Peter G. Barr-Gillespie

Molecular architecture of the chick vestibular hair bundle

Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2

Significance Our understanding of the molecular basis of our sense of hearing and balance has improved significantly, although some of the key players in sensory hair cells have yet to be identified. Sensory hair cells depend on extracellular filaments known as tip links to transduce mechanical stimuli into electrical signals. We demonstrate that the tip link protein PCDH15 interacts with two integral member proteins, TMC1 and TMC2, which have recently been put forth as candidates for the mechanotransduction channel. The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15–Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a–Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear.

Accurate label-free protein quantitation with high- and low-resolution mass spectrometers.

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well-correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10(-3) or lower were detected, with a strong falloff below 10(-4) mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate but should be limited to quantitation of proteins of intermediate to higher fractional abundance.

New treatment options for hearing loss

Hearing loss is the most common form of sensory impairment in humans and affects more than 40 million people in the United States alone. No drug-based therapy has been approved by the Food and Drug Administration, and treatment mostly relies on devices such as hearing aids and cochlear implants. Over recent years, more than 100 genetic loci have been linked to hearing loss and many of the affected genes have been identified. This understanding of the genetic pathways that regulate auditory function has revealed new targets for pharmacological treatment of the disease. Moreover, approaches that are based on stem cells and gene therapy, which may have the potential to restore or maintain auditory function, are beginning to emerge.

A Short Splice Form of Xin-Actin Binding Repeat Containing 2 (XIRP2) Lacking the Xin Repeats Is Required for Maintenance of Stereocilia Morphology and Hearing Function

Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cells mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundles proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2s role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.

Assembly of hair bundles, an amazing problem for cell biology

The hair bundle—the sensory organelle of inner-ear hair cells of vertebrates—exemplifies the ability of a cell to assemble complex, elegant structures. Proper construction of the bundle is required for proper mechanotransduction in response to external forces and to transmit information about sound and movement. Bundles contain tightly controlled numbers of actin-filled stereocilia, which are arranged in defined rows of precise heights. Indeed, many deafness mutations that disable hair-cell cytoskeletal proteins also disrupt bundles. Bundle assembly is a tractable problem in molecular and cellular systems biology; the sequence of structural changes in stereocilia is known, and a modest number of proteins may be involved.

The proteome of mouse vestibular hair bundles over development.

Development of the vertebrate hair bundle is a precisely orchestrated event that culminates in production of a tightly ordered arrangement of actin-rich stereocilia and a single axonemal kinocilium. To understand how the protein composition of the bundle changes during development, we isolated bundles from young (postnatal days P4-P6) and mature (P21-P25) mouse utricles using the twist-off method, then characterized their constituent proteins using liquid-chromatography tandem mass spectrometry with data-dependent acquisition. Using MaxQuant and label-free quantitation, we measured relative abundances of proteins in both bundles and in the whole utricle; comparison of protein abundance between the two fractions allows calculation of enrichment in bundles. These data, which are available via ProteomeXchange with identifier PXD002167, will be useful for examining the proteins present in mammalian vestibular bundles and how their concentrations change over development.

Correlation of Actin Crosslinker and Capper Expression Levels with Stereocilia Growth Phases

During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.

The Local Forces Acting on the Mechanotransduction Channel in Hair Cell Stereocilia

In hair cells, mechanotransduction channels are located in the membrane of stereocilia tips, where the base of the tip link is attached. The tip-link force determines the system of other forces in the immediate channel environment, which change the channel open probability. This system of forces includes components that are out of plane and in plane relative to the membrane; the magnitude and direction of these components depend on the channel environment and arrangement. Using a computational model, we obtained the major forces involved as functions of the force applied via the tip link at the center of the membrane. We simulated factors related to channels and the membrane, including finite-sized channels located centrally or acentrally, stiffness of the hypothesized channel-cytoskeleton tether, and bending modulus of the membrane. Membrane forces are perpendicular to the directions of the principal curvatures of the deformed membrane. Our approach allows for a fine vectorial picture of the local forces gating the channel; membrane forces change with the membrane curvature and are themselves sufficient to affect the open probability of the channel.