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 stereocilia 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. If the tip links hold a bundle together, then channels are largely in series and the opening of one relaxes the tension on channels in the adjacent shorter stereocilium. If other links hold the bundle together, then the channels are mechanically in parallel, and a given deflection of the bundle stimulates all channels equally and independently.
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 single kinocilium. Chemically cutting the tip links does not change this motion, suggesting that something other than tip links holds 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, 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 is thought to join 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.
Direct physical measurements of the unbinding force, using single-molecule force spectroscopy, 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.
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. We explored the function of several and found new proteins involved in the structure and function of the hair bundle and implicated in inherited deafness. A public database we created to serve as a general resource for the field, presenting these data and others, has been visited more than 400,000 times.
Turnover of Hair Cell Proteins
Hair cells in humans do not divide and are thought to exist for the life of the individual. At the same time, each cell must have a mechanism to degrade and replace its proteins, even as it maintains the shape of its hair bundle and the function of its mechanosensory apparatus. In collaboration with Claude Lechene (Harvard Medical School), we used a new method, multi-isotope imaging mass spectroscopy (MIMS), to study the synthesis and turnover of proteins in hair cells. MIMS forms an image representing the proportion of newly synthesized protein at each location in a sample, with resolution approaching that of electron microscopy.
We found that the stereocilia protein of adult frogs and mice is remarkably stable. In contrast to hair cell bodies, which show 40–60 percent turnover in one month, stereocilia show less than 20 percent turnover over a period of five months. A striking exception is the top half micron of each stereocilium, which displays rapid turnover. These results, and additional experiments using independent methods, are at odds with an accepted model suggesting that all the actin of a stereocilium is repolymerized in 2–3 days. Thus, the hair cell may build its stereocilia to proper tolerances and then leave them alone, except for a dynamic compartment that contains the mechanotransduction apparatus.
Some of these studies are supported by the National Institute on Deafness and Other Communication Disorders.
As of February 23, 2015