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. 2012 Oct 10;32(41):14288-93.
doi: 10.1523/JNEUROSCI.3071-12.2012.

Localization of PDZD7 to the stereocilia ankle-link associates this scaffolding protein with the Usher syndrome protein network

M'hamed Grati  1 , Jung-Bum Shin, Michael D Weston, James Green, Manzoor A Bhat, Peter G Gillespie, Bechara Kachar
Affiliations

Localization of PDZD7 to the stereocilia ankle-link associates this scaffolding protein with the Usher syndrome protein network

M'hamed Grati et al. J Neurosci. .

Abstract

Usher syndrome is the leading cause of genetic deaf-blindness. Monoallelic mutations in PDZD7 increase the severity of Usher type II syndrome caused by mutations in USH2A and GPR98, which respectively encode usherin and GPR98. PDZ domain-containing 7 protein (PDZD7) is a paralog of the scaffolding proteins harmonin and whirlin, which are implicated in Usher type 1 and type 2 syndromes. While usherin and GPR98 have been reported to form hair cell stereocilia ankle-links, harmonin localizes to the stereocilia upper tip-link density and whirlin localizes to both tip and ankle-link regions. Here, we used mass spectrometry to show that PDZD7 is expressed in chick stereocilia at a comparable molecular abundance to GPR98. We also show by immunofluorescence and by overexpression of tagged proteins in rat and mouse hair cells that PDZD7 localizes to the ankle-link region, overlapping with usherin, whirlin, and GPR98. Finally, we show in LLC-PK1 cells that cytosolic domains of usherin and GPR98 can bind to both whirlin and PDZD7. These observations are consistent with PDZD7 being a modifier and candidate gene for USH2, and suggest that PDZD7 is a second scaffolding component of the ankle-link complex.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
Detection of PDZD7 in hair cells stereocilia. A–D, Mass spectrometry analysis. A, C, Representative MS2 spectra for PDZD7 and GPR98 showing fragments that were either matched (colored) or unmatched (black) to hypothetical database spectra. The peptide sequence and statistical score indicating identification confidence are shown for each. B, D, Total peptide coverage of PDZD7 and GPR98. The width of each bar indicates the length of the identified peptide, the position of the bar along the x-axis corresponds to its position in the sequence, the height of the bar indicates the number of identical peptides identified, and the shading of the bar corresponds to log(e), the statistical significance for peptide identification, averaged over all identical peptides. The statistical score indicating identification confidence is shown for each protein. E–N, PDZD7 immunolocalization in developing rat hair cells. Confocal microscopy of inner (IHC; E, H, I, J, and N), outer (OHC; F, M), and vestibular (VHC; G, K, L) hair cells from postnatal (P) day 2 (E–I), 7 (J–L), and 10 (M, N) rats immunostained with mouse polyclonal YF-PA20973 (mAb) and rabbit polyclonal PB960 antibodies, showing localization of PDZD7 (red) at the stereocilia ankle-link region; actin (green). H, High magnification of a long and a short stereocilium from a P2 inner hair cell revealing the PDZD7 fluorescence just above the tapered base. J, Horizontal cross-section through inner HC stereocilia showing that PDZD7 immunofluorescence (red) is peripheral to the actin core (green); it is abundant between stereocilia of the same row and between stereocilia of longer and shorter rows, but excluded from the back of the tallest stereocilia. K, Oblique view of P7 vestibular stereocilia showing PDZD7 fluorescence (red, inset) at the ankle-link region. L, Absence of PDZD7 (red) at the upper tip-link insertion sites where MYO7A (blue) is clustered (arrowheads). O–Q, Maturing hair cells expressing mCherry-PDZD7 showing that PDZD7 targets the ankle-link region. Scale bars: E–G, I–Q, 5 μm; H, 300 nm.
Figure 2.
Figure 2.
PDZD7 colocalization with ankle-link proteins in maturing rat hair cells. A–C, Colocalization of PDZD7 and taperin in hair cells. A, B, Back (A) and orthogonal (B) views of stereocilia rows of P10 outer HCs colabeled for PDZD7 (blue) and taperin (green) showing the distribution of both PDZD7 (blue) and taperin (green) along the long and short stereocilia. Red, actin. C, A diagram summarizing PDZD7 (blue) and taperin (green) distribution along the stereocilia (red). D–H, PDZD7 and whirlin colocalization in developing hair cells. Whirlin is revealed with antibodies GP66 (D–F) and GP68 (G, H). D, Oblique view of P8 outer HC bundle showing immunofluorescence distribution of PDZD7 (P, green) and whirlin (W, red). Blue, actin. E, H, Oblique view of P8 VHC stereocilia showing immunofluorescence distribution of PDZD7 (green) and whirlin (blue). Red, actin. F, A view of a P2 vestibular hair bundle showing partial colocalization of PDZD7 (green) and whirlin (blue); r = 0.64 and Rr = 0.60. Red, actin. G, Orthogonal view through P2 outer HC bundles showing PDZD7 (green) and whirlin (blue) immunofluorescence at the ankle-link region. Red, actin. I–K, PDZD7 and GPR98 colocalization in P4 hair cells. I, Back view of P4 outer HC showing PDZD7 (green) and GPR98 (blue) colocalization at the ankle-link region. J, K, Oblique (K) and surface (L) views of P4 VHCs showing PDZD7 (green) and GPR98 (blue) colocalization at the ankle-link region (r = 0.73; Rr = 0.81 in K). L–N, Whirlin and GPR98 colocalization in P4 hair cells. L, A view of P4 outer HC showing whirlin (green) and GPR98 (blue) colocalization at the ankle-link region. M, A view of P4 IHC showing whirlin (green) and GPR98 (blue) colocalization at the ankle-link region. N, Front lateral view of P4 VHC showing whirlin (green) and GPR98 (blue) presence at the ankle-link region. In all three hair cell types, whirlin is also seen at the stereocilia tips (L–N). Scale bars: A, B–H, J, K, N, 5 μm; I, L, M, 2.5 μm.
Figure 3.
Figure 3.
PDZD7 colocalization with stereocilia ankle-link proteins in mature rat hair cells. A–D, PDZD7 (P) and GPR98 (G) colocalization in P15 hair cells. Both GPR98 (green) and PDZD7 (blue) are absent from mature inner HCs (IHCs; A) and outer HCs (OHCs; B), following the disappearance of ankle-links. Red, actin (A). In mature VHCs where ankle-links persist, GPR98 (green) and PDZD7 (blue) fluorescence strongly overlap, as seen through longitudinal and oblique views of the stereocilia. E–I, PDZD7 (P) and whirlin (W) colocalization in P15 hair cells. In organ of Corti, PDZD7 (blue) is absent from mature inner (E, F) and outer (G) HCs following the disappearance of ankle-links. Whirlin fluorescence (green) persists in stereocilia tips and some accumulation is visible around the ankle-link region (F, arrowhead). Longitudinal (H) and oblique (I) views of mature VHCs shows that PDZD7 fluorescence (blue) covers the regions where whirlin fluorescence (green) is present around the ankle-link area. In mature VHCs, whirlin is also seen concentrated at the stereocilia tips (H, I). J, Scanning electron micrograph of P10 whirler inner HC stereocilia showing short stereocilia that are interconnected with all sorts of links, including the ankle-links (K, arrowheads). Scale bars: A–J, 5 μm; K, 1 μm.
Figure 4.
Figure 4.
A–F, PDZD7 interactions with Usher proteins in COS7 and LLC-PK1 cells. A, In the presence of EGFP-sans (green), mCherry-PDZD7 (red) is randomly spread in the COS7 cell cytosol, associated with actin filaments (blue), or also recruited to EGFP-sans clusters, as shown by the high values of Mander's (r = 0.80) and Pearson's (Rr = 0.81) coefficients. B, EGFP-harmonin-b (green) forms plaques associated with actin (blue). The distribution of mCherry-PDZD7 (red) is unchanged in the presence of EGFP-harmonin-b, and its colocalization with EGFP-harmonin-b actin-bound plaques is nonsignificant based on the values of r = 0.80 and Rr = 0.10. C, F, Confocal cross-section scans of the apical plasma membrane of polarized LLC-PK1 cells coexpressing fusion proteins IL2α-ush2a (green in C and red in E) and IL2α-GPR98 (green in D and red in F) together with either mCherry-PDZD7 (red in C, D) or EGFP-Whirlin (green in E, F), showing that both fusion proteins are enriched in the apical plasma membrane where they highly colocalize with both whirlin and PDZD7. Scale bars, 10 μm.
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