Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

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

Research output: Contribution to journalArticle

120 Citations (Scopus)

Abstract

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 stereocilia in vivo is 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 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 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.

Original languageEnglish (US)
Pages (from-to)520-524
Number of pages5
JournalNature
Volume481
Issue number7382
DOIs
StatePublished - Jan 26 2012

Fingerprint

Stereocilia
Isotopes
Hair
Mass Spectrometry
Actins
Proteins
Newborn Animals
Microfilament Proteins
Inner Ear
Tamoxifen
Actin Cytoskeleton
Anura
Organelles
Genetic Recombination
Chickens
Protein Isoforms
Cell Culture Techniques

Cite this

Zhang, D. S., Piazza, V., Perrin, B. J., Rzadzinska, A. K., Poczatek, J. C., Wang, M., ... Lechene, C. P. (2012). Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature, 481(7382), 520-524. https://doi.org/10.1038/nature10745

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. / 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.

In: Nature, Vol. 481, No. 7382, 26.01.2012, p. 520-524.

Research output: Contribution to journalArticle

Zhang, DS, Piazza, V, Perrin, BJ, Rzadzinska, AK, Poczatek, JC, Wang, M, Prosser, HM, Ervasti, JM, Corey, DP & Lechene, CP 2012, 'Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia', Nature, vol. 481, no. 7382, pp. 520-524. https://doi.org/10.1038/nature10745
Zhang DS, Piazza V, Perrin BJ, Rzadzinska AK, Poczatek JC, Wang M et al. Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature. 2012 Jan 26;481(7382):520-524. https://doi.org/10.1038/nature10745
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. / Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. In: Nature. 2012 ; Vol. 481, No. 7382. pp. 520-524.
@article{0482af593a284b22b7119e64dbdd2bbb,
title = "Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia",
abstract = "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 stereocilia in vivo is 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 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 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.",
author = "Zhang, {Duan Sun} and Valeria Piazza and Perrin, {Benjamin J.} and Rzadzinska, {Agnieszka K.} and Poczatek, {J. Collin} and Mei Wang and Prosser, {Haydn M.} and Ervasti, {James M.} and Corey, {David P.} and Lechene, {Claude P.}",
year = "2012",
month = "1",
day = "26",
doi = "10.1038/nature10745",
language = "English (US)",
volume = "481",
pages = "520--524",
journal = "Nature",
issn = "0028-0836",
publisher = "Nature Publishing Group",
number = "7382",

}

TY - JOUR

T1 - Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

AU - Zhang, Duan Sun

AU - Piazza, Valeria

AU - Perrin, Benjamin J.

AU - Rzadzinska, Agnieszka K.

AU - Poczatek, J. Collin

AU - Wang, Mei

AU - Prosser, Haydn M.

AU - Ervasti, James M.

AU - Corey, David P.

AU - Lechene, Claude P.

PY - 2012/1/26

Y1 - 2012/1/26

N2 - 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 stereocilia in vivo is 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 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 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.

AB - 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 stereocilia in vivo is 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 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 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.

UR - http://www.scopus.com/inward/record.url?scp=84862776687&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862776687&partnerID=8YFLogxK

U2 - 10.1038/nature10745

DO - 10.1038/nature10745

M3 - Article

C2 - 22246323

AN - SCOPUS:84862776687

VL - 481

SP - 520

EP - 524

JO - Nature

JF - Nature

SN - 0028-0836

IS - 7382

ER -