Correlation of Actin Crosslinker and Capper Expression Levels with Stereocilia Growth Phases

Abstract

During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development. During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development. The vertebrate hair bundle, an excellent exemplar of intrinsic control of cellular architecture, constitutes 30–300 actin-filled stereocilia arranged in rows of increasing length but constant diameter. The elegant descriptive studies of the development of bundles from the chick cochlea (basilar papilla) from Lew Tilney and his collaborators established this preparation as ideal for studying stereocilia morphogenesis and bundle assembly (1Tilney L.G. Tilney M.S. DeRosier D.J. Actin filaments, stereocilia, and hair cells: how cells count and measure.Ann. Rev. Cell Biol. 1992; 8: 257-274Crossref PubMed Scopus (237) Google Scholar). They demonstrated that hair cells regulate stereocilia length, width, and number by segregating key steps into distinct phases, which allows the bundle to build itself sequentially; differential regulation and expression of key proteins accordingly must occur during these phases. Because these phases appear to overlap in time during mammalian hair bundle development (2Kaltenbach J.A. Falzarano P.R. Simpson T.H. Postnatal development of the hamster cochlea. II. Growth and differentiation of stereocilia bundles.J. Comp. Neurol. 1994; 350: 187-198Crossref PubMed Scopus (89) Google Scholar) but are distinct in chick, the chick cochlea is a particularly useful preparation for understanding bundle assembly. Actin filaments in developing chick cochlear stereocilia elongate in three phases (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar). In phase I, stereocilia grow to a maximum of 1.5–2.0 μm; their actin filaments are initially disordered but crosslinks visible by electron microscopy progressively form and organize filaments into tight parallel bundles (paracrystals). In phase II, elongation is minimal but new actin filaments are added laterally to the stereocilum core, increasing the number of filaments more than 4-fold in apical hair cells. Finally, in phase III, elongation resumes and stereocilia grow to their mature lengths (Figs. 1A, 1B). The discrete nature of these three phases suggests that regulation of elongation and filament number are critical for stereocilia formation. Timed expression of actin crosslinking proteins is thought to be essential for generating a uniformly packed actin paracrystal (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar, 4Stokes D.L. DeRosier D.J. Growth conditions control the size and order of actin bundles in vitro.Biophys. J. 1991; 59: 456-465Abstract Full Text PubMed Scopus Google Scholar). are with crosslinkers that appear at and an of D.J. Tilney L.G. Actin in the the of the PubMed Scopus Google Scholar). that bundle actin filaments, from the and are to this DeRosier of actin filaments by and for bundle assembly and Cell Biol. PubMed Scopus Google Scholar, of actin filament by Biol. Full Text Full Text PubMed Scopus Google Scholar, of the a of filaments and in Rev. PubMed Scopus Google Scholar). actin crosslinkers have in chick cochlea hair The that used protein protein form protein form growth mass mass mass with electron of the by in cell electron used protein protein form protein form growth mass mass mass with electron of the by in cell electron as and is present in mature and developing chick stereocilia to the that it is the in cochlear stereocilia M.S. Tilney L.G. of the stereocilia and cuticular of hair cells of the chick Cell Biol. PubMed Scopus Google Scholar). that critical for stereocilia growth Tilney L.G. The a in the the proteins of hair cell stereocilia and Full Text Full Text PubMed Scopus Google Scholar, of the proteins in the morphogenesis and of hair cell stereocilia in PubMed Scopus Google is present in chick cochlear stereocilia at of expression of the actin protein with bundle in the developing Comp. Neurol. PubMed Scopus Google Scholar). 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PubMed Scopus Google the for a hair cell is growth is by protein expression the of quantitative mass spectrometry the Tilney and DeRosier that of a actin bundle be by the expression of actin crosslinkers (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar). They that by increasing the of actin stereocilia actin be with growth by in the actin a of with a low D.L. DeRosier D.J. Growth conditions control the size and order of actin bundles in vitro.Biophys. J. 1991; 59: 456-465Abstract Full Text PubMed Scopus Google Scholar). In stereocilia, the is new filaments be readily added to the barbed-end elongation in the of levels of crosslinking proteins (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar). are with FSCN2 the actin and be by hair cells to (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar). FSCN2 expression progressively from E7 to E21, when it is the abundant in cochlear bundles Although at low protein levels at at relative to FSCN2 to stereocilia at that the of FSCN2 in the cochlea with the length of actin filaments in stereocilia at a FSCN2 levels during phase when new actin filaments are added to the stereocilia actin during this the mass of stereocilia actin filaments (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar). FSCN2 levels during phase during this the actin filament more than in stereocilia (3Tilney L.G. DeRosier D.J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate.Dev. Biol. 1986; 116: 119-129Crossref PubMed Scopus (78) Google Scholar). in a of FSCN2 present in hair cell hair cells FSCN2 located in stereocilia. The of this from to stereocilia cochlear FSCN2 of hair cells in stereocilia by hair cells FSCN2 in their at quantitation of FSCN2 in isolated hair bundles suggests that it is present at levels for actin the of FSCN2 in development have the that it from crosslinking actin and their in cell and PubMed Scopus Google Scholar). 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