Analysis of the Proteome of Hair-Cell Stereocilia by Mass Spectrometry.

Characterization of proteins that mediate mechanotransduction by hair cells, the sensory cells of the inner ear, is hampered by the scarcity of these cells and their sensory organelle, the hair bundle. Mass spectrometry, with its high sensitivity and identification precision, is the ideal method for determining which proteins are present in bundles and what proteins they interact with. We describe here the isolation of mouse hair bundles, as well as preparation of bundle protein samples for mass spectrometry. We also describe protocols for data-dependent (shotgun) and parallel reaction monitoring (targeted) mass spectrometry that allow us to identify and quantify proteins of the hair bundle. These sensitive methods are particularly useful for comparing proteomes of wild-type mice and mice with deafness mutations affecting hair-bundle proteins.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Molecular basis of mechanosensory transduction.

Mechanotransduction - a cell's conversion of a mechanical stimulus into an electrical signal - reveals vital features of an organism's environment. From hair cells and skin mechanoreceptors in vertebrates, to bristle receptors in flies and touch receptors in worms, mechanically sensitive cells are essential in the life of an organism. The scarcity of these cells and the uniqueness of their transduction mechanisms have conspired to slow molecular characterization of the ensembles that carry out mechanotransduction. But recent progress in both invertebrates and vertebrates is beginning to reveal the identities of proteins essential for transduction.

Plasma membrane Ca2+-ATPase isoform 2a is the PMCA of hair bundles.

Mechanoelectrical transduction channels of hair cells allow for the entry of appreciable amounts of Ca(2+), which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca(2+) that enters transduction channels is extruded by the plasma membrane Ca(2+)-ATPase (PMCA), a Ca(2+) pump that is highly concentrated in hair bundles and may be essential for normal hair cell function. Because PMCA isozymes and splice forms are regulated differentially and have distinct biochemical properties, we determined the identity of hair bundle PMCA in frog and rat hair cells. By screening a bullfrog saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones as well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immunoprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes and that PMCA2a is the only PMCA present in hair bundles. This complete segregation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular hair cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cells and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca(2+)-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca(2+) pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca(2+) pumping in bundles.

High-resolution structure of hair-cell tip links.

Transduction-channel gating by hair cells apparently requires a gating spring, an elastic element that transmits force to the channels. To determine whether the gating spring is the tip link, a filament interconnecting two stereocilia along the axis of mechanical sensitivity, we examined the tip link's structure at high resolution by using rapid-freeze, deep-etch electron microscopy. We found that the tip link is a right-handed, coiled double filament that usually forks into two branches before contacting a taller stereocilium; at the other end, several short filaments extend to the tip link from the shorter stereocilium. The structure of the tip link suggests that it is either a helical polymer or a braided pair of filamentous macromolecules and is thus likely to be relatively stiff and inextensible. Such behavior is incompatible with the measured elasticity of the gating spring, suggesting that the gating spring instead lies in series with the helical segment of the tip link.

Engineering of the myosin-ibeta nucleotide-binding pocket to create selective sensitivity to N(6)-modified ADP analogs.

Distinguishing the cellular functions carried out by enzymes of highly similar structure would be simplified by the availability of isozyme-selective inhibitors. To determine roles played by individual members of the large myosin superfamily, we designed a mutation in myosin's nucleotide-binding pocket that permits binding of adenine nucleotides modified with bulky N(6) substituents. Introduction of this mutation, Y61G in rat myosin-Ibeta, did not alter the enzyme's affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate. We also synthesized several N(6)-modified ADP analogs that should bind to and inhibit mutant, but not wild-type, myosin molecules. Several of these N(6)-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Ibeta; in doing so, these analogs locked Y61G myosin-Ibeta tightly to actin. N(6)-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Ibeta. Furthermore, a small fraction of inhibited Y61G molecules was sufficient to block filament motility mediated by mixtures of wild-type and Y61G myosin-Ibeta. Introduction of Y61G myosin-Ibeta molecules into a cell should permit selective inhibition by N(6)-modified ADP analogs of cellular processes dependent on myosin-Ibeta.

Localization of myosin-Ibeta near both ends of tip links in frog saccular hair cells.

Current evidence suggests that the adaptation motor of mechanoelectrical transduction in vertebrate hair cells is myosin-Ibeta. Previously, confocal and electron microscopy of bullfrog saccular hair cells using an anti-myosin-Ibeta antibody labeled the tips of stereocilia. We have now done quantitative immunoelectron microscopy to test whether myosin-Ibeta is enriched at or near the side plaques of tip links, the proposed sites of adaptation, using hair bundles that were serially sectioned parallel to the macular surface. The highest particle density occurred at stereocilia bases, close to the cuticular plate. Also, stereocilia of differing lengths had approximately the same number of total particles, suggesting equal targeting of myosin-Ibeta to all stereocilia. Finally, particles tended to clump in clusters of two to five particles in the distal two-thirds of stereocilia, suggesting a tendency for self-assembly of myosin-Ibeta. As expected from fluorescence microscopy, particle density was high in the distal 1 micrometer of stereocilia. If myosin-Ibeta is the adaptation motor, a difference should exist in particle density between regions containing the side plaque and those excluding it. Averaging of particle distributions revealed two regions with approximately twice the average density: at the upper ends of tip links in a 700-nm-long region centered approximately 100 nm above the side plaque, and at the lower ends of tip links within the tip plaques. Controls demonstrated no such increase. The shortest stereocilia, which lack side plaques, showed no concentration rise on their sides. Thus, the specific localization of myosin-Ibeta at both ends of tip links supports its role as the adaptation motor.

Myosin Ibeta is located at tip link anchors in vestibular hair bundles.

Recent studies have suggested that myosin Ibeta mediates the adaptation of mechanoelectrical transduction in vestibular hair cells. An important prediction of this hypothesis is that myosin Ibeta should be found in the side insertional plaque, an osmiophilic hair bundle structure that anchors tip links and is thought to house the adaptation motor. To determine whether myosin Ibeta was situated properly to perform adaptation, we used immunofluorescence and immunoelectron microscopy with the monoclonal antibody mT2 to examine the distribution of myosin Ibeta in hair bundles of the bullfrog utricle. Although utricular hair cells differ in their rates and extent of adaptation [Baird RA (1994) Comparative transduction mechanisms of hair cells in the bullfrog utriculus. II. Sensitivity and response dynamics to hair bundle displacement. J Neurophysiol 71:685-705.], myosin Ibeta was present in all hair bundles, regardless of adaptation kinetics. Confirming that, nevertheless, it was positioned properly to mediate adaptation, myosin Ibeta was found at significantly higher levels in the side insertional plaque. Myosin Ibeta was also present at elevated levels at the second tip link anchor of a hair bundle, the tip insertional plaque, found at the tip of a stereocilium. These data support myosin Ibeta as the adaptation motor and are consistent with the suggestion that the motor serves to restore tension applied to transduction channels to an optimal level, albeit with different kinetics in different cell types.

Plasma membrane Ca2+-ATPase extrudes Ca2+ from hair cell stereocilia.

Mechanically sensitive hair cells of the auditory and vestibular systems use Ca2+ to control adaptation of mechanical transduction, to effect frequency tuning, to trigger neurotransmitter release, and to mediate efferent synaptic signaling. To determine the role that pumps play in regulation of Ca2+ in the hair bundle, the organelle responsible for mechanoelectrical transduction, we localized and quantified the plasma membrane Ca2+-ATPase (PMCA) of the bundle. We found that each hair bundle contains approximately 10(6) PMCA molecules or approximately 2000 per square micrometer of bundle membrane and that PMCA is the principal calmodulin binding protein of the bundle. Consistent with biochemical estimates of PMCA density, we measured with extracellular Ca2+-selective electrodes a substantial Ca2+ efflux from bundles. The number of bundle Ca2+ pumps and magnitude of resting Ca2+ efflux suggested that PMCA should generate a substantial membrane current as bundles expel Ca2+. Measurement of whole-cell currents revealed a transduction-dependent outward current that was consistent with the activity of PMCA. Finally, dialysis of hair cells with PMCA inhibitors led to a large increase in the concentration of Ca2+ in bundles, which suggests that PMCA plays a major role in regulating bundle Ca2+ concentration. Our data further indicate that PMCA could elevate the extracellular Ca2+ concentration close to hair bundles above the low level found in bulk endolymph.

Unconventional myosins in inner-ear sensory epithelia.

To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Ibeta is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Ibeta is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Ibeta, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Ibeta probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

Improved electrophoresis and transfer of picogram amounts of protein with hemoglobin.

During electrophoresis and electroblotting to transfer membranes, picogram amounts of protein can react irreversibly with the polyacrylamide matrix, preventing complete electrophoresis and efficient electroblotting. Bovine hemoglobin, but not other potential carrier proteins, mitigates this protein loss by migrating with or ahead of other proteins and scavenging reactive groups. Inclusion of 5 micrograms of hemoglobin in sample wells increases by 4-fold the amount of a radiolabeled test protein, myosin I beta, found at its appropriate 120-kDa position in sodium dodecyl sulfate-polyacrylamide gels. For electroblotting, incubating the gel with 0.25 mg/ml hemoglobin prior to transfer improves mobilization of picogram amounts of radiolabeled myosin I beta out of the gel by about 6-fold. For picogram amounts of proteins, therefore, approximately 20-fold more protein transfers to a blotting membrane when hemoglobin is used during both electrophoresis and transfer. This effect is general: transfer of radiolabeled Drosophila embryo proteins is improved dramatically by including hemoglobin in the pretransfer incubation solution. We suggest that electroblot-based detection of small amounts of protein, particularly when in the absence of other potential carrier proteins, can be improved substantially by using hemoglobin.

Regeneration of broken tip links and restoration of mechanical transduction in hair cells.

A hair cell's tip links are thought to gate mechanoelectrical transduction channels. The susceptibility of tip links to acoustic trauma raises questions as to whether these fragile structures can be regenerated. We broke tip links with the calcium chelator 1,2-bis(O-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid and found that they can regenerate, albeit imperfectly, over several hours. The time course of tip-link regeneration suggests that this process may underlie recovery from temporary threshold shifts induced by noise exposure. Cycloheximide does not block tip-link regeneration, indicating that new protein synthesis is not required. The calcium ionophore ionomycin prevents regeneration, suggesting regeneration normally may be stimulated by the reduction in stereociliary Ca2+ when gating springs rupture and transduction channels close. Supporting the equivalence of tip links with gating springs, mechanoelectrical transduction returns over the same time period as tip links; strikingly, adaptation is substantially reduced, even 24 hr after breaking tip links.

Phosphate analogs block adaptation in hair cells by inhibiting adaptation-motor force production.

To ensure optimal sensitivity for mechanoelectrical transduction, hair cells adapt to prolonged stimuli using active motors. Adaptation motors are thought to employ myosin molecules as their force-producing components. We find that beryllium fluoride, vanadate, and sulfate, phosphate analogs that inhibit the ATPase activity of myosin, inhibit adaptation by abolishing motor force production. Phosphate analogs interact with a 120-kDa bundle protein, most likely myosin 1 beta, in a manner that coincides with their effects on adaptation. Features of transduction following inhibition of motor force production suggest that the gating and extent springs of the hair cell orient in parallel at rest and that the negative limit of adaptation arises when force in the stretched extent spring matches the force output of the adaptation motor.

Feeling force: mechanical transduction by vertebrates and invertebrates.

Detection of mechanical stimuli requires conversion of the signal's inherent information into neuronal electrical signals. Studies of vertebrate hair cells suggest that this is accomplished by elastic links between stereocilia that control the opening of ion channels. Molecular genetics in Caenorhabditis elegans has identified candidate proteins that may be responsible for similar functions in this organism.

Molecular machinery of auditory and vestibular transduction.

The hunt for molecules that conduct mechanoelectrical transduction in hair cells has recently intensified. A hair cell's transduction apparatus adapts to sustained stimuli, and myosin I beta and myosin VIIA have been advanced as candidates for the motor that mediates this process. The identity of the transduction channel remains unknown, although a viable suggestion proposes that it belongs to the amiloride-sensitive Na+ channel family.