Figure 7.
Localization of TPRN in hair cells lacking PTPRQ and interaction of TPRN and PTPRQ in live cells. (A and B) Localization of TPRN in P15 normal hearing heterozygous littermate (Ptprq+/−) and deaf Ptprq knockout mouse (Ptprq−/−) OHCs was examined using anti-TPRN antibodies PB913 and C9ORF75. Both antibodies gave identical staining showing TPRN is concentrated at the base of OHC stereocilia in both Ptprq+/− (A) and Ptprq−/− (B) mouse OC and visualized as green puncta. (C) Bar graph showing quantification of number of TPRN puncta in OHCs for both genotypes. Ptprq+/−n = 137/3 puncta/cells and Ptprq−/−n = 77/3 puncta/cells. Number of TPRN puncta at bases of OHC stereocilia was significantly reduced in P15 Ptprq−/− mice compared with Ptprq+/− controls as revealed by unpaired two-sided t test (***P = 0.0005). (D and E) Bar graph of mean fluorescence intensity of individual puncta showing no statistically significant differences between genotypes by unpaired two-sided t test (P = 0.4338). Imaris software spot function was used to calculate the number of puncta per cell for each genotype. ImageJ (Fiji) was used to calculate mean fluorescence intensity value of puncta for each genotype to compare the differences in TPRN expression in Ptprq+/− control and Ptprq−/− knockout mice. (F and G) TPRN localization at the base of IHC stereocilia in P15 Ptprq+/− control (F) and Ptprq−/− knockout mouse (G). (H–J) Quantification of number of TPRN puncta (H) and mean fluorescence intensity of puncta (I and J) in IHCs for both genotypes. Mean TPRN fluorescence intensity of TPRN puncta was significantly reduced in P15 Ptprq−/− knockout mouse IHCs (**P = 0.0015 by unpaired two-sided t test) but no significant differences were found in number of TPRN puncta in IHC stereocilia, 129/3, number of puncta/cells (Ptprq+/−) and 137/3 number of puncta/cells (Ptprq−/−) between the genotypes (P = 0.6489 by unpaired two-sided t test). Data are represented as mean ± SD. Puncta from three cells per genotype and three for each hair cell type were analyzed. (K) mCherry-MYO10-TPRN fusion construct (magenta) expressed in COS-7 cells targets filopodia tips and co-localizes there with EGFP-PTPRQ (green). (L) Control mCherry-MYO10HMM does not target PTPRQ protein to filopodia tips when co-expressed together, confirming that PTPRQ is delivered to the filopodia tips due to its interaction with TPRN (K). (M) Overexpressed mCherry-TPRN (magenta) interacts with EGFP-PTPRQ (green), transports PTPRQ to the nucleus, and forms a whorl-like bundle where both TPRN and PTPRQ co-localize. The experiments were performed at least three times, yielding consistent results. All scale bars are: 5 µm; scale bar in B applies to A, B, F, and G; scale bar in M applies to L and M.

Localization of TPRN in hair cells lacking PTPRQ and interaction of TPRN and PTPRQ in live cells. (A and B) Localization of TPRN in P15 normal hearing heterozygous littermate (Ptprq+/−) and deaf Ptprq knockout mouse (Ptprq−/−) OHCs was examined using anti-TPRN antibodies PB913 and C9ORF75. Both antibodies gave identical staining showing TPRN is concentrated at the base of OHC stereocilia in both Ptprq+/− (A) and Ptprq−/− (B) mouse OC and visualized as green puncta. (C) Bar graph showing quantification of number of TPRN puncta in OHCs for both genotypes. Ptprq+/−n = 137/3 puncta/cells and Ptprq−/−n = 77/3 puncta/cells. Number of TPRN puncta at bases of OHC stereocilia was significantly reduced in P15 Ptprq−/− mice compared with Ptprq+/− controls as revealed by unpaired two-sided t test (***P = 0.0005). (D and E) Bar graph of mean fluorescence intensity of individual puncta showing no statistically significant differences between genotypes by unpaired two-sided t test (P = 0.4338). Imaris software spot function was used to calculate the number of puncta per cell for each genotype. ImageJ (Fiji) was used to calculate mean fluorescence intensity value of puncta for each genotype to compare the differences in TPRN expression in Ptprq+/− control and Ptprq−/− knockout mice. (F and G) TPRN localization at the base of IHC stereocilia in P15 Ptprq+/− control (F) and Ptprq−/− knockout mouse (G). (H–J) Quantification of number of TPRN puncta (H) and mean fluorescence intensity of puncta (I and J) in IHCs for both genotypes. Mean TPRN fluorescence intensity of TPRN puncta was significantly reduced in P15 Ptprq−/− knockout mouse IHCs (**P = 0.0015 by unpaired two-sided t test) but no significant differences were found in number of TPRN puncta in IHC stereocilia, 129/3, number of puncta/cells (Ptprq+/−) and 137/3 number of puncta/cells (Ptprq−/−) between the genotypes (P = 0.6489 by unpaired two-sided t test). Data are represented as mean ± SD. Puncta from three cells per genotype and three for each hair cell type were analyzed. (K) mCherry-MYO10-TPRN fusion construct (magenta) expressed in COS-7 cells targets filopodia tips and co-localizes there with EGFP-PTPRQ (green). (L) Control mCherry-MYO10HMM does not target PTPRQ protein to filopodia tips when co-expressed together, confirming that PTPRQ is delivered to the filopodia tips due to its interaction with TPRN (K). (M) Overexpressed mCherry-TPRN (magenta) interacts with EGFP-PTPRQ (green), transports PTPRQ to the nucleus, and forms a whorl-like bundle where both TPRN and PTPRQ co-localize. The experiments were performed at least three times, yielding consistent results. All scale bars are: 5 µm; scale bar in B applies to A, B, F, and G; scale bar in M applies to L and M.

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