Work in my laboratory is focused on understanding the gating and regulation of mechanically sensitive (force-gated) ion channels. Much of this work involves the hair cells of the inner ear, which convert the mechanical stimulus of a sound wave into electrical signals that are sent to the brain. The mechanosensitive organelle of the hair cell is a bundle of stereocilia that protrudes from the top surface of the cell. Stereocilia are connected at their tips by fine filaments called tip links, which are tensioned each time the bundle is deflected in one direction by sound vibration. Tip links are thought to pull directly on ion channels in the tips of the stereocilia, which open in response to the tension and allow electric current in the form of potassium ions to flow into the hair cell to change its internal voltage.
Mechanics of the Hair Bundle
Each hair cell has a bundle of 30–300 actin-filled stereocilia and a single microtubule-based kinocilium that are connected by various links and that tend to move as a cohesive unit. The exact mechanism of their connection has important implications for the biophysical interaction between the transduction channels of a bundle.
We have used high-resolution and high-speed optical imaging to observe the movement of stereocilia upon stimulation, in hair cells from the bullfrog. In these hair cells, the stimulus is conveyed to the bundle by deflection of a single true cilium, the kinocilium. At stimulus frequencies approaching a kilohertz, all the stereocilia of a bundle move by about the same angular amount and separate by no more than 10 nm, consistent with the requirement for stereocilia to be driven synchronously by the kinocilium. Chemically cutting the tip links does not change this motion, indicating that other links hold the bundle together. These results suggest a "sliding adhesion" mechanism that allows adjacent stereocilia membranes to slide rapidly past each other by 100 nm or more, without separating by more than a few nanometers. They also indicate that transduction channels are mechanically in parallel.
We extended these studies to hair cells of the mammalian hearing organ. Hair bundles of these cochlear hair cells lack a kinocilium, and stereocilia are deflected either by direct connection to an overlying tectorial membrane or by fluid motion in the space below the tectorial membrane. We found that cochlear hair cell stereocilia are far less cohesive than in frogs. Movement of a stereocilium moves a few adjacent stereocilia, but not others, consistent with a stimulus delivered in a more distributed manner to many stereocilia.
Structure of the Tip Link
The major proteins of the tip link are two cadherins that are mutated in hereditary deafness. A parallel dimer of protocadherin 15 extending up from the tip of one stereocilium joins a parallel dimer of cadherin 23 coming down from the side of the adjacent taller stereocilium. To understand the junction between these two dimers, we synthesized and crystallized the N terminus of cadherin 23 by itself and also together with the N terminus of protocadherin 15. These two cadherins join by binding of their N termini in an "extended-handshake" configuration. With steered molecular dynamics simulations, we explored the force needed to unfold a single strand and the force for unbinding. For unfolding, molecular dynamics simulations highlighted the importance of three Ca2+ ions situated between each of the 27 extracellular domains. Many mutations that cause inherited deafness occur in the acidic residues that bind these Ca2+ ions.
We used single-molecule force spectroscopy to make direct physical measurements of the unbinding force. These measurements confirmed the molecular dynamics predictions but indicated a relatively modest unbinding force that might be exceeded by loud noise. Modeling suggests that the arrangement of parallel dimers in the tip link considerably increases unbinding force and allows tip links to remain intact for many days, and this idea is being tested.
Identification of New Proteins Important for Hair Cell Function
To identify new proteins of the mechanotransduction apparatus, we used RNA sequencing to determine the pattern of expression of all genes during development in hair cells. From an engineered mouse expressing green fluorescent protein in hair cells, we used fluorescence-activated cell sorting to isolate pure fractions of hair cells and surrounding cells at ages before and during acquisition of mechanosensitivity and sequenced their mRNA. In similar experiments, we identified mRNAs specifically undergoing translation. Selecting a few hundred genes expressed in hair cells but not surrounding cells, with expression increasing during differentiation, revealed many proteins known to be involved in hair cell function and known to be mutated in human inherited deafness. Additional proteins of this group may be similarly involved in hearing and deafness. A public database we created as a general resource for the field has been visited more than 700,000 times.
Some of these studies are supported by the National Institute on Deafness and Other Communication Disorders.
As of March 23, 2016