Research ArticleDEVELOPMENTAL BIOLOGY

The release of surface-anchored α-tectorin, an apical extracellular matrix protein, mediates tectorial membrane organization

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaay6300
DOI: 10.1126/sciadv.aay6300
  • Fig. 1 α-Tectorin is widely expressed in TM-producing cells and tethered to the plasma membrane via a GPI anchorage.

    (A and B) Toluidine blue–stained radial sections of the developing mouse cochlea at postnatal day 2 (P2) (A) and P7 (B). The inner sulcus (IS) is formed between P2 and P7, when the TM becomes detached from the surface of columnar supporting cells. IHC, inner hair cell; OHC, outer hair cell; Co, columnar cells; Cu, cuboidal cells; SL, spiral limbus. Scale bar, 50 μm. (C) In situ hybridization of Tecta mRNA in the P2 mouse cochlea. Tecta is expressed in cochlear supporting cells including interdental cells (ID) of the spiral limbus, inner supporting cells of Kölliker’s organ (Ko) including columnar cells, and outer supporting cells including pillar cells (PC), Deiters’ cells (DC), and Hensen’s cells (Hs) but not in inner hair cell and outer hair cell. Scale bar, 50 μm. (D) Schematic of Myc-tagged TECTA structure (top) and cellular localization. Red bars indicate a potential cleavage site of proteolytic sheddases. A blue arrow indicates the cleavage site of bacterial phosphatidylinositol-phospholipase C (PI-PLC) and potential GPI-anchored lipases. N, N terminus; C, C terminus; ER, endoplasmic reticulum; PM, plasma membrane. (E) Myc-TECTA was expressed in human embryonic kidney (HEK) 293T cells, and its localization was determined by Western blots using an anti-Myc antibody. Treatment of TECTA-expressing cells with PI-PLC, which cleaves a GPI anchor, facilitates the release of TECTA into the media (top) and removes surface TECTA as determined by surface biotinylation assay (bottom). (F) Surface expression of TECTA is absent in PI-PLC–treated cells as shown by live cell surface staining of TECTA (green, anti-Myc antibody raised in rabbit), followed by total permeabilized staining (red, anti-Myc antibody raised in mouse). Scale bar, 20 μm.

  • Fig. 2 GPI anchorage of TECTA is required for the surface localization of TECTA and the organization of the TM.

    (A) Schematic of TECTAWT and TECTAS (top) and their cellular localization (bottom). TECTAS is generated by replacing the GPI anchorage site (ω) with a stop codon (X). (B and C) TECTAS is constitutively secreted into the media (B, top) and not retained on the plasma membrane as shown by surface biotinylation (B, bottom), and live cell surface staining (C). Scale bar, 20 μm (C). (D) A mouse line expressing TECTAS was generated by knocking a stop codon into the ω-site of the Tecta gene (fig. S1A). Radial sections of the mature cochlea (P28) stained with Toluidine blue show that the TM is severely disorganized and detached from the organ of Corti in TectaS/S mice. Arrows indicate the TM. Scale bars, 100 μm (D) and 50 μm (inset b). (E and F) Threshold sound pressure level (SPL) for auditory brainstem response (ABR) (E) and distortion product otoacoustic emission (DPOAE) (F) of TectaS/S mice are significantly elevated at all tested frequencies (8, 12, 16, 22, and 32 kHz) in comparison with Tecta+/+ mice at P28 (*P < 0.01, t test; means ± SEM).

  • Fig. 3 Ultrastructural analysis of the developing TM.

    (A to E) TEM of radial sections of the developing mouse cochlea (P2). (A) The wild-type TM grows on the apical surface of supporting cells and is composed of the top covernet layer (CN), the central body, and the bottom border layer. M ↔ L indicates medial-lateral axis of the cochlea. Scale bar, 20 μm. (B, inset b) The apical surface of polarized columnar cells displays densely arrayed microvilli, on which collagen fibril bundles (arrow) are attached. The collagen bundles project up from the epithelial surface and are directed medially in the radial plane. Fine filaments (arrowhead) are intermingled with collagen bundles. Scale bars, 1 μm. (C) Immuno-EM of wild-type cochlea with anti-TECTA antibody shows that TECTA is localized to the microvillus surface of supporting cells where collagen fibril bundles are associated (arrows), as well as in between parallel collagen fibrils distal to the apical surface (arrowhead). Scale bar, 200 nm. (D) The central body of the TM is composed of parallel collagen fibrils oriented along the radial plane (arrow) and intermingled thinner filaments (arrowhead). Scale bar, 1 μm. Inset: (D′) A longitudinal section of the TM shows a regular spacing of collagen fibrils. Scale bar, 0.5 μm. (E) Collagen fibrils are not associated with the apical surface of the inner supporting cells of TectaS/S mice. Scale bar, 1 μm. (F) The collagen fibrils in the luminal space of the scala media that are not associated with the apical surface of the epithelium of TectaS/S mice form wavy and disorganized collagen clusters. The orientation of collagen fibrils is disrupted as shown by the different length of cross-sectioned collagen fibrils (arrows). Scale bar, 1 μm. (G to J) SEM of the developing mouse cochlea (P2). (G) The major bundles of the covernet (arrows) are longitudinally oriented along the apical-basal (A↔B) axis of the cochlea of wild-type mice. GER, greater epithelial ridge. Scale bar, 20 μm. (H) The reticular branches of the covernet (arrow) lie atop parallel collagen fibrils within the central body of the wild-type TM, which are arranged along the radial axis with an apically directed slant (arrowhead). Scale bar, 1 μm. (I and J) The apical surface of the TectaS/S cochlea is smooth due to the lack of collagen fibril bundles associated with microvilli of inner supporting cells. Scale bars, (I) 10 μm and (J) 1 μm.

  • Fig. 4 Col II is not associated with the surface membrane but aggregated in the luminal space of the scala media in TectaS/S mice.

    (A and B) Radial sections of P2 Tecta+/+ and TectaS/S cochleae were stained with antibodies against TECTA (red) and Col II (cyan) as well as with PSA lectin (green) and Hoechst (blue) and then analyzed by high-resolution Airyscan fluorescence confocal microscopy. (A) The TM of wild-type mice exhibits a multilayered organization. TECTA, Col II, and PSA staining are detected both immediately adjacent to the apical cell surface of supporting cells (dotted line) and in upper TM layers. Scale bars, 20 μm (A) and 5 μm (insets a and b). (B) Col II does not accumulate on the apical surface of the TectaS/S cochlea. The disorganized collagen located in the luminal space of the scala media is weakly associated with TECTAS protein and lacks PSA staining. Scale bars, 20 μm (B) and 5 μm (insets a and b). (C and D) Western blots of the TectaS/S mouse cochlea (P0) with the littermate control. (C) TECTAWT (arrowheads) appears as a monomer of 260 kDa, as well as higher–molecular weight, oligomeric bands (left). TECTAS protein is present only as a monomer. The total amount of TECTA protein is reduced in the TectaS/S mouse cochlea. The treatment of PNGase F reveals that the high molecular bands of TectaWT are oligomers or hyperglycosylated monomers (right). (D) Steady-state levels of OTOG (arrowheads) protein in the TectaS/S cochlea (P0) are reduced, as compared to the wild-type control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 5 GPI-dependent release of TECTA is required for the growth of the TM.

    The TectaT allele, encoding a TECTAT, was generated by replacing sequences encoding the TECTA C-terminal GPI anchorage signal with those encoding the transmembrane domain of CD2 (fig. S5A). (A) Airyscan-processed images of the developing TectaT/T cochlea (P2) following TECTA (red), Col II (cyan), PSA (green), and Hoechst (blue) staining. TECTAT protein is expressed on the apical surface of cochlear supporting cells (inset a) (dotted line) and is not detected in the lumen of the cochlear duct (inset b). Coll II accumulates on the cell surface but does not form a multilayered architecture. Excessive Col II not associated with the surface forms an aggregate in the luminal space, which lacks TECTAT and PSA signal. Scale bars, 20 μm (A) and 5 μm (insets a and b). (B and C) TEM of the developing TectaT/T cochlea (P2). Microvilli of supporting cells are associated with electron-dense materials (B) (arrows). Collagen fibrils in the luminal space are disorganized and randomly oriented as shown by the different length of cross-sectioned fibrils (C) (arrows). Scale bars, 1 μm. (D and E) SEM of the apical surface of the developing cochlea of TectaT/T (D) and Tecta+/+ (E) mouse (P2). The TM is removed to reveal the cochlear epithelial cell surface of wild-type mice. Microvilli of TectaT/T mice are covered with electron-dense materials and horizontally orientated fibers (arrows), while those of Tecta+/+ mice show a bare surface except for the top, where ring-shaped electron-dense materials are observed. Scale bars, 1 μm. (F) Double immuno-EM of TECTA (6-nm gold particle; red arrows) and Col II (12 nm-gold particle; blue arrowheads) of the apical surface of polarized columnar cells of the TectaT/T cochlea (P2). Collagen fibrils that were not organized into bundles were associated with the microvillus surface. Scale bar, 200 nm. (G) Radial semithin sections of the mature Tecta+/+ cochlea (P28) stained with toluidine blue. Scale bar,100 μm. (H) (inset h) Radial semithin sections of the mature TectaT/T cochlea (P28) stained with Toluidine blue. In TectaT/T mice, the TM (arrow) is severely disorganized and aggregated in the scala media (arrow) and is not observed over the organ of Corti. Scale bars, 100 μm (H) and 50 μm (inset h).

  • Fig. 6 Proposed model for the morphogenesis of the TM, a layered ECM structure: 3D printing model.

    The TM is a multilayered structure: top covernet layer (pink), body layer (orange), and bottom layer (green). (A) An extracellular assembly model. The ECM architecture is formed by self-assembly of secreted and released proteins. (B) A 3D printing model. Printing of a new layer on the surface (1) and the simultaneous release of the preestablished layer (2) builds an organized, multilayered ECM structure.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaay6300/DC1

    Supplementary Materials and Methods

    Fig. S1. Generation and characterization of TectaS mice.

    Fig. S2. The function of the hair cells is normal in TectaS/S mice.

    Fig. S3. The GPI anchorage of TECTA plays roles in multimerization and stability of the protein.

    Fig. S4. TECTAT is expressed on the cell surface but is not released by PI-PLC.

    Fig. S5. Generation and characterization of TectaT mice.

    Fig. S6. Incorporation of OTOG, a secreted TM component, into the disorganized collagen network is impaired in TectaS/S and TectaT/T mice.

    Fig. S7. The function of the hair cells is unaffected in TectaT/T mice.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Generation and characterization of TectaS mice.
    • Fig. S2. The function of the hair cells is normal in TectaS/S mice.
    • Fig. S3. The GPI anchorage of TECTA plays roles in multimerization and stability of the protein.
    • Fig. S4. TECTAT is expressed on the cell surface but is not released by PI-PLC.
    • Fig. S5. Generation and characterization of TectaT mice.
    • Fig. S6. Incorporation of OTOG, a secreted TM component, into the disorganized collagen network is impaired in TectaS/S and TectaT/T mice.
    • Fig. S7. The function of the hair cells is unaffected in TectaT/T mice.

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