MECHANISMS AND MOLECULES MAINTAINING SENSITIVITY AND DURABILITY OF MECHANO-ELECTRICAL TRANSDUCTION IN MAMMALIAN AUDITORY HAIR CELLS

Author

• Abigail K. Dragich, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0000-0002-5181-3074

Date Available

5-15-2028

Year of Publication

2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Physiology

Faculty

Gregory I. Frolenkov

Faculty

A. Catalina Velez-Ortega

Abstract

Mechanosensory hair cells (HCs) in the cochlea contain actin-based stereocilia that are arranged in rows of increasing heights and harbor MET channels at their tips. To detect soft sounds, MET channels are gated by extracellular tip links that are tensioned at rest. Classical models postulate that this tension is generated by myosin motors pulling the upper end of the tip link upwards. Here, an alternative mechanism of tip link tensioning is tested by MET-dependent remodeling of stereocilia F-actin, which requires the upper end of the tip link to have limited mobility. MET-dependent stereocilia shortening was evoked with different stimuli that block Ca²⁺ influx through the MET channels. Each produced similar increases in the resting MET current, which is maintained by resting tip link tension. Drugs stabilizing actin filaments inhibited these effects while drugs affecting actin polymerization prolonged their recovery. These data are the first to demonstrate the role of actin remodeling in regulation of resting MET currents, showing that the same mechanism can simultaneously maintain precise stereocilia heights.

While this project suggests that resting MET current is regulated by a myosin-independent mechanism, the well-established role of myosins in other properties of MET cannot be ignored. G-α Interacting Protein C-terminus 3 (GIPC3) is a small cytosolic adaptor protein linking myosin motors and receptors. Mutations in GIPC3 cause mild to profound deafness in humans, but its role in HCs is unknown. Our collaborator, Dr. Craig Vander Kooi, generated two new Gipc3 mutant mouse strains, one with complete loss of GIPC3 (Gipc3^-) and another with a mutation p.W301X (Gipc3^W301X) recapitulating human deafness. Here, I demonstrated GIPC3 localization to the stereocilia, along with no changes in resting MET current. Interestingly, loss of even one copy of Gipc3 decreases the extent of slow adaptation of MET currents, suggesting that dynamic regulation of tip link tensioning is disrupted. Additionally, predicted GIPC3 interacting partner myosin VIIA (MYO7A) is mis-localized from Gipc3^-/- stereocilia along with the disappearance of an electron dense region anchoring the upper end of the tip link to the stereocilia core, leading to a MET apparatus more susceptible to mechanical damage. Thus, we proposed that GIPC3 is proposed as a novel component of the MET machinery anchoring the upper end of the tip link to the actin cytoskeleton through a MYO7A based complex, a mechanism critical for durability of the MET apparatus during continuous cycles of sound-induced stimuli.

A separate role of GIPC3 and MYO6 has been demonstrated in shaping the apical region of HCs. This is not surprising, given that GIPC3 is a pleiotropic adaptor with the ability to link different myosins. GIPC3 and MYO6 are enriched at the cuticular plate, a site of vesicle trafficking and endocytosis. Here, I explored the role of a MYO6-GIPC3 complex in endocytosis and stereocilia protein recycling in HCs. Loss of GIPC3 causes membrane blebbing around the cuticular plate, accumulation of intracellular vesicles, and mis-localization of early endosomal marker APPL2 (a top interacting partner of GIPC3). Live cell imaging showed that loss of GIPC3 blocks the endocytosis of FM3-25 dye, and this effect can be recapitulated in wildtype HCs by applying a MYO6 inhibitor. GIPC3 is predicted to interact with stereocilia-specific plasma membrane Ca²⁺-ATPase (PMCA2). Loss of GIPC3 resulted in loss of PMCA2 from the stereocilia. These data suggests that a GIPC3-MYO6-based complex is required for endocytosis and trafficking of stereocilia proteins in HCs. Together, these data suggest a novel model where a GIPC3-based complex anchors the upper end of the tip link, provides the MET machinery with resilience to mechanical stimuli, and allows MET-dependent stereocilia remodeling to maintain resting MET tension, and thus the sensitivity of hearing.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2026.302

Archival?

Archival

Funding Information

This study was supported by the National Institute of Deafness and Communication Disorders Grant (F31DC020639) from 2022-2026.

Recommended Citation

Dragich, Abigail K., "MECHANISMS AND MOLECULES MAINTAINING SENSITIVITY AND DURABILITY OF MECHANO-ELECTRICAL TRANSDUCTION IN MAMMALIAN AUDITORY HAIR CELLS" (2026). Theses and Dissertations--Physiology. 75.
https://uknowledge.uky.edu/physiology_etds/75