Figure 1.
TPRN protein domains, motifs, antibody epitopes, and localization in mouse cochlear hair cells. (A) Illustration of a mechanosensory stereocilia bundle on the apical surface of a hair cell. The three rows of actin-based stereocilia are interconnected by tip links and anchored to the hair cell body by F-actin rootlets (magenta). The tapered region of stereocilia is indicated by a bracket and outlined by green lines. (B) Schematic of mouse TPRN protein encoded by four exons indicated by different colors and number of aa encoded by each exon. Below the TPRN schematic, colored bars indicate predicted domains using ELM (http://elm.eu.org) unless otherwise indicated. The gray bars indicate two small regions of aa sequence percentage identity to phostensin (aa 15–107 and 590–646). Purple bar is an EH ligand (aa 61–65) containing an NPF motif, the light green bars show three Y-based endocytic sorting motifs (Yxxphi) (aa 85–88, 593–596, and 605–608), blue bar is a NLS (aa 617–627, https://nls-mapper.iab.keio.ac.jp), light blue bar is a PP1-binding site with KISF motif (aa 624–627), violet bar is a nuclear export signal, NES (aa 598–609), and an arrow indicates a PDZ ligand (aa 744–749). Locations of epitopes of custom rabbit polyclonal anti-TPRN antibodies (red bars above the schematic) PB913 and PB939 and a RabmAb against the same epitope to raise antiserum PB939. The brown bar—location of epitope of the commercial anti-TPRN (C9ORF75) antibody (HPA020899, RRID:AB_1845835; MilliporeSigma). (C) Confocal microscopy image showing TPRN localization (green) at bases of stereocilia spanning the taper region of all rows. (D) Stereocilia side view image. Unless otherwise stated, rhodamine-phalloidin (magenta) was used to counterstain F-actin in most panels. (E) Localization of TPRN (green) in relation to ANKRD24 (blue) in P20 WT OHC stereocilia. (F) Localization of ANKRD24 (blue) in relation to its interacting partner TRIOBP-5 (yellow) in P14 WT IHCs. (E′–F′) Line graphs of normalized fluorescence intensity for each protein measured from the stereocilia pivot point (0) to 1-μm down (1) and 1-μm up (−1) the stereocilium. ANKRD24 (cyan) concentrates at the pivot point of every stereocilium (magenta). A fainter signal of ANKRD24 (F′) distributes along the rootlet portion highlighted by TRIOBP-5 in (F). (G–J) STED super-resolution images at the pivot points of OHC stereocilia in P18 C57BL/6J mouse revealed rootlet insertions into cuticular plate stained with Star Red phalloidin (G) and surrounding ring patterns of TRIOBP-4 (H), TRIOBP-5 (I), and ANKRD24 (J). Inserts in H–J show mean intensity projection of aligned cross sections through the pivot point of row 1 OHC stereocilia double stained with F-actin (phalloidin, magenta) and TRIOBP-4 (n = 25), TRIOBP-5 (n = 52), and ANKRD24 (n = 64), correspondingly (turquoise). (K) A similar ring pattern at P6 stereocilia taper region just above the apical surface of IHC is revealed in STED image by a secondary nanobody together with anti-TPRN antibody (green). (L) Image along the longitudinal axis of P6 IHC stereocilia showing a funnel-like pattern (white arrowheads) of TPRN staining (green) at the taper region. (M) STED image of P6 OHC showing similar TPRN ring pattern (green). (N) Enlarged images of a rectangular area depicted in M of individual OHC stereocilia outlined by TPRN rings (green). (O) Diameters of ring staining for ANKRD24, TRIOBP-4, and TRIOBP-5 in the longest row (Row 1) stereocilia of OHCs. Number of stereocilia/cells: ANKRD24, n = 39/3; TRIOBP-5, n = 57/3; TRIOBP-4, n = 54/4. Asterisks show statistical significance of the differences between proteins: overall one-way ANOVA (P < 0.0001) followed by Tukey’s post hoc comparison test (***P < 0.0001). The graph compares the average ring diameters for each protein using one-way ANOVA (P < 0.0001) followed by Tukey’s post hoc comparison test (***P < 0.0001). (P) Examples of intensity profiles along a line through the center of a stereocilium rootlet cross section revealed by F-actin staining, which were used to determine the diameters of TRIOBP-4, TRIOBP-5, and ANKRD24 staining patterns in panel O. (Q) Similar plot of TPRN fluorescence intensity also confirms the ring appearance of TPRN staining in P6 stereocilia cross sections. (R) Left, mean intensity projection of 20 aligned longitudinal sections through the base of row 1 IHC stereocilia stained with F-actin (magenta) and TPRN (green). Right, intensity profiles of F-actin and TPRN staining across stereocilium at different distances from the pivot point (0). Images in C-E and F were obtained using Zeiss LSM880 Airyscan confocal system and in G–J using a FACILITY microscope system (Abberior Inc). Inserts in H-J and images in K-N and R were obtained using a STEDYCON super-resolution system (Abberior Inc) on an Eclipse Ti2 microscope (Nikon). Scale bars are 2 μm in C–J, 1 μm in K–N, 500 nm in L and R, and 200 nm in H–J inserts. Scale bar in G applies to G–J.

TPRN protein domains, motifs, antibody epitopes, and localization in mouse cochlear hair cells. (A) Illustration of a mechanosensory stereocilia bundle on the apical surface of a hair cell. The three rows of actin-based stereocilia are interconnected by tip links and anchored to the hair cell body by F-actin rootlets (magenta). The tapered region of stereocilia is indicated by a bracket and outlined by green lines. (B) Schematic of mouse TPRN protein encoded by four exons indicated by different colors and number of aa encoded by each exon. Below the TPRN schematic, colored bars indicate predicted domains using ELM (http://elm.eu.org) unless otherwise indicated. The gray bars indicate two small regions of aa sequence percentage identity to phostensin (aa 15–107 and 590–646). Purple bar is an EH ligand (aa 61–65) containing an NPF motif, the light green bars show three Y-based endocytic sorting motifs (Yxxphi) (aa 85–88, 593–596, and 605–608), blue bar is a NLS (aa 617–627, https://nls-mapper.iab.keio.ac.jp), light blue bar is a PP1-binding site with KISF motif (aa 624–627), violet bar is a nuclear export signal, NES (aa 598–609), and an arrow indicates a PDZ ligand (aa 744–749). Locations of epitopes of custom rabbit polyclonal anti-TPRN antibodies (red bars above the schematic) PB913 and PB939 and a RabmAb against the same epitope to raise antiserum PB939. The brown bar—location of epitope of the commercial anti-TPRN (C9ORF75) antibody (HPA020899, RRID:AB_1845835; MilliporeSigma). (C) Confocal microscopy image showing TPRN localization (green) at bases of stereocilia spanning the taper region of all rows. (D) Stereocilia side view image. Unless otherwise stated, rhodamine-phalloidin (magenta) was used to counterstain F-actin in most panels. (E) Localization of TPRN (green) in relation to ANKRD24 (blue) in P20 WT OHC stereocilia. (F) Localization of ANKRD24 (blue) in relation to its interacting partner TRIOBP-5 (yellow) in P14 WT IHCs. (E′–F′) Line graphs of normalized fluorescence intensity for each protein measured from the stereocilia pivot point (0) to 1-μm down (1) and 1-μm up (−1) the stereocilium. ANKRD24 (cyan) concentrates at the pivot point of every stereocilium (magenta). A fainter signal of ANKRD24 (F′) distributes along the rootlet portion highlighted by TRIOBP-5 in (F). (G–J) STED super-resolution images at the pivot points of OHC stereocilia in P18 C57BL/6J mouse revealed rootlet insertions into cuticular plate stained with Star Red phalloidin (G) and surrounding ring patterns of TRIOBP-4 (H), TRIOBP-5 (I), and ANKRD24 (J). Inserts in H–J show mean intensity projection of aligned cross sections through the pivot point of row 1 OHC stereocilia double stained with F-actin (phalloidin, magenta) and TRIOBP-4 (n = 25), TRIOBP-5 (n = 52), and ANKRD24 (n = 64), correspondingly (turquoise). (K) A similar ring pattern at P6 stereocilia taper region just above the apical surface of IHC is revealed in STED image by a secondary nanobody together with anti-TPRN antibody (green). (L) Image along the longitudinal axis of P6 IHC stereocilia showing a funnel-like pattern (white arrowheads) of TPRN staining (green) at the taper region. (M) STED image of P6 OHC showing similar TPRN ring pattern (green). (N) Enlarged images of a rectangular area depicted in M of individual OHC stereocilia outlined by TPRN rings (green). (O) Diameters of ring staining for ANKRD24, TRIOBP-4, and TRIOBP-5 in the longest row (Row 1) stereocilia of OHCs. Number of stereocilia/cells: ANKRD24, n = 39/3; TRIOBP-5, n = 57/3; TRIOBP-4, n = 54/4. Asterisks show statistical significance of the differences between proteins: overall one-way ANOVA (P < 0.0001) followed by Tukey’s post hoc comparison test (***P < 0.0001). The graph compares the average ring diameters for each protein using one-way ANOVA (P < 0.0001) followed by Tukey’s post hoc comparison test (***P < 0.0001). (P) Examples of intensity profiles along a line through the center of a stereocilium rootlet cross section revealed by F-actin staining, which were used to determine the diameters of TRIOBP-4, TRIOBP-5, and ANKRD24 staining patterns in panel O. (Q) Similar plot of TPRN fluorescence intensity also confirms the ring appearance of TPRN staining in P6 stereocilia cross sections. (R) Left, mean intensity projection of 20 aligned longitudinal sections through the base of row 1 IHC stereocilia stained with F-actin (magenta) and TPRN (green). Right, intensity profiles of F-actin and TPRN staining across stereocilium at different distances from the pivot point (0). Images in C-E and F were obtained using Zeiss LSM880 Airyscan confocal system and in G–J using a FACILITY microscope system (Abberior Inc). Inserts in H-J and images in K-N and R were obtained using a STEDYCON super-resolution system (Abberior Inc) on an Eclipse Ti2 microscope (Nikon). Scale bars are 2 μm in C–J, 1 μm in K–N, 500 nm in L and R, and 200 nm in H–J inserts. Scale bar in G applies to G–J.

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