Confocal-microscopic visualization of membrane addition during synaptic exocytosis at presynaptic active zones of hair cells.
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Animals↗
Biomedical subjects
A J Hudspeth
Publications and source records attributed to A J Hudspeth.
Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.
A hair cell, the sensory receptor of the internal ear, transduces mechanical stimuli into electrical responses. Transduction results from displacement of the hair bundle, a cluster of rod-shaped stereocilia extending from the cell's apical surface. Biophysical experiments indicate that, by producing shear between abutting stereocilia, a bundle displacement directly opens cation-selective transduction channels. Specific models of gating depend on the location of these channels, which has been controversial: although some physiological and immunocytochemical experiments have situated the transduction channels at the hair bundle's top, monitoring of fluorescence signals from the Ca2+ indicator fura-2 has instead suggested that Ca2+ traverses channels at the bundle's base. To examine the site of Ca2+ entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of < 1 micron and a temporal resolution of < 2 ms, to observe hair cells filled with the indicator fluo-3. An unstimulated hair cell showed a "tip blush" of enhanced fluorescence at the hair bundle's top, which we attribute to Ca2+ permeation through transduction channels open at rest. Upon mechanical stimulation, individual stereocilia displayed increased fluorescence that originated near their tips, then spread toward their bases. Our results confirm that mechanoelectrical transduction occurs near stereociliary tips.
Animals↗
Modeling the active process of the cochlea: phase relations, amplification, and spontaneous oscillation.
The high sensitivity and sharp frequency selectivity of acoustical signal transduction in the cochlea suggest that an active process pumps energy into the basilar membrane's oscillations. This function is generally attributed to outer hair cells, but its exact mechanism remains uncertain. Several classical models of amplification represent the load upon the basilar membrane as a single mass. Such models encounter a fundamental difficulty, however: the phase difference between basilar-membrane movement and the force generated by outer hair cells inhibits, rather than amplifies, the modeled basilar-membrane oscillations. For this reason, modelers must introduce artificially either negative impedance or an appropriate phase shift, neither of which is justified by physical analysis of the system. We consider here a physical model based upon the recent demonstration that the basilar membrane and reticular lamina can move independently, albeit with elastic coupling through outer hair cells. The mechanical model comprises two resonant masses, representing the basilar membrane and the reticular lamina, coupled through an intermediate spring, the outer hair cells. The spring's set point changes in response to displacement of the reticular lamina, which causes deflection of the hair bundles, variation of outer hair cell length and, hence, force production. Depending upon the frequency of the acoustical input, the basilar membrane and reticular lamina can oscillate either in phase or in counterphase. In the latter instance, the force produced by hair cells leads basilar-membrane oscillation, energy is pumped into basilar-membrane movement, and an external input can be strongly amplified. The model is also capable of producing spontaneous oscillation. In agreement with experimental observations, the model describes mechanical relaxation of the basilar membrane after electrical stimulation causes outer hair cells to change their length.
Animals↗
Gating-spring models of mechanoelectrical transduction by hair cells of the internal ear.
A sensory receptor of the internal ear, or hair cell, responds to sound or acceleration when this mechanical stimulus deflects the cell's mechanosensitive organelle, or hair bundle. The gating-spring model posits that mechanoelectrical transduction occurs as mechanical force is transmitted through an elastic element, or gating spring, to the molecular gate of each transduction channel; increased tension in the gating spring then promotes the channel's transition from a closed to an open state. Electrophysiological and micromechanical data from a variety of hair cells, both in vivo and in vitro, confirm that the stimulus dependence of channel open probability and bundle stiffness are quantitatively consistent with the model. The results accord still better, however, with an extended formulation including channel transitions among one open and two closed states. In addition to providing a derivation of this three-state model, this review delineates several experimentally testable predictions of gating-spring models.
Acoustic Stimulation↗
Molecular cloning of a myosin I beta isozyme that may mediate adaptation by hair cells of the bullfrog's internal ear.
The internal ear's sensory receptor, or hair cell, responds when stimuli deflect its mechanoreceptive hair bundle. As a hair cell adapts to sustained stimulation, mechanical adjustments within the bundle reset its position of sensitivity. Because several lines of experimentation suggest that a form of myosin I mediates adaptation, we endeavored to clone cDNAs encoding this motor molecule. By using degenerate oligonucleotide primers based upon the deduced amino acid sequence for mammalian myosin I beta, we performed reverse transcription and polymerase chain reactions (PCRs) to produce a candidate cDNA from polyadenylylated mRNA isolated from the frog's brain. The resultant product was used to probe a cDNA library, from which were isolated clones encoding an approximately 119-kDa isozyme of myosin I beta. PCR amplification disclosed the presence of mRNA encoding the same isozyme in tissue from the bullfrog's sacculus, an organ of the internal ear. When expressed as a bacterial fusion protein, a domain from the tail region of this form of myosin I was recognized by monoclonal antibodies that react with myosin I in hair bundles. This cloned approximately 119-kDa isozyme of myosin I is accordingly a candidate to be the motor molecule responsible for the adaptation of mechanoelectrical transduction by hair cells.
Adaptation, Physiological↗
Clustering of Ca2+ channels and Ca(2+)-activated K+ channels at fluorescently labeled presynaptic active zones of hair cells.
Electrical resonance, which in some hair cells provides a mechanism for frequency tuning, is mediated by clusters of Ca2+ channels and Ca(2+)-activated K+ channels that have been proposed to occur at presynaptic active zones. To localize Ca2+ channels on the cellular surface, we loaded hair cells from the frog's sacculus with the Ca2+ indicator fluo-3 and imaged them by fluorescence confocal microscopy. When a cell was depolarized, we observed on its basolateral surface several foci of transiently enhanced fluorescence due to local Ca2+ influx. After protracted recording, each cell displayed on average 18 brightly and permanently fluorescent spots at the same positions. We mapped these spots in four hair cells and compared their locations with those of presynaptic active zones, as determined from transmission electron micrographs of serial sections through the same cells. The results demonstrated that enhanced fluo-3 fluorescence marks active zones. Measurement of currents through membrane patches at fluorescently labeled active zones demonstrated that both voltage-activated Ca2+ channels and Ca(2+)-activated K+ channels occur there. These results confirm that the ion channels involved in electrical tuning and synaptic transmission by hair cells cluster together at presynaptic active zones.
Aniline Compounds↗
Pulling springs to tune transduction: adaptation by hair cells.
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Acclimatization↗
Auditory illusions and the single hair cell.
Like our other senses, the auditory system can produce illusions. Prominent among these are distortion products: when listening to two tones, one of frequency f1 and the second of a higher frequency f2, an individual may hear not only these primary tones, but also a difference tone of frequency f2 - f1, a sum tone of frequency f2 + f1, and combination tones of frequencies such as 2f1 - f2 and 2f2 - f1. Discovered by Tartini early in the eighteenth century, these illusory sounds are sufficiently conspicuous that they were employed to carry melodies in classical compositions. Distortion products originate within the cochlea, where they manifest themselves in the basilar membrane's vibration. Here we demonstrate distortion products in individual hair cells of the bullfrog's sacculus, where they emerge from a nonlinearity inherent in the mechanoelectrical transduction process. In addition to offering an explanation for cochlear distortion products, our results suggest that the mechanical properties of hair bundles significantly influence the basilar membrane's motion.
Animals↗
Adenine nucleoside diphosphates block adaptation of mechanoelectrical transduction in hair cells.
By adapting to sustained stimuli, hair cells in the internal ear retain their sensitivity to minute transient displacements. Because one model for adaptation asserts that this process is mediated by a myosin isozyme, we reasoned that we should be able to arrest adaptation by interfering with myosin's ATPase cycle though introduction of ADP into hair cells. During tight-seal, whole-cell recordings of transduction currents in cells isolated from bullfrog (Rana catesbeiana) sacculus, dialysis with 5-25 mM ADP gave variable results. In half of the cells examined, the rate of adaptation remained unchanged or even increased; adaptation was blocked in the remaining cells. Because we suspected that the variable effect of ADP resulted from the conversion of ADP to ATP by adenylate kinase, we employed the ADP analog adenosine 5'-[beta-thio]diphosphate (ADP[beta S]), which is not a substrate for adenylate kinase. Adaptation consistently disappeared in the presence of 1-10 mM ADP[beta S]; in addition, the transduction channels' open probability at rest grew from approximately 0.1 to 0.8 or more. Both effects could be reversed by 2 mM ATP. When used in conjunction with the adenylate kinase inhibitor P1,P5-bis(5'-adenosyl) pentaphosphate (Ap5A), ADP had effects similar to those of ADP[beta S]. These results suggest that adaptation by hair cells involves adenine nucleotides, and they lend support to the hypothesis that the adaptation process is powered by a myosin motor.
Adenosine Diphosphate↗
Calmodulin and calmodulin-binding proteins in hair bundles.
Calcium ion plays an important role in the hair cell's mechanoelectrical transduction process; in particular, Ca2+ controls adaptation to protracted mechanical stimuli. Because calmodulin is a ubiquitous intracellular receptor for Ca2+ and has been shown to accumulate at the tips of stereocilia, we determined its concentration and identified the proteins with which it interacts in the hair bundle. By performing quantitative immunoblot analysis on isolated bundles, we ascertained that the average concentration of calmodulin within each stereocilium is approximately 70 microM. Extraction experiments disclosed that, in the presence of 20 microM Ca2+, 50% of the calmodulin is bound to detergent-soluble receptors. To distinguish these receptors, we developed an assay that utilizes calmodulin crosslinked to alkaline phosphatase. This technique is approximately 100-fold more sensitive than calmodulin-binding assays that employ 125I- or biotin-labeled calmodulin. When used with chemiluminescence detection in a blot-overlay assay, the calmodulin-alkaline phosphatase conjugate identified hair-bundle proteins of molecular masses 25, 35, 145, 175, 240, and 350 kDa. We examined the subcellular distribution of these receptors; all but the 240-kDa molecule are soluble in a nonionic detergent. The relatively high concentration of calmodulin and the presence of several calmodulin-binding proteins provide evidence for a role of calmodulin in hair bundles.
Alkaline Phosphatase↗
Displacement-clamp measurement of the forces exerted by gating springs in the hair bundle.
Mechanical stimuli applied to the hair bundle of a hair cell are communicated to the transduction channels by gating springs, elastic elements that are stretched when the bundle is displaced toward its tall edge. To quantify the magnitude and time dependence of the forces exerted by gating springs, we have developed a displacement-clamp system that constrains a bundle's motion while measuring the forces that the bundle produces during adaptation to mechanical stimuli, in response to channel blockage, and upon destruction of the gating springs. Our results suggest that each gating spring exerts a tension of approximately 8 pN in the resting bundle and can sustain at least 4-13 pN of additional tension. The experiments provide further evidence that the gating springs account for at least one-third of the hair bundle's dynamic stiffness and that a force of approximately 100 fN is sufficient to open a single transduction channel.
Animals↗
Hair-bundle stiffness dominates the elastic reactance to otolithic-membrane shear.
Efficient transduction by acousticolateralis organs requires that a stimulus force principally deflect hair bundles, rather than flex other structural elements. Hair bundles might therefore be expected to provide a large fraction of the impedence to shear motions of otolithic membranes and other accessory structures. We measured the stiffness for shear motions of the bullfrog's saccular otolithic membrane, and determined the stiffness due to a single hair bundle and its associated extracellular filaments; this component is termed the elemental stiffness. Stiffness measurements were made by displacing the base of a flexible probe whose tip was coupled to the otolithic membrane, and simultaneously measuring the flexion of the probe and the displacement of the membrane. The average elemental stiffness, about 1350 microN.m-1, only modestly exceeded the stiffness of individual hair bundles. The hair bundles therefore provide the dominant component of stiffness in the bullfrog's sacculus, and thus account for a significant component of impedance to otolithic-membrane shear. As a corollary, stiffness changes or active movements in hair bundles should influence the mechanical responses of this and other receptor organs.
Animals↗
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 bullfrog's 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.
Adenosine Triphosphate↗
To fuse or not to fuse?
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Biophysical Phenomena↗
Hair-bundle mechanics and a model for mechanoelectrical transduction by hair cells.
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Animals↗
Chemiluminescence detection of proteins from single cells.
The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, chemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.
Alkaline Phosphatase↗
Localization of the hair cell's transduction channels at the hair bundle's top by iontophoretic application of a channel blocker.
In order to understand how the hair cell's mechanoelectrical transduction channels are gated during mechanical stimulation, it is essential to determine their location with respect to the hair bundle's constituent stereocilia. We localized the transduction channels by focally blocking receptor currents with iontophoretically ejected gentamicin, an aminoglycoside antibiotic that acts as a reversible channel blocker. The drug was most effective when directed at the top of a hair bundle, whereas application at the bundle's bottom or at the cuticular plate had little or no effect. Computer simulations of blocking agreed with experimental data only when the transduction channels were hypothesized to occur near the bundle's top. These results confirm that the hair cell's transduction channels are located near the stereociliary tips.
Animals↗
High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins.
The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.
Animals↗