On Friday, May 1, 2026, Abigail Dragich successfully defended her dissertation and earned her doctoral degree in physiology. Congratulations, Dr. Dragich!
MECHANISMS AND MOLECULES MAINTAINING SENSITIVITY AND DURABILTIY OF MECHANO-ELECTRICAL TRANSDUCTION IN MAMMALIAN AUDITORY HAIR CELLS
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, we tested an alternative mechanism of tip link tensioning by MET-dependent remodeling of stereocilia F-actin, which requires the upper end of the tip link to have limited mobility. We evoked MET-dependent stereocilia shortening with different stimuli that block Ca2+ influx through the MET channels; each produced similar increases in the resting MET current, which is proportional to resting tip link tension. Drugs stabilizing actin filaments inhibited these effects while drugs affecting actin polymerization prolonged their recovery. Our data is 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 our data suggests that resting MET current is regulated by a myosin-independent mechanism, we cannot ignore the well-established role of myosins in other features of MET. G-a 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. Here, we localized GIPC3 to the stereocilia of HCs and generated new Gipc3 mutant mouse strains, one with complete loss of GIPC3 (Gipc3-) and another with a mutation p.W301X (Gipc3W301X) recapitulating human deafness. Both mutants have progressive deafness and disrupted stereocilia bundles 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 fragile MET apparatus. We propose GIPC3 is 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. In the last part of this Ph.D. thesis, we explored whether a MYO6-GIPC3 complex can also mediate endocytosis and stereocilia protein recycling in HCs. We found that 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. We propose that a GIPC3-MYO6-based complex is required for endocytosis and trafficking of stereocilia proteins in HCs.
Our data suggests 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.
