GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region

ABSTRACT GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

postnatal stages and that its absence in mouse model causes misshaping and distortion of inner and outer hair cell stereocilia bundle visible as early at P6-7 but preserving MET. Authors also identified MYO6 as GIPC3 partner both colocalizing at hair cell apical junction at the pericuticular necklace area and that GIPC3 localization depends on MYO6, and not vice versa. Very interestingly, GIPC3-ko inner hair cells presented severe cuticular plate defects and altered apical junctions. Besides, the authors identified GIPC3 interactome in hair cells several elements of which including MYO18A were localized in hair cells, therefore further demonstrating its effect on dimensions of cuticular plate and apical surfaces. The scarcity of publications describing these types of molecular complexes and the dissection of their structural involvement clearly justifies the extreme technical and conceptual complexities of performing these studies. This study is extremely well-designed and beautifully executed, and convincingly demonstrates for the first time the role of GIPC3 and its molecular network in the shaping of hair cell cuticular plate and their apical junctions that are crucial in the stabilization, shaping, integrity and therefore excitability of mechanosensory stereocilia hair bundle.

Comments for the author
After a very thorough examination of the manuscript that is quite long and rich and utilized an impressive number of techniques and unique resources, I found the manuscript extremely wellwritten with beautiful figure panels that were nicely assembled into very informative sets of figures. I strongly recommend the immediate publication of this manuscript by JCS without any modifications or corrections. I congratulate in advance the authors for this impactful scientific achievement!

Reviewer 2
Advance summary and potential significance to field Chatterjee et al.'s manuscript titled "GIPC3 couples to MYO6 and PDZ domain proteins and shapes the hair cell apical region" is a tour de force of cell biology, biochemistry, genetics, and functional analyses. Herein they show that GIPC3 localizes to the cuticular plate, a poorly understood organelle of hair cells, which deserves attention. The authors show that Gipc3KO/KO have defects in the cuticular plate and hair bundle morphology. They then use biochemistry/ cell biology to identify binding partners of GIPC3 and identify MYO6 and other proteins, such as MYO18A, that harbor PDZ binding motifs in their primary amino acid sequences. They produce an elegant model in
Line 48: consider changing "a N-terminal" to "an N-terminal".
Line 287: consider removing "the" before binary

First revision
Author response to reviewers' comments

Editor:
As you will see, the reviewers gave favourable reports, raising however some minorpoints that will require amendments to your manuscript. I hope that you will be able to carry these out because I would like to be able to accept your paper.
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The last three minor points raised by Review #2 were corrected and highlighted with yellow in the revised text. The new version of the Supplemental Material has the text noted by Reviewer #2 removed.
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Reviewer #1:
Reviewer 1 Advance Summary and Potential Significance to Field: The manuscript by Chatterjee et al identifies and characterizes inner ear hair cell molecular network involving GIPC3, a poorly studied protein with very important suspected function(s). The authors first found that GIPC3 is enriched at the apical pole of sensory hair cells since early postnatal stages and that its absence in mouse model causes misshaping and distortion of inner and outer hair cell stereocilia bundle visible as early at P6-7 but preserving MET. Authors also identified MYO6 as GIPC3 partner both colocalizing at hair cell apical junction at the pericuticular necklace area and that GIPC3 localization depends on MYO6, and not vice versa. Very interestingly, GIPC3-ko inner hair cells presented severe cuticular plate defects and altered apical junctions. Besides, the authors identified GIPC3 interactome in hair cells several elements of which including MYO18A were localized in hair cells, therefore further demonstrating its effect on dimensions of cuticular plate and apical surfaces. The scarcity of publications describing these types of molecular complexes and the dissection of their structural involvement clearly justifies the extreme technical and conceptual complexities of performing these studies. This study is extremely well-designed and beautifully executed, and convincingly demonstrates for the first time the role of GIPC3 and its molecular network in the shaping of hair cell cuticular plate and their apical junctions that are crucial in the stabilization, shaping, integrity and therefore excitability of mechanosensory stereocilia hair bundle. Reviewer 1 Comments for the Author: After a very thorough examination of the manuscript that is quite long and rich and utilized an impressive number of techniques and unique resources, I found the manuscript extremely well-written with beautiful figure panels that were nicely assembled into very informative sets of figures. I strongly recommend the immediate publication of this manuscript by JCS without any modifications or corrections. I congratulate in advance the authors for this impactful scientific achievement! We thank the reviewer for this favorable assessment.

Reviewer #2:
Chatterjee et al.'s manuscript titled "GIPC3 couples to MYO6 and PDZ domain proteins and shapes the hair cell apical region" is a tour de force of cell biology, biochemistry, genetics, and functional analyses. Herein, they show that GIPC3 localizes to the cuticular plate, a poorly understood organelle of hair cells, which deserves attention. The authors show that Gipc3KO/KO have defects in the cuticular plate and hair bundle morphology. They then use biochemistry/ cell biology to identify binding partners of GIPC3 and identify MYO6 and other proteins, such as MYO18A, that harbor PDZ binding motifs in their primary amino acid sequences. They produce an