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首页> 外文期刊>Nature >Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia
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Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

机译：多同位素成像质谱分析显示毛细胞立体纤毛中的蛋白质更新缓慢

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摘要

Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles1. 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 studied2 by transfecting neonatal rat hair cells in culture with a P-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 lengths3. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells4'5. In contrast, turnover in chick stereocilia in vivo is much slower6. 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 a ~(15)N-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 vivo and in 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 P-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.
机译：内耳的毛细胞在动物的生命中通常不会被替换，并且必须不断更新其各种细胞器的组成部分1。其中的是纤毛纤毛，每条纤毛的芯由数百根肌动蛋白丝构成，这些肌动蛋白丝从其顶表面伸出，并在其尖端处带有机械转导装置。以前已经通过用P-肌动蛋白-GFP融合体转染培养物中的新生大鼠毛细胞来研究立体睫毛肌的肌动蛋白更新2，并且发现证据表明肌动蛋白在2-3天内由上至下被替换。肌动蛋白结合蛋白espin的过度表达会导致12-24小时内的纤毛延长，这也提示了纤毛长度的快速调节3。同样，在鸡和哺乳动物的毛细胞中裂解后的5到10小时内，机械感官的“尖端连接”也就被替换了4'5。相比之下，雏鸡体内的立体睫毛的周转要慢得多。可能是只有纤毛纤毛的某些成分才能快速翻转，这种快速更新仅在新生动物中，仅在培养物中发生，或者仅在诸如破损或肌动蛋白过表达的挑战时才发生。在这里，我们通过给动物饲喂〜（15）N标记的前体氨基酸并使用多同位素成像质谱法来测量新蛋白质的出现来定量蛋白质更新。出人意料的是，在成年蛙和小鼠以及新生小鼠体内和体外，立体纤毛都非常稳定，每天以<10％的速度掺入新合成的蛋白质。只有立体纤毛尖端具有快速的翻转并且没有观察到跑步运动。其他方法也证实了这一点：在表达P-actin-GFP的毛细胞中，我们将基准线在发束上漂白，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，除了尖端，肌动蛋白同工型都没有离开纤毛。因此，立体纤毛的快速周转仅在尖端发生，而不是通过跑步机过程发生。

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  • 来源
    《Nature》 |2012年第7382期|p.520-524|共5页
  • 作者

    Duan-Sun Zhang; Valeria Piazza; Benjamin J. Perrin; Agnieszka K. Rzadzinska; J. Collin Poczatek; Mei Wang; Haydn M. Prosser; James M. Ervasti; David P. Corey; Claude P. Lechene;

  • 作者单位

    Department of Neurobiology, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.;

    Department of Neurobiology, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.;

    Department of Biochemistry, Molecular Biology and Biophysics,Universityof Minnesota, Minneapolis, Minnesota 55455, USA.;

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB101SA, UK.;

    National Resource for Imaging Mass Spectrometry, Division of Genetics, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts 02139, USA;

    National Resource for Imaging Mass Spectrometry, Division of Genetics, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts 02139, USA;

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB101SA, UK.;

    Department of Biochemistry, Molecular Biology and Biophysics,Universityof Minnesota, Minneapolis, Minnesota 55455, USA.;

    Department of Neurobiology, Harvard Medical School and Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.;

    National Resource for Imaging Mass Spectrometry, Division of Genetics, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts 02139, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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