an animal’s life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a β-actin–GFP fusion, and evidence was found that actin is replaced, from the top down, in 2–3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12–24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory ‘tip links’ are replaced in 5–10 hours after cleavage in chicken and mammalian hair cells,. In contrast, turnover in chick stereociliain vivois much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a15N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice,in vivoandin vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing β-actin–GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of β- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.
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影响因子：
56.9
作者：
Lechene, Claude P.;Luyten, Yvette;Distel, Daniel L.
通讯作者：
Distel, Daniel L.
影响因子：
--
作者：
Lechene C;Hillion F;McMahon G;Benson D;Kleinfeld AM;Kampf JP;Distel D;Luyten Y;Bonventre J;Hentschel D;Park KM;Ito S;Schwartz M;Benichou G;Slodzian G
通讯作者：
Slodzian G
影响因子：
--
作者：
Rzadzinska, A;Schneider, M;Kachar, B
通讯作者：
Kachar, B
DOI：
10.1073/pnas.86.13.4973
发表时间：
1989-07-01
影响因子：
11.1
作者：
SHEPHERD, GMG;BARRES, BA;COREY, DP
通讯作者：
COREY, DP
影响因子：
16.2
作者：
Shin, Jung-Bum;Streijger, Femke;Gillespie, Peter G.
通讯作者：
Gillespie, Peter G.