Neuron,

Vol. 7, 985-994, December,

1991, Copyright

0 1991 by Cell Press

Tip-link Integrity and Mechanical Transduction in Vertebrate Hair Cells John A. Assad,*+ Gordon M. G. Shepherd,* and David P. Corey*511 *Department of Neurobiology *Program in Neuroscience Harvard Medical School Boston, Massachusetts 02115 SDepartment of Neurology Massachusetts General Hospital Boston, Massachusetts 02114 IINeuroscience Group Howard Hughes Medical Institute

Summary An attractive hypothesis for hair-cell transduction is that fine, filamentous “tip links” pull directly on mechanically sensitive ion channels located at the tips of the stereocilia. We tested the involvement of tip links in the transduction process by treating bundles with a BAPTAbuffered, low-Ca*+ saline (1O-v M). BAPTA abolished the transduction current in a few hundred milliseconds. BAPTA treatment for a few seconds eliminated the tip links observed by either scanning or transmission electron microscopy. BAPTA also eliminated the voltagedependent movement and caused a positive bundle displacement of 133 nm, in quantitative agreement with a model for regulation of tension. We conclude that tip links convey tension to the transduction channels of hair cells. Introduction When the mechanosensitive bundle of a vertebrate hair cell is displaced in the positive direction (toward the taller stereocilia), transduction channels open and allow the flow of positive ions into the cell. The opening of a channel is thought to result from an increase of mechanical tension on the channel protein itself. The principal evidence for direct mechanical gating of these channels is that the transduction process is extremely rapid (Corey and Hudspeth, 1979a), that the opening and closing rates depend on the size of the stimulus (Corey and Hudspeth, 1983; Crawford et al., 1989), and that the mechanical complianceof a bundle includes a component that matches the opening of the channels (Howard and Hudspeth, 1988). In this view, the open probability is a direct function of tension, conveyed by an elastic “gating spring.” A peculiarity of the gating kinetics is that channel opening is progressively speeded by larger positive displacements, whereas the closing rate is independent of the stimulus for sufficiently large negative displacements. + Present address: Department of Physiology and Center for Visual Science, University of Rochester, Rochester, New York 14642.

This suggests that the gating springs are not rigid elements, but can be slack-that they can pull but not push on the channels (Corey and Hudspeth, 1983). The structural correlate of this process has not been well established. A simple model has evolved from several independent observations. First, measurement of current flow near moving bundles indicated thatthetransductionchannelsareatornearthetipsof the stereocilia (Hudspeth, 1982). While this has been challenged by measurements with a Ca*+ indicator dye (Ohmori, 1988), two additional experirnents have corroborated the localization of the channels at the tips (Huang and Corey, 1990, Biophys. Sot., abstract; Jaramillo and Hudspeth, 1991). Second, the discovery of fine filaments between the tips of adjacent stereocilia led to the suggestion that these “tip links”were the actual mechanical linkages to the channels (Pickles et al., 1984). The geometry of the bundle is such that excitatory displacements would stretch the tip links and apply tension to the channels; inhibitory displacements would relax them. All vertebrate hair cells so far examined-from different species and different organs whose stereociliary morphology may otherwise vary-possess tip links. The tip-lr,nk hypothesis for transduction is thus extremely attractive, yet direct evidence for it is limited. In this paper we directly implicate the tip links in the transduction process, with the finding that a brief treatment of low extracellular Ca2+ destroys both the tip links and the mechanical sensitivity. In dissociated cells, moreover, low Ca*+ both abolishes the voltagedependent bundle movement driven by the cells’ active regulation of gating spring tension and causes the bundle to relax forward by 133 nm, 1ir-rquantitative agreement with the idea that low Ca2+ destroys transduction by cutting the attachments to the ion channels.

Results Abolition

of Transduction

by low Ca*+

It has long been recognized that Ca*+ in the solution bathingthe hair bundles is required for hair-cell transduction (Sand, 1975; Corey and Hudspeth, 1979b; Crawford et al., 1991). While earlier experiments suggested that Ca*+ might carry the receptor current (Sand, 1975), more recent work views Ca2+ as a necessary cofactor for the transduction apparatus (Crawford et al., 1991). We have reexamined the Ca2+ dependence with whole-cell voltage clamp, direct bundle stimulation, and rapid application of tes,t solutions. Figure 1 shows transduction currents of single cells in response to a triangle-wave stimulus of 1.0 pm peakto-peak amplitude; this was a saturating stimulus for these cells. The bath contained a normal frog saline with 4 m M Ca*+. Halfway through the record, a lowCa*+ saline (lOmg M; buffered with 5 m M BAPTA) was

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I

I

I

Time Figure 1. Abolition

of Transduction

1

(set)

by Low Ca’+

(A-C) Transduction current in 3 different cells elicited by a 1 pm peak-to-peak, tnangk-wave displacement ot the bundle (bottom trace) A 5 m M BAPTA solution was delivered by a pressure pipette positioned about 20 @ m from the cell. at the time Indicated by the bar In all 3 cases, transduction current was abolished by the BAPTA treatment and did not return for the duration ot the recording, usually several minutes after the exposure to low Ca2+. Membrane potential was maintained at -80 mV by the patch clamp. The bath contained the external recording solution, with 4 m M Caz+.

puffed onto the cell from a distance of about 20 pm. The bundlewas held firmly bythe stimulus probeand continued to be driven by the triangle-wave stimulus. As the wave of low Ca2+ reached the bundle, the transduction currentwas initially increased by about lOO%, a consequence of relieving a voltage-dependent block of the channels by Caz+ (Assad and Corey, unpublished data; Crawford et al., 1991). In as little as 50 ms, however, the transduction currentwas abolished, and it was not restored even several minutes after the BAPTA solution diffused away. In Figures IA and IB the total membrane current increased, at least transiently, during the exposure to low Ca*+. This increase is due, at least in part, to an observed shift in the activation of basolaterally situated voltage-dependent Ca2+ channels (Roberts et al., 1990) to more negative potentialsperhaps as a result of alleviation of screening charge-coupled with an increased monovalent ion flux through these channels (Assad and Corey, unpublished data; Hess et al., 1986). We and others have found that lowering the Ca2+ concentration inside the stereocilia shifts the I(X)

curve-which relates receptor current to displacements-toward more negative positions, so that more channels are open at rest (Assad et al., 1989; Crawford et al., 1989, 1991). In extreme cases, it is possible that such a shift is so large that all the channels are open with zerodisplacement and that a cell appears insensitive to mechanical stimuli, while in fact a sufficiently large negative displacement could close channels. That does not appear to account for the loss of sensitivity in this experiment, because there was not evidence of a progressive shift of the I(X) curve, because the stimulus included large negative excursions that were ineffectual in closing channels, and because the effect was not reversible upon return to normal Ca*concentration. It seems more likely that some part of the transduction apparatus was actually broken by lowering the Ca*+ concentration. Abolition of Tip links by low CaZ+ To determinewhat part of the transduction apparatus was broken, hair cells were similarly treated with low Ca*+ and prepared for scanning electron microscopy

Hair Cell Transduction 987

Control

Figure 2. Scanning Electron Micrographs of Stereocilia Treated with CaZ+ or BAPTA Ca2+-treated sacculi (left) were dissected in a 0.1 m M Caz+ solution and then placed in 4 m M Ca*+ before fixation in 4 m M Caz+. BAPTA-treated sacculi (right) were dissected in the same way, but placed in a 5 m M BAPTA solution for 10 s before fixation in 4 m M Caz+. Bar, 500 nm.

(SEM). Sacculi were dissected as for physiological experiments and treated to remove their otolithic membranes, but were not dissociated. Experimental sacculi were transferred to a solution buffered with 5 mM BAPTA for about 10 s and then immediately returned to normal saline. Control sacculi were transferred to normal saline in the same manner. Both sets were fixed with glutaraldehyde and 0~0~ and processed for field emission SEM. In order to photograph a representative sample of both control and experimental bundles, maculae were viewed at low magnification (3000x) and bundles were chosen more or less randomly from all regions. At the low magnification it was not possible to observe tip links; thus, their presence could not bias the choice of a cell. Bundles were photographed at high magnification (50,000x), from an angle approximately perpendicular to the bevel of the tips. Twelve to20 bundles were photographed from each of 4 maculae. Figure 2 shows representative images of control (left) and BAPTA-treated (right) bundles taken with an accelerating voltage of 4 kV. The stereocilia in both samples appearwell preserved, with no bending. The lumpy or convoluted appearance of the stereocilia may represent condensed glycocalyx or membrane wrinkled bydifferential shrinkage,although transmission electron microscopy (TEM; see below) supports the former interpretation. At higher accelerating voltages (35 kV), these same stereocilia appeared smooth and slightly translucent and tip links were less distinct. In control bundles, tip links can be observed extending from the tips of most stereocilia to the sides of the tallest adjacent stereocilia. Links are arranged along columns of stereocilia, parallel to the physiological axis of the bundle, as previously described (Pickles et al., 1984, 1989). The tip links viewed by SEM appear thicker and shorterthan those viewed by TEM (see below), possibly because of the gold-palladium coating or the condensation of the glycocalyx onto the membrane. However, the difference raises the concern that the links observed in SEM are not bona fide tip links, but perhaps tubes of membrane drawn fromthetipsof stereocilia.Toexcludethispossibility, several sacculi were treated with a detergent (Triton X-100,2%) to remove cell membranes after the glutaraldehyde fixation (Figure 3). Triton-treated stereocilia differed from untreated bundles in that their surfaces were smooth, perhaps representing the underlying actin cores. Yet the tip links of treated bundles were, if anything, more apparent. Thus the links in field emission SEM images most likely represent the tip links observed with TEM and are not a lipid artifact. It is immediately apparent from Figure 2 that the main morphological consequence of BAPTA treatment was the loss of tip links. In each control bundle, roughly half of the stereocilia have tip links, whereas tip links are largely absent in BAPTA-treated bundles. No other systematic difference was noticed; in partic-

Figure 3. Intact

Tip Links following

Detergent

Treatment

Specimen was dissected in 0.1 mM CaI+ and then maintained in control saline containing 4 mM Ca2+ at all times prior to fixation. Triton X-100 (2%) was added to the fixative for the final 20 min of fixation. Bar, 500 nm.

ular, the bundles were not splayed or otherwise disrupted by the BAPTA treatment. The presence of tip links was more quantitatively assessed by measuring the proportion of stereocilia bearing tip links in each image. Sixty-three photographic images were coded, randomized, and judged by four observers. The proportion of tip links was calculatedastheaveragenumberoftiplinkscounted, divided by the number of tips for which links could have been seen. Altogether, 420 tip links were counted, out of more than 1800 stereociliary pairs. The results are presented as a histogram in Figure 4, showing the percentage of tip links per bundle. In controls, tip links were generally abundant; the average was 39% rf- 25% (SD, N = 38). In BAPTA-treated bundles, tip links were rarely observed (1.4% k 1.9%, N = 25). Moreover, those that were observed were questionable: in no case did all four observers agree that a BAPTA-treated bundle contained a tip link. Analysis of similarly treated maculae by TEM yielded similar results. Figures 5a-5d show representative tip links between stereociliary pairs from control bundles. The links characteristically inserted into osmophilic densities at either end. Representative BAPTA-treated stereocilia are shown in Figures 5f-5j. The proportion of tip links was scored by photographing every stereociliary pair for which a tip link might have been observed, as judged by the presence of both upper and lower osmophilic densities. Because the densities are thicker than the tip link and may occur in several sections, the actual proportion of tip links was probably underestimated. The scored results are shown in Figure 5e. Tip links were almost never seen in BAPTA-treated specimens (1.3% * 1.8% SD; N = 58),whiletheyappeared in control specimens in about half the stereociliary pairs where they might have been seen (48% + 4% SD; N = 74). A common observation with TEM is membrane

Hair Cell Transduction 989

Control

a, P

0

L 2

25 -,

5 &

20 -

f

15-

10

20

30

40

50

60

70

80

90

100

I 90

I 100

“tenting’at the lower site of attachment of the tip link, where the membrane is seen rising away by approximately 15 nm from the underlying osmophilic density that caps the actin core (Figure 5). This effect is presumably due to tension in the tip link. The plroportion of stereocilia displaying tenting was similarly affected by treatment with BAPTA: tenting was observed in 61% + 5% (SD, N = 74) of control samples and 14% k 10% (SD, N = 58) of BAPTA-treated samples. In addition to tip links, stereocilia are connected by upper and lower lateral links, which span from each stereocilium to its 6 neighboring stereocilia (BaggerSjobick and Wersall, 1973; Kimura, 1966). We observed an effect of BAPTA treatment only on the tip links. This was particularly evident when hair bundles were stained with 8% tannic acid to improve visualization of the various links. In both control ancl test bundles, the upper lateral links were readily identified, while the lower lateral links were barely evident (data not shown). The poor definition of the lower links may indicate their susceptibility to degradation in the enzyme treatment used to dissociate the otolithic membrane from the kinocilia. This treatment, which leaves mechanical transduction intact, removes the lower links (Jacobs and Hudspeth, 1990).

5 m M BAPTA 2 lo500

10

I 20

, 30

1 40

I 50

( 60

I 70

, 80

% tip links Figure 4. Abolition

of Tip Links by Low CaL+

The number of tip links in each micrograph was counted in a blind assay by four observers, and the average was calculated for each micrograph. Data were expressed as a percentage of the number of stereociliary tips for which tip links could have been seen. The histogram shows the distribution of this percentage for the control bundles (top) and the BAPTA-treated bundles (bottom). A total of 63 bundles were analyzed, 38 control and 25 BAPTA-treated. Bin width, 5%.

b

igure 5. Transmission

Electron

Abolition of Active Bundle Movement by Low Ca2+ It thus seems that a principal consequence of BAPTA

d

I

Micrographs

of Stereocilia

Treated

with

:a2+-treated sacculi (a-d) were dissected in a 0.1 m M CaZ+ solution APTA-treated sacculi (f-j) were dissected in the same way, but placed esults of scoring tip-link disposition, expressed as a proportion of the pparent, are shown in (e). Error bars represent standard error of the

Ca*+ or BAPTA

and then placed in 4 m M CaZ+ before fixation in 4 m M Ca2+. in a 5 m M BAPTA solution for 3-4 s before fixation in 4 m M Ca’+. total number of stereociliary pairs forwhich both densities were mean.

NeUrO”

990

Figure 6. Abolition of Acttve Bundle Movement by Low Ca>+ tn Three Different Cells

I

I

20 m M

BAPTA

4 m M BAPTA

2 mM

BAPTA

Active bundle movement was elicited by 1 s depolarizations from -80 to +80 mV, repeated every 2 s, as indicated by the trace labeled V,,. Bundle movement was analyzed using the automated procedure described in Experimental Procedures. The negative direction is shown as downward. At the time denoted by the bar in each experiment, a low-Cal’ solution, buffered with the indicated concentration of BAPTA, was gently puffed onto the cell from a pres surepipettepositionedatleast6Oumaway. In each case, the voltage-dependent movementwas irreversibly blocked within about 5 s after the initiation of the puff, and the bundle moved to a more positive position. The amplitude of the positive relaxation was measured 2 s after the initial movement of the bundle. In some cases the position then began to drift gradually in the negative direction, although the drift was not qualitatively different from the baseline drift normally observed in other active-bundle movement experiments (Assad and Corey, 1992). The significance nt the large transient movements, occurring in either the positive (A) or the negative (B and C) direction at the initiation of the forward relaxation, was unclear. However, it is unlikely that they were due solely to mechanical artifacts, since their direction did not always correlate with the directton of flow of the puff.

set

treatment is the simultaneous loss of mechanical sensitivity and the loss of tip links, strongly suggesting that the tip links are an essential component of the transduction mechanism. The simplest interpretation is clearly that the tip links are the gating springs that convey stress to the channels. Yet BAPTA could destroy mechanical sensitivity by disrupting some other element of the transduction apparatus, so it is important to assess the integrity of the gating springs by an additional method. Tension in the gating springs is manifested electrophysiologically, by the opening of ion channels; the first experiment showed that BAPTA abolished the transduction current. It is also apparent mechanically, in that altered tension on channels can move a free-standing bundle by a fraction of a micrometer (Assad et al., 1989). Our current model of adaptation supposes that an intracellular “motor” element maintains constant resting tension in the gating springs (Howard and Hudspeth, 1987; Hacohen et al., 1989; Assad and Corey, 1992). This tension exertsaslight negative pull on the hair bundle of about 130 nm; the pull can be increased bydepolarization, to generate a movement of free-standing bun-

dles by about 70 n m (Assad et al., 1989; Assad and Corey, 1992). If the gating springs were cut, in this view, the voltage-dependent movement would be abolished and the bundle would relax forward by about 130 n m to its rest position in the absence ot gating spring tension. Bundle position is thus a mechanical assay of gating spring integrity. An experiment designed to test these predictions was performed on voltage-clamped single cells, by gently puffing a BAPTA solution on them while the membrane potential alternated between +80 and -80 m V (Figure 6). The puffer pipettewas positioned about 60 p m from the hair bundle to minimize turbulence. In Figure 6A it was oriented with the stream directed at the short stereocilia. In Figures 6B and 6C the stream was directed at the kinocilium so that the puff would tend to push the bundle in the negative direction. The movementofthetipofthe bundlewasmeasuredfrom the video record using the automated routine described in Experimental Procedures. In each case, the effect of BAPTA was to abolish the voltage-dependent movement irreversibly. Regardless of the direction of the puff, the bundle then

Hair Cell Transduction 991

moved in the positive direction to a new resting position. The average movement, measured relative to the rest position at -80 mV, was 133 + 70 nm (SD, N = 5). Discussion The experiments reported here support the notion that tip links are part of the mechanical chain that conveys tension to the transduction channels. LowCa*+ saline (lOmg M; buffered with BAPTA) irreversibly eliminated the tip links, as observed with both scanning and transmission electron microscopy.The same treatment irreversiblydestroyed thegatingsprings,or some element mechanically in series with the gating springs, as shown by the loss of mechanical sensitivity, and the abolition of voltage-dependent bundle movement with concurrent positive displacement of free-standing bundles. The average positive displacement with BAPTA treatment closely matched that predicted from aquantitative model for regulation of tension in the gating springs. It remains possible that the elimination of tip links and the loss of tension in the gating springs are coincidental-that both are effects of low Ca*+, but are otherwise unrelated. However, both were irreversible and both occurred with exposures of a few seconds or less. While these experiments strongly suggest that tip links convey tension to the channels, it does not necessarily follow that they are the elastic elements that were inferred from physiological measurements and that are termed the gating springs. It may be that the tip links are themselves relatively inextensible and that some other element in series is stretched with positive displacements. The tenting of stereociliary tips might represent the gating springs, for instance. However, the tip links are both longer and thinner than other candidate structures and might be expected to stretch the most. It seems most plausible that the morphologically described tip links are the physiologically defined gating springs. Several other groups have used electron microscopy to examine the vulnerability of tip links to various treatments, although none has directly correlated the effects with the physiology of the hair cells. Acoustic trauma apparently disrupts tip links, but onlywhen the bundlesare nonspecificallydamaged aswell (Pickles et al., 1987). A variety of proteolytic enzymes have no detectable effect on the tip links (Pickles et al., 1990; Osborne and Comis, 1990). For instance, a protease was used in these experiments to loosen the otolithic membrane, but it did not disrupt mechanical transduction or tip links. A possible exception is elastase (Osborne and Comis, 1990). However, elastase causes splaying of the bundle as well (Osborne and Comis, 1990), which might secondarily disrupt the tip links. The brief BAPTA exposures used in our experiments caused no apparent change in the gross morphology of the bundle: stereocilia remained unbent and continued to touch at their tips.

The confirmation of tip links as part of the transduction chain would obviously limit the possible placement of the transduction channels. As in the original suggestion of Pickles et al. (1984), channels almost certainly occur near the point where the tip links contact the stereociliary membrane. As yet unclear is whether channels are at the upper or lower end of the tip link, or at both. The estimate of Howard and Hudspeth (1988), that there are roughly 85 channels per cell, is consistent with a channel at either one or both ends of each tip link, given an average of 60 stereocilia per bundle (Jacobs and Hudspeth, 1990). Forge et al. (1988) and Jacobs and Hudspeth (1990) have described particles in freeze fracture replicas at thetipsof stereocilia. lfthese particles,which number 4-6 per stereocilium or about 300 per cell, represent channels, then it may be that not all of them are mechanicallyconnected. If the mechanical chain extends from the actin core of one stereocilium to the actin core of the other, then there must be transmembrane elements at both ends of a tip link. It is thus possible that the freeze-fracture particles represent integral membrane proteins that are not channels, but that help to tie the tip link to the cytoskeleton. The confirmation of tip links as part of the chain would also constrain the possible placement of the motor element that underlies adaptation. A variety of physiological evidence has indicated that a cytoplasmic motor maintains the resting tension in the gating spring, perhaps by moving one end of the gating spring (Howard and Hudspeth, 1987; Eatock et al., 1987; Hacohen et al., 1989; Assad and Corey, 1992). If the tip link is the gating spring, then one of its attachments must move. Howard and Hudspeth (1987) have speculated that the upper attachment point can slip to relax tension following positive displacements that stretch tip links, and can climb to restore tension after negative displacements that relax the links. We might then expect to find the osmophilic density of the upper tip-link insertion to be lowerwhen fixed after positive displacements and higher after negative displacements. We might also expect that relieving tension by cutting tip links with BAPTAwould allow the densities to climb toward the tips of stereocilia. In preliminary experiments, this seems to be the case (Shepherd et al., 1991, Sot. Cen. Physiol., abstract). What is the biochemical nature of the tip links? First, the persistence of tip links after solubilization of the cell membrane with detergent argues that the structures are not membranous and that they are linked in some way to the underlying cytoskeleton. We expect that the electron-dense plaques, situated between the membrane and the actin core where the links insert, represent this connection. Second, the extremely rapid action of low Ca2+ on the tip links suggests that they are directly sensitive to Ca*+. It is possible that the tip link is a dimeric structure held together by Ca*+-dependent interactions between apposing components, analogous to other Ca*+-dependent cell adhesion molecules, such as cadherins (Jes-

NeLlrOn 992

sell, 1988). Alternatively, the tip links may be polymers of smaller subunits, like actin or tubulin. Howard et al. (1988) have pointed out that the 5 nm diameter of the tip links is similar to that of elastin filaments and that elastin can stretch to twice its length, as would be required if tip links were the gating springs. The CaZ+ dependence of the tip links may provide a means for their biochemical identification and characterization. Experimental

Procedures

Physiological Recording Dissociation Single hair cells were dissociated from the sacculi ot adult bullfrogs, as described elsewhere (Assad and Corey, 1992). Briefly, a saline resembling perilymph (120 m M NaCI, 2 m M KCI, 0.1 m M CaC12, 3 m M dextrose, 5 m M HEPES [pH 7.251) was buffered to a free Ca2+ concentration of IO-‘M with 1 m M EDTA and dripped onto sacculi that had been surgically exposed. While the EDTA solution had access to the basolateral surfaces of cells, tight junctions of the intact labyrinth presumably prevented the solution from reaching the hair bundles. In addition, otoconia in each sacculus may have helped maintain a normal endolymphatic Ca2+ concentration near the bundles. After 15 min, sacculi were removed from the animal and further dissected, then treated for 30 min with a protease solution (75 Kg/ml; type XXIV; SigmaChemical Co.) in salinecontaining0.1 m M CaCI,, to loosen the attachments to the otolithic membranes. Following removal ofthe otolithic membranes, cells were flicked out of the maculae into saline containing 1 m M CaCI>, 0.4 m M M&I,, and 40 pg/ml DNAse I (Worthington Biochemical Co.) and were allowed to settle onto the clean glass bottom of the recording chamber. All steps of the dissociation were performed at room temperature. Recording Membrane currents were measured with standard whole-cell, patch-clamp methods, as described elsewhere (Assad et al., 1989; Assad and Corey, 1992). The internal solution contained Cs’ to block most of the current through Ca2+-activated K’ channels. Composition was 85 m M CsCI, 2 m M MgCI,, 10 m M EGTA, 2 m M Na2ATP, and 5 m M HEPES (pH 7.25). The external solution was a normal frog saline, with the Caz+ concentration slightly elevated and Cs’ added to block inwardly rectifying K’ channels. Composition was 120 m M NaCl, 2 m M KCI, 4 m M CaC12, 5 m M CsCI. 3 m M dextrose, and 5 m M HEPES (pH 7.25). The series resistance of the patch pipette was routinely compensated by the patch clamp (Yale Mk V with 1 Gn headstage), so that residual resistance was less than 4-6 MR. Stimuli were generated and rcsponses recorded with a PDP II/73 computer equipped with an INDEC interface. Mechanical Stimulation Single hair bundles were moved along their morphologlcal axis with a two-dimensional piezoelectric bimorph stimulator, of the “pi” configuration (Corey and Hudspeth, 1980). Cells tended to settle on the chamber bottom sideways, so that the kinocilium was at the far left or right edge of a bundle, as viewed from the top; only these cells were studied. For most experiments, the glass stimulus probe was fabricated from a hollow pipette of about 0.7 pm tip diameter. Suction was applied to the back end of the pipette through a fine polyethylene tube (Holton and Hudspeth, 1986); under constant suction, the bulb of the kinocilium adhered tightly to the tip of the stimulus probe. The rise time of the stimulator, measured with a photocell in an image plane of the microscope, was less than 1.5 ms (IO%-90%). Pressure Application of Low Cap Ca2+ was buffered to approximately 10~’ M with the Cal’ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid; Tsien, 1980). Except when noted, the buffer solution contained 5 m M BAPTA, 120 m M NaCI, 2 m M KCI, 0.1 m M CaCI,, 3 m M dextrose, and 5 m M HEPES. The solution was delivered by solenoid-controlled pressure ejection from a pipette (ejection pressure, *IO kPa).

Video Microscopy Camera and Recording Hair cells were observed with a 63x objective and DIC optics on a Zeiss IM-35 inverted mlcroscope. An image of the field wa\ projected at high magnification onto the faceplate of a Hamamatsu Newviron video camera (C2400 series). so that the video field subtended about 20 pm. The camera output was observed on an oscilloscope, and gain and offset were adjusted to just below saturation. The video signal was recorded on a standard VHS video cassette recorder and, for shorter segments, directly onto optical memory disk (Panasonic TQ-2026). Measurement of Active Bundle Movement The spontaneous movements of a hair bundle that tallow depolarizing voltage steps were quantified as described elsewhere (Assad et al., 1989; Assad and Corey, 1992). Intensity profiles 01 up to 10 lines drawn across the bundle image, each about 50 pixels long and 5 pixels wide, were measured with an ITI-151 image processor and recorded on optical memory disk for up to several thousand frames, using a C-language program written bv P. L. Huang and N. Hacohen. These data files werethen analyzed with a QuickBASIC program on an 80486 computer to determine the frame-to-frame movement. For each line, intensity profile5 were averaged for several initial frames to create reference profiles. Then, for each new frame, the reference profile was shifted laterally to match each new profile, mlnimlzing the squared ditferences wtth Newton’s method. This method matched profiles within about 0.5 nm; the overall noise In the method, measured with static images, was typically 4 nm. The shift needed to match profiles was then taken as the movement ot the bundle.

Scanning Electron Microscopy Preparation and Fixation Both sacculi were removrd tram the dn~mal and enzymatlcall\ treated to tooben the otolithic membranes, as described abovr. After the membranes had been peeled away, the sacculi were transferred for about 10 s to either a control saline solution con taining 4 m M Ca:‘, or a saline solution buffered to 10 ’ M Ca-’ with 5 m M BAPTA. The two samples were then transterred 10 normal saline solution containing 4 m M Ca” and mounted next to each other on a glass toverslip that had been coated threr times with Cell-Tak (Collaborative Research. Inc.). Specimen5 were then imrnedlately prepared for SEM. The sacculi were fixed for 60 min with 2% glutaraldehydr (Trd Pella, Inc.) in a buffer containing 80 m M sodium cacodylate and 4 m M CaC&. All step\ of the fixation were done on ice. In \omr case\, $acculi were transferred tor the final 20 min of the Inc-ubation Into a solution containing the \ame concentration ot glutaraldehyde, plus 2’~~ Triton X-100 detergent, to sotubitize the cell membranes. After several rinse5 with buffer, all tissues were postfixed for 30 mln with 1% 050; tied Pelta, Inc.) In the same buffer. The saccull were again rinsed with buffer at room temperature and dehy drated in an ethanol series. The specimens were critical-point dried from liquid CO> and sputter-c-oated with gold-palladium Microscopy Specimens were observed on d field emlsblon scdnnlng rlectrc,n microscope (Amray 1860FE, Hitachi S-4000, or JEOL. 6300F). Thr sacculi were orlented so that the bevel of the hair bundles could be viewed en face. The macular epithetium was rapidly scanned at low power (about 3000x 1 in order to locate bundles that werr undamaged and optimally oriented. tmages were photographed directly or rec.orded onto optical memory disk via the micrc scopes’ RS-170 video output\. Analysis of SFM Data Photographs or video prints ot the bundle\ wer$x coded as eitht,l BAPTA-treated or control, the code obscured, and the entire set of pictures numbered at random. Analysis was performed blind by four observers. Each observer was instructed to record the total number of tip links visible in each photograph, excludtng broken or partially obscured tip links. The data were expressed as a fraction of the total number of stereociliary tips for which tip links could have been seen in each photograph and averaged among the four observers. Because tips of adjacent stereocilia

Hair Cell Transduction 991

were occasionally fused, this procedure underestimated centage of tip links present in each bundle.

the per-

Transmission Electron Microscopy Preparation and Fixation Maculae were prepared and treated with Ca2+ or BAPTA as described for SEM. Five maculae were treated with 4 m M Ca2+ and 3 with 5 m M BAPTA. Samples were transferred to test and control solutions for 3-4 s and then returned to saline containing 4 mM Ca*+ for 60 s. Maculae were fixed by immersion for 1 hr in 1% glutaraldehydefled Pella, Inc.) in saline buffered to pH 7.25with 20 m M HEPES. This and subsequent steps, except when noted, were performed at room temperature. Maculae were next washed in 100 m M cacodylate buffer (pH 6.3). To enhance preservation and visualization of the actin cytoskeleton of stereocilia, maculae were treated for 15 min with 5 PM phalloidin (Calbiothem) in cacodylate buffer containing 0.1% Triton X-100. The wash step was repeated, followed by postfixation in 1% OsOa (Ted Pella, Inc.) in 0.1 M phosphate buffer, for 30 min on ice. Maculae were washed thoroughly with distilled HZ0 and stained en bloc with 0.5% uranyl acetate for 3 hr. After dehydration in a methanol series, samples were embedded in Spurr’s embedding plastic (Polysciences, Inc.), which was polymerized for 24 hr at 65°C. Approximately 75 thin sections, cut parallel to the saccular nerve, were collected from each macula onto EM grids and stained with saturated uranyl acetate and 0.2% lead citrate. Microscopy Specimens were examined using a JEOL JEM IOOCX-II electron microscope operated at an accelerating voltage of 80 kV. Thin sections were systematically examined such that every seemingly well-oriented bundle where a tip link might be seen was photographed at low (6,700x or 10,000x) and higher (20,000x) magnification. Analysis of TFM Data The criterion for evaluating the disposition of tip links was that onlythose pairs of stereocilia inwhich both sites oftip-link insertion could be discerned were included in the analysis. These insertions are evident as pronounced osmophilic densities at the tip of the lower and side of the taller stereocilia. After these optimally sectioned stereociliary pairs were selected, negatives and prints were coded as either BAPTA-treated or control, the code was obscured, and theentire set of pictures was numbered at random. Analysis was performed blind by four observers instructed to record the presence or absence of tip links in each selected stereociliary pair, using the negativeor positive images as desired. In the same way, observers were instructed to score the presence of tenting, as defined in Results. The number of tip links was expressed as a proportion of the total number of stereociliary pairs for which both densities were apparent and was averaged among the observers. Acknowledgments We thank Drs. Bruce Bean, Steven Block, and Jonathon Howard for comments on the manuscript, Robin Pinto and Karen Rock for electron microscopy, and Susan Cronin for laboratoryadministration. We are especially indebted to Dr. Bechara Kachar and Marianne Parakkal for assistance with transmission electron microscopy and use of their microscope facility at the National Institutes of Health, for the experiment in Figure 5. This work was supported by grants from the NIH (DC-00304) and the Office of Naval Research (NOO14-91-J-1159), by the Howard Hughes Medical Institute, and by an NSF predoctoral fellowship to J. A. A. D. P. C. is an Associate Investigator of the Howard Hughes Medical Institute, and J. A. A and G. M. G. S. are Ryan Fellows at Harvard Medical School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

1, 1991; revised

September

13, 1991.

References Assad, J. A., and Corey, adaptation by vertebrate

D. P. (1992). An active motor mediates hair cells. J. Neurosci., in press.

Assad, J. A., Hacohen, N., and Corey, D. P. (1989). Voltage dependence of adaptation and active bundle movement in bullfrog saccular hair cells. Proc. Natl. Acad. Sci. USA 86, 2918-2922. Bagger-SjBbLk, D., and Werslll, J. (1973). The sensory hairs and tectorial membrane of the basilar papilla in the lizard Calotes versicolor. J. Neurocytol. 2, 329-350. Corey, D. P., and Hudspeth, A. J. (1979a). Response vertebrate hair cells. Biophys. J. 26, 499-506.

latency

of

Corey, D. P., and Hudspeth, A. J. (197913). Ionic basis of the recep tor potential in a vertebrate hair cell. Nature 287, 675-677. Corey, D. P., and Hudspeth, A. J. (1980). Mechanical and micromanipulation with piezoelectric bimorph Neurosci. Meth. 3, 183-202. Corey, D. P., and Hudspeth, current in bullfrog saccular

stimulation elements. J.

A. J. (1983). Kinetics of the receptor hair cells. J. Neurosci. 3, 962-976.

Crawford, A. C., Evans, M. C., and Fettiplace, R. (1989). Activation and adaptation of transducer currents in turtle hair cells. J. Physiol. 479, 405-434. Crawford, A. C., Evans, M. G., and Fettiplace, R. (1991). The actions of calcium on the mechano-electrical transducer current of turtle hair cells. J. Physiol. 434, 369-398. Eatock, R. A. Corey, D. P., and Hudspeth, A. J. (1987). Adaptation of mechanoelectrical transduction in hair cells of the bullfrog’s sacculus. J. Neurosci. 7, 2821-2836. Forge, A., Davies, S., and Zajic, C. (1988). Characteristics of the membrane of the stereocilia and cell apex in cochlear hair cells. J. Neurocytol. 77, 325-334. Hacohen, N., Assad, J. A., Smith, W., and Corey, D. P. (1989). Regulation of tension on hair-cell transduction channels: displacement and calcium dependence. J. Neurosci. I), 3988-3997. Hess, P., Lansman, J. B., andTsien, R. W. (1986). Calcium channel selectivity for divalent and monovalent cations. J. Gen. Physiol. 88, 293-319. Holton, T., and Hudspeth, A. J. (1986). The transduction channel of hair cells from the bull-frog characterized by noise analysis. J. Physiol. 375, 195-227. Howard, the hair duction USA 84,

J., and Hudspeth, A. J. (1987). Mechanical relaxation of bundle mediates adaptation in mechanoelectrical transby the bullfrog’s saccular hair cell. Proc. Natl. Acad. Sci. 3064-3068.

Howard, J., and Hudspeth, A. J. (1988). Compliance of the hair bundle associated with gating of the mechanoelrctrical transduction channels in the bullfrog’s sacculal hair cell. Neuron 7, 189-199. Howard, J., Roberts, W. M., and Hudspeth, A. J. (11988). Mechanoelectrical transduction by hair cells. Annu. Rev. Biophys. Biophys. Chem. 77, 99-124. Hudspeth, A. J. (1982). Extracellular current flow and the site of transduction by vertebrate hair cells. J. Neurosci. 2, I-IO. Hudspeth, 397404.

A. J. (1989). How the ear’s works

work.

Nature

347,

Jacobs, R. A., and Hudspeth, A. 1. (1990). Ultrastructural correlates of mechanoelectrical transduction in hair cells of the bullfrog’s internal ear. Cold Spring Harbor Symp. Quant. Biol. 55, 547-561. Jaramillo, F., and Hudspeth, A. J. (1991). Localization of the hair cell’s transduction channels at the hair bundle’s top1 by iontophoretie application of a channel blocker. Neuron 7, 409-420. Jessell, T. M. (1988). Adhesion neural development. Neuron

molecules 7, 3-13.

Kimura, R. S. (1966). Hairs of the cochlear attachment to the tectorial membrane. 55-72.

and the’ hierarchy

of

sensory cells and their Acta Otolaryngol. 67,

NellrCOl 994

Ohmori, H. (1988). Mechanical stimulation and fura- fluorescence in the hair bundle of dissociated hair cells of the chick. J. Physiol. 399, 115-137. Osborne, M. P., and Comis, S. D. (1990). Action of elastase, collagenase and other enzymes upon linkages between stereocilia in the guinea-pig cochlea. Acta Otolaryngol. 770, 37-45. Pickles, J. O., Comis, S. D., and Osborne, M. P. (1984). Cross-links between stereocilia in the guinea pig organ of Corti, and their possible relation to sensory transduction. Hearing Res. 75. 103112. Pickles,J. O., Osborne, M. P., and Comis, S. D. (1987). Vulnerability of tip links between stereocilia to acoustic trauma rn the guinea pig. Hearing Res. 25, 173-183. Pickles, J. O., Brix, J., Comis, 5. D., Cleich, O., Koppl, C., Manley, G. A., and Osborne, M. P. (1989). The organization of tip links and stereocilia on hair cells of bird and lizard basilar papillae. Hearing Res. 41, 31-42. Pickles, J. O., Brix, J., and Manley, C. A. (1990). Influence of collagenase on tip links in hair cells of the chick basilar papilla. Hearing Res. 50, 139-143. Roberts, W. M., Jacobs, R. A., and Hudspeth, A. J. (1990). Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J. Neurosci. 70, 3664-3684. Sand, 0. (1975). Effects of different ionic environments on the mechanosensitivity of lateral line organs in the mudpuppy. J. Comp. Physiol. 702, 27-42. Tsien, R. Y. (1980). New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry 79, 23962404.

Tip-link integrity and mechanical transduction in vertebrate hair cells.

An attractive hypothesis for hair-cell transduction is that fine, filamentous "tip links" pull directly on mechanically sensitive ion channels located...
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