Article Figures & Data
Figures
- Figure 1.
Hair-bundle structural domains and lipid composition. A, Lipid composition of chick utricle hair bundles. The inner light blue pie graph indicates distribution of phospholipids, sphingomyelin, and cholesterol (mol% indicated); “Other” includes ceramide, hexosylceramide, and phosphatidylglycerol. Dark blue pie graphs indicate acyl chain compositions for the indicated lipid species (for phospholipids, the sum of the two acyl chains (denoted by number of total C atoms in both fatty acids/number of total double bonds in both fatty acids). In addition to the indicated acyl chain, sphingolipids also contain a C18 sphingosine backbone. B, Species distributions for hair bundles and utricular epithelium. The “Mol%” in the lipid class panel is calculated as moles of indicated lipid divided by moles of all lipids analyzed; percentage sign in individual lipid panels refers to moles of indicated acyl chain species divided by total moles of that lipid class. Values are given as mean ± SEM (n = 6 for each).
- Figure 2.
Membrane domains in bullfrog hair bundles. A, Little CTB labeling without neuraminidase treatment. B, Pretreatment with neuraminidase greatly enhances CTB labeling, particularly in basal taper region (delineated by arrows); hair-cell apical surface labeling was also enhanced. CTB antibody used for detection in A and B. C, PI(4,5)P2 antibody labeling. In this and other figures, PI(4,5)P2 is abbreviated PIP2. D, Filipin labeling. A–D show isolated bullfrog hair cells; panel widths are all 12.5 μm. E, Approximate positions of regions used for profile averaging. F, Profile averages for CTB (n = 7), actin (n = 7), and PI(4,5)P2 (n = 6). Colored lines indicate means, and the gray shading shows ±SEM. Profiles of individual cells were aligned to the dip in actin staining at the taper; this point was defined to be zero on the abscissa. G, Profile averages for filipin-stained cells (n = 11). Profiles were aligned to the peak of actin staining, which is near stereocilia tips; this point was defined to be zero on the abscissa.
- Figure 3.
Ganglioside domain controls. A, Taper domain is visible with antibody against GM1 ganglioside (after neuraminidase treatment). As seen with some cells labeled with CTB (e.g., D, E), the lower half of the kinociliary bulb was labeled. B, After neuraminidase treatment, the taper ganglioside domain is visible in live hair cells treated with rhodamine-phosphatidylethanolamine (Rh-PE) to label membranes and Alexa 488-CTB (488-CTB) to label gangliosides. C, Additional treatment of cell in B with anti-cholera toxin antibody does not change the 488-CTB pattern appreciably. D, Control hair cell labeled with CTB after neuraminidase treatment. CTB antibody used for detection. E, Hair cell treated with 10 mm MβC for 40 min to extract cholesterol. The taper ganglioside domain is not disrupted. As seen occasionally, the lower halves of the kinociliary bulbs of the cells in D and E were also labeled with CTB.
- Figure 4.
Hair-bundle membrane proteins and PI(4,5)P2 pathway. In all panels, proteins indicated by blue-colored short names were detected in purified hair bundles by mass spectrometry. A, Chick hair-bundle membrane proteins. Proteins detected by liquid chromatography (LC)-MS/MS were quantified using intensity-weighted spectral counting. The size of each slice is proportional to molar abundance. Red, Proteins enriched in bundles over epithelium more than fivefold; green, less than fivefold. Only proteins enriched 0.1-fold or greater are plotted. B, Peptide coverage of PI(4,5)P2-transport and metabolizing proteins in LC-MS/MS of chick hair bundles. Peptides detected by X! Tandem analysis of the complete dataset of chick bundles were plotted against residue number. Height of plotted bars corresponds to the number of identical peptides detected; width corresponds to peptide length, plotted at the position in sequence. Gray shading indicates peptide log(e) (log of expectation) score, indicating statistical confidence in protein identification (key is shown in PITPNA panel). C, PI pathway in hair bundles. D, Ceramide, sphingolipid, and ganglioside pathways. Green indicates lipids analyzed by mass spectrometry (Fig. 1); glucosylceramide and galactosylceramide are analyzed together as hexosylceramide. Italicized enzyme short names correspond to proteins not detected in bundles. All sialyltransferase (SAT) reactions can be reversed by endogenous or exogenous neuraminidase. Polysialylated gangliosides that could be converted to GM1 ganglioside with neuraminidase treatment are indicated with gray shading. E, Bundle abundance (green) and bundle-to-epithelium enrichment (magenta) of PI(4,5)P2 and ganglioside metabolic proteins.
- Figure 5.
PMCA2 and PTPRQ segregate to distinct domains delineated by CTB labeling. A, PMCA2 localization in isolated cell labeled with F2a antibody. Note near absence of PMCA2 in basal taper region (arrows), which appears red in the color merge panel. No neuraminidase, CTB, or CTB antibody were used. B, PMCA2 absence from basal taper region is also clear in whole-mount tissue (arrows). C, Reciprocal PMCA2 and CTB labeling. D, PTPRQ and CTB labeling overlap. CTB antibody was used for detection in C and D. Panel widths: A, C, D, 12.5 μm; B, 65 μm.
- Figure 6.
Radixin is activated at the PI(4,5)P2–PTPRQ boundary. A, Radixin and PTPRQ are located in similar, but not entirely coextensive, patterns at the base of the hair bundle. B, Antibody for activated ERM proteins (including pRDX) only labels above the stereocilia tapers. Note pRDX punctae throughout bundle. Arrows indicate the pRDX-free taper region. C, pRDX labeling is shifted toward stereocilia tips from CTB labeling. Arrows indicate the pRDX-free taper region. D, The MYO7A band is more basal than the pRDX band. Arrows indicate the basal MYO7A band. E–G, Intensity profiles for RDX/pRDX (n = 5; E), RDX/PTPRQ (n = 5; F), and CTB/pRDX (n = 5; G) averaged from individual cells aligned at the apical-surface actin dip. Panel widths: A–C, 12.5 μm.
- Figure 7.
Radixin activation depends on polyphosphatidylinositols. A, B, RDX and pRDX (detected with anti-pERM antibody) immunolabeling without (A) and with (B) 30 μm PAO for 1 h. pRDX and RDX both decline after PAO treatment. C–F, PMCA2 and PTPRQ distribution and intensity are not affected by PAO treatment. G, H, PI(4,5)P2 immunolabeling with (G) and without (H) 30 μm PAO (1 h). PAO completely abolishes PI(4,5)P2 immunoreactivity. I, Mean ± SEM for whole-bundle pixel intensity; regions of interest used for measurement included the entire bundle. Significance: **p < 0.01; ***p < 0.001. Panel widths: A–H, 12.5 μm.
- Figure 8.
Membrane domains in hair bundles. A, Relationship between radixin, phosphoradixin, and PI(4,5)P2 profiles; data are from different sets of cells, aligned at the apical surface (small peak in each profile). The black curve is obtained by multiplying the square of the relative radixin concentration by the square of the relative PI(4,5)P2 concentration and then normalizing to a peak of 1.0. The approximate stereocilia profile is indicated by diagram. B, Scanning electron micrograph of bullfrog hair bundle pseudocolored to indicate structural and functional domains. Purple, Taper domain; green, shaft/tip domain; yellow, kinocilium domain. Inset, Transmission electron micrograph of taper region.
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