Myosin and Adaptation by Hair Cells
Authors: Peter G. Gillespie, David P. Corey
Journal: Neuron (1997)

Auditory hair cells respond to mechanical deflections with astonishing sensitivity: at the threshold of human hearing, the hair bundle, the structure responsible for mechanoelectrical transduction, moves only about a tenth of a nanometer. Vestibular hair cells are nearly as sensitive. How do these cells accomplish this feat? Excitatory mechanoelectrical transduction ensues when hair bundles are deflected, opening ion channels called transduction channels that pass cations, including Ca2+ (Figure 1 and Figure 2; reviewed by10Pickles J.O. Corey D.P. Trends Neurosci. 1992; 15: 254-259Abstract Full Text PDF PubMed Scopus (137) Google Scholar). Stimulus energy is directed to these channels by elastic gating springs; tip links, which connect from the tip of a stereocilium to the side of its tallest neighbor, are probably the gating springs. At rest, gating springs bear a modest tension, which ensures that transduction channels are optimally poised for sensitive and rapid modulation. Resting tension may be supplied by the same mechanism that mediates adaptation, the process by which the hair cell restores its sensitivity during a sustained stimulus (reviewed by9Hudspeth A.J. Gillespie P.G. Neuron. 1994; 12: 1-9Abstract Full Text PDF PubMed Scopus (212) Google Scholar).Figure 2Hair-Bundle StructureShow full caption(A) hair bundle from the bullfrog sacculus comprises 50–60 stereocilia and a single kinocilium. Stereocilia range in height from 4–8 μm. In this differential-interference-contrast micrograph, about a dozen stereocilia and a stimulus probe are visible, attached to the bulb of the kinocilium.(B) Phalloidin labeling of another hair cell, to show the distribution of actin in the bundle and apical cell body.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) hair bundle from the bullfrog sacculus comprises 50–60 stereocilia and a single kinocilium. Stereocilia range in height from 4–8 μm. In this differential-interference-contrast micrograph, about a dozen stereocilia and a stimulus probe are visible, attached to the bulb of the kinocilium. (B) Phalloidin labeling of another hair cell, to show the distribution of actin in the bundle and apical cell body. Two models have been advanced for adaptation. One model invokes adaptation motors, each a cluster of myosin molecules located in series with a transduction channel and its gating spring. Adaptation occurs when motors respond to changes in gating-spring tension by moving up or down the actin cytoskeleton, returning tension to near its resting level (7Howard J. Hudspeth A.J. Proc. Natl. Acad. Sci. USA. 1987; 84: 3064-3068Crossref PubMed Scopus (287) Google Scholar, 1Assad J.A. Corey D.P. J. Neurosci. 1992; 12: 3291-3309Crossref PubMed Google Scholar). In the second model, adaptation arises when Ca2+ that enters through an open transduction channel stabilizes a closed state of the channel (3Crawford A.C. Evans M.G. Fettiplace R. J. Physiol. 1989; 419: 405-434PubMed Google Scholar, 4Crawford A.C. Evans M.G. Fettiplace R. J. Physiol. 1991; 434: 369-398PubMed Google Scholar). Unlike the motor model, this model suggests that the sensitivity of the channel to force changes during adaptation. Specific tests of the two models in bullfrog vestibular hair cells have favored the motor model, although adaptation in the turtle's auditory system is sufficiently different to suggest both processes may be at work (11Ricci A.J. Fettiplace R. J. Physiol. (Lond.). 1997; 501: 111-124Crossref Scopus (118) Google Scholar). In this minireview, we employ the motor model to dissect adaptation and describe recent data that have strengthened the case that myosin Iβ is the active component of the adaptation motor. The motor model supposes that tension in a single gating spring is controlled by several dozen myosin molecules, yoked together to generate high force, to reduce noise, and to ensure that the motor never detaches from the cytoskeleton (Figure 3). During negative deflections of a hair bundle, which slacken its gating springs, external force on an adaptation motor is reduced, and a motor expends ATP to climb toward barbed ends of stereociliary actin filaments. Sensitivity is regained—adaptation takes place—because the motor restores gating-spring tension to near its resting level. Adaptation to positive deflections occurs because force exerted on an adaptation motor exceeds the motor's resting force production; myosin molecules slip down actin filaments while still progressing through their ATPase cycles (1Assad J.A. Corey D.P. J. Neurosci. 1992; 12: 3291-3309Crossref PubMed Google Scholar). A useful way to model slipping adaptation considers it as a form of "molecular friction," where the velocity of motor movement depends both on the external force and on the number of attached myosin molecules (Yamoah and Gillespie, 1996 and references therein). This velocity should be inversely proportional to the number of myosin molecules per motor (m), the probability that any given myosin molecule is attached (p), the time each myosin molecule spends during its ATPase cycle in the attached state (ta), and the stiffness of each attached myosin molecule in the motor complex (κm). Differences in slipping adaptation rate—for example, between auditory and vestibular organs—could arise from changes in value of any of these parameters. What properties of adaptation assist us in the characterization of the motor? Because adaptation takes place within actin-based stereocilia, active motor components would logically belong to the myosin superfamily. Stereociliary actin is polarized, with barbed ends at the distal tip, consistent with a model in which myosin molecules climb toward the tip when a gating spring is slackened (1Assad J.A. Corey D.P. J. Neurosci. 1992; 12: 3291-3309Crossref PubMed Google Scholar). Like other myosin ensembles, this climbing velocity should not depend on m, beyond a certain minimum. Each adaptation motor can maximally generate ∼20 pN of force, suggesting that p·m, the number of myosin molecules exerting force at any one time, may be ten or fewer. In bullfrog hair cells, a motor is restrained in positive and negative directions, producing incomplete adaptation (12Shepherd G.M.G. Corey D.P. J. Neurosci. 1994; 14: 6217-6229PubMed Google Scholar, 15Yamoah E.N. Gillespie P.G. Neuron. 1996; 17: 523-533Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); the motor may be tethered by an elastic extent spring. Adaptation can also be incomplete in turtle cochlear hair cells; completeness of adaptation depends on the concentration of intracellular Ca2+ buffer (11Ricci A.J. Fettiplace R. J. Physiol. (Lond.). 1997; 501: 111-124Crossref Scopus (118) Google Scholar), an observation that is not easily reconciled with the concept of an extent spring. Adaptation slows when hair cells are treated with nucleotide or phosphate-analog inhibitors of myosin's ATPase cycle (5Gillespie P.G. Hudspeth A.J. Proc. Natl. Acad. Sci. USA. 1993; 90: 2710-2714Crossref PubMed Scopus (55) Google Scholar, 15Yamoah E.N. Gillespie P.G. Neuron. 1996; 17: 523-533Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In muscle, ADP slows contraction and increases isometric force by increasing the duration of myosin's strongly bound state; a similar effect on ta is seen in hair cells, where ADP and the poorly metabolized adenosine 5′-[β-thio]diphosphate (ADPβS) slow adaptation and increase resting tension. Phosphate analogs, such as vanadate and beryllium fluoride, stabilize a weakly bound state of muscle myosin, slowing ATP hydrolysis and reducing force production by myosin molecules; in hair cells, these analogs both reduce motor force production and slow adaptation (probably by affecting both ta and p). By binding to calmodulin, intracellular Ca2+ accelerates adaptation (14Walker R.G Hudspeth A.J. Proc. Natl. Acad. Sci. USA. 1996; 93: 2203-2207Crossref PubMed Scopus (49) Google Scholar and references therein). When depolarization is used to reduce Ca2+ entry, adaptation to positive or negative displacements is slowed. Likewise, intracellular dialysis with Ca2+ chelators reduces adaptation's rate (11Ricci A.J. Fettiplace R. J. Physiol. (Lond.). 1997; 501: 111-124Crossref Scopus (118) Google Scholar). The model that invokes Ca2+-dependent channel closure easily explains not only these results (3Crawford A.C. Evans M.G. Fettiplace R. J. Physiol. 1989; 419: 405-434PubMed Google Scholar, 4Crawford A.C. Evans M.G. Fettiplace R. J. Physiol. 1991; 434: 369-398PubMed Google Scholar) but also bundle twitches, which may be manifestations of high-sensitivity deflection detection (2Benser M.E. Marquis R.E. Hudspeth A.J. J. Neurosci. 1996; 16: 5629-5643PubMed Google Scholar). The motor model also accounts for the effects of Ca2+, with at least two possible mechanisms. Ca2+ entry could trigger adaptation-motor slipping by decreasing ta (9Hudspeth A.J. Gillespie P.G. Neuron. 1994; 12: 1-9Abstract Full Text PDF PubMed Scopus (212) Google Scholar). Alternatively, Ca2+ could speed adaptation by reducing the stiffness of myosin's force-generating lever arm (8Howard J. Spudich J.A. Proc. Natl. Acad. Sci. USA. 1996; 93: 4462-4464PubMed Google Scholar), thereby reducing κm. Consistent with this mechanism, agents that displace calmodulin from myosin block adaptation without increasing force production (14Walker R.G Hudspeth A.J. Proc. Natl. Acad. Sci. USA. 1996; 93: 2203-2207Crossref PubMed Scopus (49) Google Scholar). Most hair cells respond optimally to a specific stimulus frequency, and the rate of adaptation in the turtle cochlea increases as best frequency increases (11Ricci A.J. Fettiplace R. J. Physiol. (Lond.). 1997; 501: 111-124Crossref Scopus (118) Google Scholar). Adaptation could speed up if higher frequency cells allowed greater Ca2+ entry, perhaps by having more transduction channels per adaptation motor (11Ricci A.J. Fettiplace R. J. Physiol. (Lond.). 1997; 501: 111-124Crossref Scopus (118) Google Scholar). The protein-friction model provides an alternate explanation: m, p, ta, or κm could systematically decrease. An attractive explanation suggests that the number of myosin molecules per adaptation motor decreases as best frequency increases; under constant Ca2+ conditions, force production per adaptation motor therefore may be smaller for high-frequency cells than for those at the low-frequency end of the turtle cochlea. The adaptation motor may be subject to modulation. Intracellular cyclic nucleotides apparently decrease resting tension (11Ricci A.J. Fettiplace R. J. Physiol. (Lond.). 1997; 501: 111-124Crossref Scopus (118) Google Scholar); the motor model might incorporate this observation by suggesting that phosphorylation of myosin molecules within the adaptation motor decreases their force production. Because of their compact size and membrane-binding properties, proteins of the myosin I class are particularly appropriate as adaptation-motor candidates. A degenerate-primer, RT-PCR strategy revealed that the bullfrog's sacculus possesses at least 13 distinct myosin transcripts from 10 genes; six classes (I, II, V, VI, VII, and X) were represented (13Solc C.K. Derfler B.H. Duyk G.M. Corey D.P. Auditory Neurosci. 1994; 1: 63-75Google Scholar). Two members of the myosin I class were observed: myosin Iβ PCR products were abundant, whereas myosin Iα products were rare. Photoaffinity labeling identified bundle myosin molecules of 120, 160, and 230 kDa; these masses correspond closely to those of myosin Iβ, myosin VI, and myosin VIIa, each found in bundles in sufficient numbers to carry out adaptation (6Hasson T. Gillespie P.G. MacDonald R.B. Garcia J. Zhao Y.-d. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (431) Google Scholar and references therein). Although myosin Iα and members of the myosin II and V classes apparently are not present in bundles, other isozymes might have escaped detection so far. Are any of these myosin isozymes situated within stereocilia in a location appropriate for a proposed motor function? Immunofluorescence localization with isoform-specific antibodies showed that myosins Iβ, VI, and VIIa are present in the cell bodies of hair cells, with differences in distribution that presumably relate to functions other than transduction. Modest amounts of myosin VI are distributed throughout stereocilia but show no special concentration in any one region. Myosin VIIa is also distributed throughout stereocilia and is particularly concentrated in a band near the bases of stereocilia, far from the tip links. By contrast, most myosin Iβ is in the distal two-thirds of the stereocilia and is strikingly concentrated at their tips, making this isozyme the best candidate for an adaptation motor (6Hasson T. Gillespie P.G. MacDonald R.B. Garcia J. Zhao Y.-d. Yee A.G. Mooseker M.S. Corey D.P. J. Cell Biol. 1997; 137: 1287-1307Crossref PubMed Scopus (431) Google Scholar and references therein). Furthermore, electron-microscopic localization of myosin Iβ showed it to be particularly concentrated in a zone within 150 nm of the lower and, to a lesser extent, the upper end of the tip link in bullfrog sacculus and utriculus (D. P. C. et al. and R. Baird et al., unpublished data). While location at the upper end of each tip link is expected for an adaptation motor, location at the lower end as well is consistent with a symmetrical transduction complex, in which an ion channel at each end of the tip link is bound to the cytoskeleton by the same protein complex. Although immunocytochemical localization data concur with a role in adaptation for myosin Iβ, we covet still more conclusive evidence. Isozyme-specific, inhibitory antibodies might block adaptation selectively if they could be delivered in high concentration to stereociliary tips, not a simple technical prospect. Depletion of myosin Iβ from hair cells—for example, by supplying antisense oligonucleotides or by knocking out its gene—could perturb adaptation but might also eliminate transduction, complicating interpretation. Similar problems could be faced if dominant-negative forms of myosin Iβ—for example, the tail alone—are introduced into hair cells, either through a recording electrode or by direct expression. Most decisive would be a conditional knockout of myosin Iβ, where motor activity ceased only when a specific inhibitor was supplied. Because convincing experiments have not been reported, identification of the adaptation motor as myosin Iβ must still be seen as tentative. 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.