The Nanomechanics of Protocadherin 15, A Protein Essential for Human Hearing

Author

Camila M. Villasante

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hudspeth Laboratory

Abstract

Mechanical force controls the opening and closing of mechanotransductive ion channels atop the hair bundles in the inner ear. A mechanical element called the gating spring modulates the mechanotransduction channel’s open probability by changing the force transmitted to the channels. The molecular identity of the gating spring is yet unconfirmed, but a leading candidate is the filamentous tip link connecting the mechanotransduction channel to the tallest neighboring stereocilium. The tip link is essential to mechanotransduction: when it is broken, mechanotransduction is abolished, and when it is allowed to regenerate, mechanotransduction returns. Each tip link comprises four protein molecules: a dimer of protocadherin 15 and a dimer of cadherin 23, both of which are stabilized by Ca²⁺ binding. Further underscoring the role of the tip link in hearing, there are numerous mutations of its constituent proteins that result in deafness. My thesis work has focused on protocadherin 15, the lower portion of the tip link that connects at its C-terminus to the mechanotransduction channel. I was interested in answering several questions: does protocadherin 15 have the appropriate properties to be a component of the gating spring? What factors control its mechanical response? What is its stiffness? How does it soften under force? And how do these answers change in the case of a deafness-causing mutation in protocadherin 15? In order to answer these questions, I used an optical-trap system with sub-nanometer spatial resolution and microsecond temporal resolution to investigate the mechanics of protocadherin 15 at a single-molecule level. To augment this approach I also undertook electron microscopic studies to investigate the structure of wildtype and mutated protocadherin 15. I found that both the mechanics and structure of protocadherin 15 are dependent on Ca²⁺ and that protocadherin 15 undergoes limited unfolding at a physiological level of Ca²⁺. My experimentally determined stiffness for protocadherin 15 accords with published values of the gating spring’s stiffness, which implies that protocadherin 15 is able to modulate its stiffness without undergoing large unfolding events in physiological Ca²⁺ conditions. In the case of a point mutation that causes non-syndromic hearing loss, the structure of protocadherin 15 is more conformationally heterogenous, and the protein undergoes frequent unfolding events at all levels of Ca²⁺. The frequent unfolding events suggest that the mutated protocadherin 15 has lost the ability to maintain appropriate tension under physiological forces, which could prevent the proper opening of the mechanotransduction channel and result in deafness fort those with this mutation. This work shows that the maintenance of appropriate tension in the gating spring is critical to the appropriate conveyance of force to the mechanotransduction channel.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Recommended Citation

Villasante, Camila M., "The Nanomechanics of Protocadherin 15, A Protein Essential for Human Hearing" (2023). Student Theses and Dissertations. 780.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/780