Fig 2: Model for GIPC3 coupling of apical cell junctions to the cuticular plate. (A) Tracings of averaged TJP1-labeled apical cell borders at indicated ages (adapted from fig. 2 of Etournay et al., 2010 with permission). (B,C) Key complexes and processes in P1 IHC. The cuticular plate is rounded up early in development. Apical junctions in blue, cuticular plate in dark orange, stereocilia in gray. The pericuticular necklace is the gap between the apical junctions and the cuticular plate. MYO18A is not shown at this age. (D,E) P7.5 IHC. The cuticular plate is flattened later in development. GIPC3–MYO6 complexes are in red; the MYO18A structure is in green; MYO18A at the apical junction region is not shown. (F) As the apical circumference is remodeled between P1 and P7.5, the IHC narrows along the lateral-medial axis. Inside the cell, active force generated along the cochlear axis stretches the cuticular plate; maintaining constant volume, the cuticular plate passively shrinks along the lateral-medial axis. GIPC3–MYO6 complexes either generate or are simply coupled to the force production that stretches the cuticular plate. Loss of GIPC3 prevents the elongation of the cuticular plate. The MYO18A structure underneath the cuticular plate might assist in its flattening; GIPC3 could couple MYO18A there with MYO6, ACTN1 or ACTN4 in the cuticular plate. B and D show medial-lateral sections through IHC centers; C, E and F show cross-section of IHCs at level of dashed line in B and D. The pericuticular necklace was removed for clarity in C–F, and the apical junctions were removed in F.

Fig 3: Model for GIPC3 coupling of apical cell junctions to the cuticular plate.A, Tracings of averaged TJP1-labeled apical cell borders at indicated ages (adapted from Fig. 2 of Etournay et al., 2018). B-C, Key complexes and processes in P1 IHC. Cuticular plate is rounded up early in development. Apical junctions in blue, cuticular plate in dark orange, stereocilia in gray. The pericuticular necklace is the gap between the apical junctions and the cuticular plate. MYO18A is not shown at this age. D-E, P7.5 IHC. Cuticular plate is flattened later in development. GIPC3-MYO6 complexes in red; MYO18A structure in green; MYO18A at apical junction region is not shown. F, As the apical circumference is remodeled between P1 and P7.5, the IHC narrows along the lateral-medial axis. Inside the cell, active force generated along the cochlear axis stretches the cuticular plate; maintaining constant volume, the cuticular plate passively shrinks along the lateral-medial axis. GIPC3-MYO6 complexes either generate or are simply coupled to the force production that stretches the cuticular plate. Loss of GIPC3 prevents the elongation of the cuticular plate. The MYO18A structure underneath the cuticular plate may assist in its flattening; GIPC3 could couple MYO18A there with MYO6, ACTN1, or ACTN4 in the cuticular plate. B and D show medial-lateral sections through IHC centers; C and E-F show cross-section of IHCs at level of dashed line in B and D. The pericuticular necklace was removed for clarity in C-F, and the apical junctions were removed in F.

Fig 4: MYO18A is located in the hair cell apical domain. (A) Top, sequence logo for binding of ligands to GIPC1 PDZ domain; bottom, C-terminal ten amino acids of APPL2, MYO18A, ACTN1 and ACTN4. (B) Immunolocalization of MYO18A in P14.5 mouse cochlea; slices from a three-dimensional image stack. Transects for other image axes are shown in yellow; the X and Y transects in the main X-Y image show the locations for the Y-Z and X-Z images. Arrow indicates concentration of MYO18A immunoreactivity below the IHC cuticular plate. IHC, inner hair cell; IPC, inner pillar cell; OPC, outer pillar cell; OHC, outer hair cell. Immunolocalization experiments for MYO18A were performed more than five times. (C,D) MYO18A immunoreactivity in P15.5 IHCs using lattice SIM imaging. (C) Image showing four IHCs at the stereocilia/cuticular plate level. (D) Image showing a single IHC (labeled with asterisk in C) at the cuticular plate level (different plane than in C). Arrows delineate the gap between cuticular plate actin and the circumferential actin belt. (E,F) MYO18A immunoreactivity in P14.5 IHCs from folded cochleas using Airyscan imaging. E is from a Gipc3KO/+ mouse and F is from a Gipc3KO/KO mouse. (G) NanoSPD of MYO18A–GIPC3. Example of filopodial targeting of mCherry–GIPC3 by GFP–FL-MYO18A, mediated by MYO10NANOTRAP. (H) Expression of GFP–FL-MYO18A and mCherry–FL-GIPC3 constructs in HeLa cells (no MYO10NANOTRAP expressed). Arrows indicate large cytoplasmic aggregates containing GFP and mCherry. squeezing. Images in C–H representative of at least three repeats. (I) GFP–MYO18A constructs. ‘Motor’, actin- and ATP-binding domains are homologous to myosin motor domains in active myosins; IQ, isoleucine/glutamine calmodulin-binding; PBM, PDZ-binding motif. (J) mCherry–GIPC3 constructs. mCh, mCherry; GH1, GIPC-homology 1; GH2, GIPC-homology 2. (K) Prey (mCh–GIPC3) fluorescence with GFP–MYO18A constructs or GFP control. Mean±s.d. plotted in K and L. One-way ANOVA comparisons to no-bait condition with Dunnett correction for multiple comparisons. Sample sizes (n) were GFP–FL-MYO18A (105 filopodia), GFP–IQ-PBM-MYO18A (109), GFP–CC-PBM-MYO18A (116), GFP–C-PBM-MYO18A (129), GFP–?PBM-MYO18A (127), GFP (208). (L) Prey (mCh–GIPC3 constructs or mCh–MYO6) fluorescence with GFP-MYO18A constructs or GFP control. One-way ANOVA comparisons to no-bait condition with Dunnett correction for multiple comparisons. Sample sizes (n) were mCh–D1-GIPC3 (128 filopodia for GFP–FL-MYO18A and 88 for GFP alone), mCh–D3-GIPC3 (180 and 47) and mCh–MYO6 (107 and 93). Panel widths: B, 37.5 µm for X-Y plot (same scale applies to Y-Z and X-Z panels); C, 50 µm; D, 12 µm; E-F, 45 µm; G,H, 15 µm.