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.

Figure 1. Incorporation of ¹⁵N into frog saccular epithelium

a, left, Mass-26 image for a hair cell (HC) and supporting cells (SC) at day 32. CP: cuticular plate, HB: hair bundle, OM: otolithic membrane. right, Image of mass27/mass26 ratio, revealing incorporation of ¹⁵N. Color scale represents 0-30% incorporation. b, top, Mass-26 image of the shortest stereocilium in a. bottom, Profile of incorporation along its length. c, Turnover for days 1, 2, 4, 8, 21 and 32. Mean ± SE; N was typically 8-10.

Figure 2. Incorporation of ¹⁵N into adult mouse hair cells

a, Mass-26 image of utricle; day 56. b, Mass 27/mass26 ratio: low incorporation in stereocilia. c, Projection of a 3D stack of a. d, Ratio image from c: high turnover at tips. e, Incorporation, days 1-56. SC-supporting cell; HC-hair cell; cutic-cuticular plate; cyto-cytoplasm. f, Cochlear inner hair cell; day 56; mass 26. g, Ratio image. h, 3D reconstruction from 2200 images. i, Incorporation along axis of stereocilia from h. Bar: plotted length. Line widths: 95% confidence. j, Incorporation after 8, 32, 56 or 150 days. Color scale 0-100%; scale bars in μm.

Figure 3. Tracking treadmilling by bleaching GFP-tagged β-actin

a, Neonatal utricular macula in culture, hair cells expressed β-actin:GFP. Bleached bundles indicated (*); scale in μm. b Projection of confocal planes before and after bleaching. c, 3D stack rotated to show oblique bleached line. d, Same bundle on days 3, 5, and 7. A shift in bundle orientation partially obscured the line. e, Rotation of the day-7 stack revealed the bleached line. f, Line positions for three bundles, as a proportion of bundle height. g, Average movement of bleached lines, as a proportion of bundle height. N=24; mean ± SE.

Figure 4. Loss of actin from tips following knockout of actin genes

Adult (three-week) cochlear inner hair cells, labeled with antibodies to β-actin (green) or γ-actin (red). a, Floxed β-actin mouse crossed to Cagg-CreER, without tamoxifen (control), or 1, 3 or 34 weeks after tamoxifen. Loss of β-actin occurred only at tips of stereocilia. In control mice, a few hair cells displayed loss of β-actin at tips without tamoxifen, suggesting a leaky promoter. b, Floxed γ-actin mouse 18 weeks after tamoxifen. Loss of γ-actin occurred only at tips of stereocilia.