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. 2013 Mar;16(3):365-74.
doi: 10.1038/nn.3312. Epub 2013 Jan 20.

Molecular Architecture of the Chick Vestibular Hair Bundle

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Free PMC article

Molecular Architecture of the Chick Vestibular Hair Bundle

Jung-Bum Shin et al. Nat Neurosci. .
Free PMC article

Abstract

Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Figures

Figure 1
Figure 1
Quantitative analysis of chick hair-bundle proteins. (a) Top, proteins identified in bundles and epithelium (2 or more experiments). Bottom left, representation of bundle proteins as bundle-enriched, unenriched, and epithelium-enriched (by protein frequency). Bottom right, same as middle except the molar fractions of proteins in each class were summed. (b) Calibration curve relating mole fraction of human protein standards spiked into Escherichia coli extract to riBAQ value. The number of identified proteins is indicated for each data point (mean ± s.d.). The points corresponding to mole fractions of 10−2, 10−3, and 10−4 were fit with a line constrained through the 0,0 point (y = 1.02×; R = 0.999). (c) Calibration curve relating mole fraction determined from riBAQ values to mole fraction measured using quantitative immunoblots with purified proteins as standards; data points are mean ± s.e.m. and are fit by y = 0.98× (R = 0.97). Data for CKB and GAPDH were from ref. . Dotted lines in a and b correspond to the unity line. (d) Abundance distribution of bundle and epithelium proteins. Single Gaussian fits. (e) Enrichment distribution of proteins detected in bundles and epithelium; single Gaussian fit. (f) Cumulative protein molar abundance from highest to lowest abundance proteins. The most abundant 8–9 proteins in bundles and epithelium are indicated. (g) Mole fractions of proteins in epithelium (left) and bundle (right); the slope of the line connecting them represents bundle-to-epithelium enrichment. Proteins most highly enriched in the epithelium are indicated at left, while bundle-enriched proteins are at right. Hue represents relative enrichment (power coefficient of fit connecting points) for each protein. Far right, proteins detected only in bundles.
Figure 2
Figure 2
Protein composition of chick hair bundles. (a) Hair bundle proteins ranked in order of abundance. Data-point color indicates protein class (key in panel b); symbol size represents bundle-to-epithelium enrichment. Red callouts indicate the most abundant actin-associated proteins; proteins significantly enriched over the contamination level are indicated by bold symbols. Blue and magenta callouts highlight proteins known to be in 1:1 stoichiometry. (b) Bundle proteins ranked in order of enrichment. Color indicates protein class, while symbol size indicates abundance. Proteins encoded by deafness genes are indicated; deafness proteins significantly enriched over the contamination level are indicated by bold symbols, those detected in 2 or fewer epithelium runs (hence not subject to statistical analysis) are indicated by italic symbols.
Figure 3
Figure 3
Electron tomography of chick stereocilia. (a–c) Tomogram from a stereocilium oriented longitudinally with respect to the plane of section. (a) Two-dimensional 0° tilt projection image recorded for tomographic reconstruction. (b) Single ~0.8 nm slice of unfiltered three-dimensional reconstruction. (c) Single ~0.8 nm slice of bilaterally filtered density map. Scale bars in a–k are 100 nm. (d–e) Stereocilia model from longitudinal tomogram. Red lines represent actin, blue lines represent actin-actin crosslinkers, orange lines represent actin-membrane connectors, and membrane density is depicted in light green. (d) Overview of stereocilia model overlaid on the surface-rendered density map (6.4 nm thick). (e) Overview of model alone and segmented membrane density. (f–h) Transverse stereocilia tomogram. (f) Two-dimensional 0° tilt projection of transverse image recorded for tomographic reconstruction. (g) Single 0.8 nm slice of unfiltered three-dimensional reconstruction. (h) Single 0.8 nm slice of bilaterally filtered density map of transverse orientation, allowing precise measurements of actin-actin distances. (i–k) Oblique stereocilia tomogram. (i) Two-dimensional 0° tilt projection of oblique image used for tomographic reconstruction (stereocilia longitudinal axis is at an angle of ~18° with respect to sectioning plane). (j) Single 0.8 nm slice of unfiltered three-dimensional reconstruction. (k) Single 0.8 nm slice of bilaterally filtered three-dimensional density map. (l–m) Scaled views of density and model in mid-shaft region; from longitudinal stereocilia orientation. (l) Density map only. (m) Model overlaid on the density map. (n) Model alone. Scale bars in l–q are 20 nm. (o–q) Close-up views of density map in a region adjacent to the membrane. (o) Density map only. (p) Model overlaid on the density map. (q) Model alone. (r) Histogram showing distribution of actin-actin distances at sites of cross-bridges. The data were fit with the sum of three Gaussians (red), with equal σ (width) for each. Individual fits for 8, 11, and 15 nm peaks are shown in gray. (s) Close-up view of model. Scale bar is 50 nm.
Figure 4
Figure 4
Interaction network for hair-bundle proteins. Symbols (nodes) represent bundle proteins or second messengers; only nodes with two or more interactions are plotted, with exceptions of OCM and CALB2. Underline labels indicate deafness proteins. Node colors indicate functional classification (same key as in Fig. 2b), while node symbol size represents protein abundance in bundles. Ca2+, PI(4,5)P2, and cAMP are indicated by diamond node symbols. Solid links (lines between nodes) represent interactions validated with literature citations; Supplementary Table 3 lists all interactions and evidence. Dotted links correspond to interactions involving paralogs of bundle proteins; dashed links represent hypothetical interactions (e.g., SLC9A3R2 interactions from Table 3). The layout of the plot is controlled by the density of links between nearby nodes. The distribution of nodes and links in the plot is fit well by a power law, which indicates that the plot contains a few highly connected nodes (hubs) and many other less-connected nodes. Supplementary Fig. 5 reproduces this figure with each link hyperlinked to a PubMed reference supporting the interaction.
Figure 5
Figure 5
Identification of RDX and SLC9A3R2 in hair bundles. (a) Protein immunoblotting with purified hair bundles. Lanes from left to right for each blot: Hair bundles, bundles from 40 chick ears (~0.6 µg total protein); Agarose, agarose equivalent to that in the bundles sample; Epithelium, utricle sensory epithelium from 4 chick ears (~10 µg protein). Antibodies used are indicated at left. Both anti-RDX and anti-pERM (which recognizes phosphorylated ezrin, radixin, and moesin) detected bands both at the expected size (~70 kD) and at ~80 kD (asterisk). (b) RDX-SLC9A3R2 interaction. Epitope-tagged chick WT RDX, T564D-RDX, and SLC9A3R2 were expressed in HEK cells in the indicated combinations. Tagged SLC9A3R2 was immunoprecipitated, and associated RDX was detected by immunoblotting. (c) RDX and pERM immunocytochemistry. RDX and pERM co-localize except in the taper region (double arrows in bottom panel). Inset, magnification of apical surfaces of supporting and hair cells. Note that pERM immunoreactivity is absent from bases of microvilli (MV), as in stereocilia (SC). Scale bar in panel c is 10 µm and applies to panels c-f. (d) RDX and SLC9A3R2 immunocytochemistry. RDX and SLC9A3R2 overlap throughout the bundle, but SLC9A3R2 is more concentrated towards stereociliary bases (double arrows in bottom panel), including the tapers, than is RDX. (e) MYO7A immunocytochemistry. MYO7A is concentrated towards stereociliary tips and in a band above the taper region (asterisks). (f) RHOA immunocytochemistry. Staining is seen in stereocilia (arrow) and the kinocilium or the tallest stereocilia of the bundle (arrowheads). RHOA is also substantially enriched in hair cells over supporting cells.

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