Peter G. Gillespie

Molecular basis of mechanosensory transduction

Mechanotransduction — a cells conversion of a mechanical stimulus into an electrical signal — reveals vital features of an organisms 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.

Cadherin 23 is a component of the tip link in hair-cell stereocilia

Mechanoelectrical transduction, the conversion of mechanical force into electrochemical signals, underlies a range of sensory phenomena, including touch, hearing and balance. Hair cells of the vertebrate inner ear are specialized mechanosensors that transduce mechanical forces arising from sound waves and head movement to provide our senses of hearing and balance; however, the mechanotransduction channel of hair cells and the molecules that regulate channel activity have remained elusive. One molecule that might participate in mechanoelectrical transduction is cadherin 23 (CDH23), as mutations in its gene cause deafness and age-related hearing loss. Furthermore, CDH23 is large enough to be the tip link, the extracellular filament proposed to gate the mechanotransduction channel. Here we show that antibodies against CDH23 label the tip link, and that CDH23 has biochemical properties similar to those of the tip link. Moreover, CDH23 forms a complex with myosin-1c, the only known component of the mechanotransduction apparatus, suggesting that CDH23 and myosin-1c cooperate to regulate the activity of mechanically gated ion channels in hair cells.

A chemical-genetic strategy implicates myosin-1c in adaptation by hair cells

Myosin-1c (also known as myosin-Ibeta) has been proposed to mediate the slow component of adaptation by hair cells, the sensory cells of the inner ear. To test this hypothesis, we mutated tyrosine-61 of myosin-1c to glycine, conferring susceptibility to inhibition by N(6)-modified ADP analogs. We expressed the mutant myosin-1c in utricular hair cells of transgenic mice, delivered an ADP analog through a whole-cell recording pipette, and found that the analog rapidly blocked adaptation to positive and negative deflections in transgenic cells but not in wild-type cells. The speed and specificity of inhibition suggests that myosin-1c participates in adaptation in hair cells.

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 Ca2+, which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca2+ that enters transduction channels is extruded by the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ 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 Ca2+-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca2+ pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca2+pumping in bundles.

Identification of a 120 kd hair-bundle myosin located near stereociliary tips

By adapting to sustained stimuli, hair cells of the internal ear maintain their optimal sensitivity to minute displacements. Biophysical experiments have suggested that adaptation is mediated by a molecular motor, most likely a member of the myosin family. To provide direct evidence for the presence of myosin isozymes in hair bundles, we used photoaffinity labeling with vanadate-trapped uridine and adenine nucleotides to identify proteins of 120, 160, and 230 kd in a preparation of hair bundles purified from the bullfrogs sacculus. The photoaffinity labeling properties of these proteins, particularly the 120 kd protein, resembled those of other well-characterized myosins. A 120 kd hair-bundle protein was also recognized by a monoclonal antibody directed against a vertebrate myosin I isozyme. Immunofluorescence microscopy localized this protein near the beveled top edge of the hair bundle, the site of mechanoelectrical transduction and adaptation.

Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.

Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells.

Hair Cells Require Phosphatidylinositol 4,5-Bisphosphate for Mechanical Transduction and Adaptation

After opening in response to mechanical stimuli, hair cell transduction channels adapt with fast and slow mechanisms that each depend on Ca(2+). We demonstrate here that transduction and adaptation require phosphatidylinositol 4,5-bisphosphate (PIP(2)) for normal kinetics. PIP(2) has a striking distribution in hair cells, being excluded from the basal region of hair bundles and apical surfaces of frog saccular hair cells. Localization of a phosphatidylinositol lipid phosphatase, Ptprq, to these PIP(2)-free domains suggests that Ptprq maintains low PIP(2) levels there. Depletion of PIP(2) by inhibition of phosphatidylinositol 4-kinase or sequestration by aminoglycosides reduces the rates of fast and slow adaptation. PIP(2) and other anionic phospholipids bind directly to the IQ domains of myosin-1c, the motor that mediates slow adaptation, permitting a strong interaction with membranes and likely regulating the motors activity. PIP(2) depletion also causes a loss in transduction current. PIP(2) therefore plays an essential role in hair cell adaptation and transduction.

Myosin and adaptation by hair cells

Conclusive identification of the adaptation motor is not entirely academic. For example, a rational approach to identifying other members of the transduction apparatus is first to identify one component—the adaptation motor seems most likely—and then to identify interacting proteins using biochemical and molecular biological methods. Furthermore, once we identify the active component of the motor, we can dissect adaptation by studying purified proteins in biochemical or mechanical assays. To understand how Ca2+ and force regulate adaptation-motor activity, it may be best to examine directly the in vitro effects of these modulators both on ATPase activity and on single-molecule mechanics of the myosin isozyme responsible. The time is right to begin this analysis with myosin Iβ.***To whom correspondence should be addressed.

Harmonin Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells

In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement.