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School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK
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Abstract |
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(Received 31 March 2004;
accepted after revision 18 June 2004;
first published online 24 June 2004)
Corresponding author Ian J. Russell: School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK. Email: i.j.russell{at}sussex.ac.uk
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Methods |
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Three- to 14-day-old chicks (ISA Brown breed, Hubbard ISA, Peterborough, Northants, UK) were used in this study. Animals were killed with CO2 and decapitated. Pieces of skull containing the inner ear were placed in cold (+4°C) Hank's balanced salt solution without Phenol Red (HBSS, mM: CaCl2, 1.26; KCl, 5.36; KH2PO4, 0.44; MgCl2, 0.49; NaCl, 136.9; NaHCO3, 4.17; Na2HPO4, 3.38; and glucose, 5.56; Invitrogen, Paisley, UK) buffered with 10 mM Hepes, pH 7.3. Utricular maculae were dissected and otolitic membranes carefully removed with fine forceps. Maculae were transferred into a recording chamber, folded in half and fixed under a fine nylon strand on the bottom of the chamber so that the hair bundles protruded horizontally from the folded edge. The line of folding of the macula was orientated in the mediolateral direction in the middle one-third of the sensory epithelium.
Particulate debris was removed from the tissue by perfusing the preparation with filtered (0.22 µm pores) HBSS at a rate of 500–1000 µl min–1 before data collection. Perfusion was maintained throughout the entire experiment, except during the recording periods that lasted for approximately 20 s. A stock solution of subtilisin (Protease type XXIV, Sigma, UK) in HBSS (1 g l–1) was prepared on the day of the experiment and diluted to obtain a final concentration of 50 mg l–1. A 5 mM solution of BAPTA (Sigma) was prepared from calcium- and magnesium-free Hank's balanced salt solution without Phenol Red (mM: KCl, 5.33; KH2PO4, 0.44; NaHCO3, 4.00; NaCl, 137.93, Na2HPO4, 0.30 and glucose, 5.60; Invitrogen, Paisley, UK), adding 0.9 mM MgCl2 and 10 mM Hepes, and adjusting the pH to 7.3 with NaOH. BAPTA application and consequent wash out of the preparation with HBSS were undertaken using the same perfusion system. Concanavalin A (Calbiochem, Nottingham, UK) was used as a 0.3 or 0.6 µM solution in HBSS.
To assess viability of the hair cells during experiments, a 5 µM solution of vital dye FM1-43 (Molecular Probes, Eugene, OR, USA) in HBSS was injected through the tip of a 40 µm diameter glass pipette over the apical surface of the epithelium for 1 s. Fluorescence of cell bodies after the dye application indicates permeation of the dye into hair cells by fast endocytosis (Meyer et al. 2001) or through transduction channels (Gale et al. 2001). FM1-43 staining was still observed after up to 45 min of treatment with 50 mg l–1 subtilisin (Fig. 2), but was not detected after a 10 min treatment with BAPTA. To check that cells that had loaded with FM1-43 did not have damaged membranes, we applied a 500 µM solution of the nucleic acid stain SYTOX® Green (Molecular Probes) in the bath. By use of an FITC filter set with a long-pass 515 nm emission filter (Zeiss 488009, Welwyn Garden City, Herts, UK) we were able to observe the fluorescence of both dyes. Different distribution patterns, colour and intensity of the dyes' fluorescence allowed sequential use of the dyes on the same preparation. Fluorescence of SYTOX-stained cell nuclei was observed only on the periphery of the macula in a small number of cells, presumably damaged during dissection, in swollen cells, or in hair cells with clearly visible broken hair bundles.
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Interferometry and data processing
The interferometer employed in this study (Fig. 3A) shares the same methodology and is similar in design to that used by Denk & Webb (1990). In essence, a laser beam was split in two and the separation width controlled by a DIC slider on the condenser. The beams formed 0.5 µm spots in the focal plane of the microscope. The upper quadrant of a hair bundle (viewed in profile, Fig. 3B) was positioned, by moving the tissue stage, so that the spots were on either side of the bundle. As the hair bundle moved, the difference of refraction indices in the spots changed, producing a differential signal between the beams that was detected by the photodiodes in the system. In detail, the beam of a 633 nm, 5 mW helium–neon laser was circularly polarized using a rotating polariser and a quarterwave plate, spatially filtered using a x10 microscope objective with 6 µm pinhole, and collimated with a single lens of 35 mm focal length. The beam was then combined with the conventional illumination of a Zeiss Axioskop microscope using a 20 mm beam splitter cube (BS1 on Fig. 3A, Coherent, Ely, Cambs, UK). A water immersion 40 x 0.75 NA objective equipped with a DIC slider (Zeiss, Welwyn Garden City, Herts, UK) replaced the condenser lens. The objective and the slider focused the laser light and split it into two partly overlapping 0.5 µm points with perpendicular polarization. At the same time, the objective worked as a condenser and provided DIC illumination of the specimen. Adjustments were made to provide simultaneous Köhler illumination of the specimen and focusing of the laser light into a diffraction-limited point in the focal plane of the microscope. The specimen was fixed in a 0.4 ml volume, glass-bottomed Perspex chamber and viewed through an identical 40 x 0.75 NA objective with a DIC slider. The second slider combined the images of the perpendicularly polarized points. The resulting image and the conventional microscopic image were separated with a beamsplitter BS2 (Coherent) situated inside the custom-built microscope camera port. The laser light was directed into an additional perpendicular flange of the camera port, where a polarizing beam splitter BS3 (Coherent) and two photodiodes with integrated preamplifiers (Burr-Brown OPT-210) were mounted. The photodiodes provided intensity signals for two perpendicularly polarized components of the laser beam. Green illumination was used to obtain the conventional microscope image. Four green filters (Zeiss 467803 or Hoya 533) were used to filter the incandescent illumination, and to protect the experimenter's eyes and the CCD camera (Retiga 1300) from the laser light. A polariser and two separate analysers produced conventional DIC images for the camera and eyepieces. The microscope binocular tube was switched to the camera position during measurements, and a DIC image of the specimen was projected on the camera chip using a x4 adapter (452985, Zeiss). The final magnification on the PC screen was x4500 with an object resolution of 42 nm per pixel. At the same time, the photodiodes on the camera port provided the interferometric signal.
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Displacement of a small object (e.g. a stereocilium) results in the interferometer voltage output signal V, which is proportional to the displacement and can be found according to the equation of Denk & Webb (1990):
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| (1) |
The apical quarter of the bundle was positioned in the focused beam of the interferometer so that the long axis of the bundle was perpendicular to the direction of the DIC shadow. In order to maximize the signal-to-noise ratio of the hair bundle Brownian motion, measurements reported in this paper were made from the tallest rows of stereocilia. These findings are likely to be representative also of the movements due to Brownian motion of the shortest rows of stereocilia, because the linkages are present between all stereocilia in any given hair bundle type. Maximum oscilloscope signal was obtained by small variations of the calibration movement direction and the bundle position when using 15–80 nm, peak-to-peak movements in a direction parallel to that of the DIC shadow. Recording duration was about 8 s or 220 samples at a sampling rate of 125 kHz. Eight-pole Bessel anti-aliasing filters with –3 dB at 50 kHz were the only signal filters used in the data acquisition and processing. A computer programme written in C controlled data acquisition and generated signals for the calibration movement. The time series obtained were converted to the frequency domain by direct application of FFT and scaled to displacement units using the peak of the calibration movement of known amplitude. The resulting spectrum, after deletion of the calibration peak and zero-frequency component, was filtered with a 128-point binomial filter and every 64th point kept. All data processing was carried out using procedures in Igor software (Wavemetrics, Lake Oswego, OR, USA). Usually three or four consecutive recordings were made with a minimum interval of 1–2 min required for data saving and preview.
To test the instrument, glass fibres of 4 µm diameter, which were reduced to 0.3 µm in diameter within 1 µm of their tips, were pulled using a P-2000 puller (Sutter Instruments, Novato, CA, USA). The combined beams of the interferometer were focused on the tip of the glass fibre and the Brownian motion spectrum of the fibre was recorded. As expected, the spectra had typical Lorentzian characteristics within the 10 Hz to 30 kHz frequency range.
The data were processed in the framework of a single oscillator model of the hair bundle. The displacement power spectral density (PSD, 
) of the hair bundles is given by
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| (2) |
x2
is the mean square displacement and f is frequency. For the single oscillator model, the hair-bundle spectrum 
resembles (Denk et al. 1989) a Lorentzian overdamped oscillator spectrum (S):
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| (3) |
is viscous damping, kB is the Boltzmann constant and T is the absolute temperature. To reveal changes in the mechanical properties of the hair bundles, the ratio 
of the experimentally obtained displacement density spectra after, 
, and before, 
, treatment was calculated as:
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| (4) |
and 
are the corresponding experimental displacement PSD. Assuming that treatment of the overdamped oscillator changed only its stiffness from value K1 to a new value K2 and that the viscous damping, , of the medium was constant, the theoretical frequency-dependent ratio of displacement spectral densities is
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| (5) |
) are the displacement density, displacement PSD and the roll-off frequency before the treatment; and X2, S2 and f2=K2/(2) are the same quantities after the treatment, respectively. An additional scaling parameter, a, constrained to 0.7 < a < 1.3, was used during fitting of the experimental data to accommodate small calibration errors.
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| (6) |
is 1 and for f0 is
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| (7) |
K2/K1 is relative stiffness. Therefore, to obtain a rough estimate of relative stiffness we averaged the experimentally obtained 
in the frequency regions from 2 to 8 kHz and from 20 to 80 Hz and took the quotient of the average values. Then we performed a direct fit of the experimental data to eqn (6) that led to similar values of .
The absolute stiffness of the hair bundle was calculated using the equipartition theorem for a single spring with stiffness K:
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| (8) |
x2 is the mean square displacement. x2 was calculated as a numeric integral of the experimental squared displacement spectra 
over F1= 20 Hz to F2= 10 kHz frequency range. |
| (9) |
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| (10) |
Immunofluorescence and transmission electron microscopy
Utricular maculae were dissected as described above for the stiffness measurements and treated with 5 mM BAPTA and/or 50 mg l–1 subtilisin for periods ranging from 5 to 40 min. Control maculae were incubated in HBSS for the same amount of time. Immunofluorescence samples were prepared as described by Goodyear & Richardson (1999). Each macula was treated for a particular time period, then washed in HBSS and fixed in ice-cold 3.7% formaldehyde buffered with 0.1 M sodium phosphate, pH 7.2. After fixation, the maculae were washed in 150 mM NaCl and 10 mM sodium phosphate (PBS) solution, pH 7.2, preblocked with 10% (v/v) heat-inactivated horse serum (HS) in TBS (150 mM NaCl, 10 mM Tris-HCl, pH 7.4) for 40 min and incubated for 3 h with monoclonal anti-hair cell antigen (HCA) tissue culture supernatant diluted 1:100 in 10% HS/TBS (Goodyear & Richardson, 1992). Maculae were washed with PBS again, stained with FITC-conjugated rabbit anti-mouse Ig (Dako, High Wycombe, Bucks, UK) or with two layers of FITC-conjugated antibodies (rabbit anti-mouse and swine anti-rabbit Ig, Dako). Maculae were finally washed and mounted in Vectashield (Vector Laboratories, Peterborough, Cambs, UK). The sample images were acquired using a fluorescent microscope equipped with a CCD camera (Photometric SPOT) and processed with Matlab Image Processing Toolbox (Mathworks, Natick MA, USA). Background intensity was obtained using the morphological opening function of the Matlab toolbox, with a disk-shaped structuring element 32 pixels in diameter. Bundles were detected as bright objects on a manually selected region of the striola. Immunofluorescence intensity was obtained as an average of the maximum intensity of individual bundles in the selected region using the original image with the background subtracted. The intensity in three manually selected rectangular regions, covering more than 80% of striola, was averaged for each macula.
For transmission electron microscopy (TEM), the maculae were prepared as described for fluorescence measurements, treated with BAPTA or subtilisin for 10 or 25 min, respectively, washed three times with HBSS, and fixed with 2.5% (v/v) gluteraldehyde in 0.1 M sodium cacodylate with 1% (w/v) tannic acid, pH 7.2. The maculae were then washed three times in 0.1 M cacodylate buffer, fixed for 1 h in 1% (w/v) osmium tetroxide in 0.1 M sodium cacodylate, dehydrated through an ethanol series, equilibrated in propylene oxide and embedded in Taab 812 resin (Taab Laboratories, Reading, Berks, UK). Blocks were polymerized at 60°C for 24 h. Sections of 100 nm thickness were cut on a Reichert Ultracut E microtome and examined using a Hitachi 7100 transmission electron microscope.
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Results |
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The hair bundles can be clearly seen in profile protruding from the folded edge of the maculae (Fig. 3B). A significant number of them usually lie within the focal plane of the microscope. The striolar region can be recognized by a change in the predominant direction of hair-bundle polarization. For the measurements, we chose type I or II hair bundles situated within 60 µm of the point of polarity change. Bundles with irregular profiles or those that were inclined, bent or split were rejected. The plane of polarity of the hair bundles in this region was close to the focal plane of the microscope. The choice of long bundles with high contrast and sampling of bundles situated within 60 µm of the line of polarity change may have introduced a statistical bias in the data. An additional requirement for the successful observation of a particular bundle was the absence of any obstruction (e.g. other hair bundles or tissue debris) in the path of the laser light. The only indication of cell viability during an experiment was the appearance of the tissue. Throughout the experiment the cells looked healthy, with intact hair bundles. If a bundle moved, inclined or split due to tissue deterioration, data acquisition was stopped and the data were excluded from the subsequent analysis. At the end of each measurement session the preparation was checked using fluorescence microscopy with SYTOX to confirm that the hair cells were still viable.
Control experiments
The interferometer signal, V (eqn (1)), proportional to hair-bundle displacement was calculated and stored for all hair bundles. The distribution of bundle position was estimated for seven of the bundles. The calibration movement was either switched off during these experiments, or the sinusoidal component at the calibration frequency was filtered out during later analysis using the software. The distributions were almost ideally Gaussian after high-pass filtering at 10 Hz for all seven bundles. Hence, the force versus displacement curve is linear in the range of displacements used.
A set of control experiments with glass fibres of 0.5–20 µm diameter was performed to eliminate the possibility that the hair-bundle spectra were influenced through the movement of the cuticular plate or fluid flow associated with spontaneous cell membrane fluctuations. The fibres were positioned along the long axis of the bundle and within 1 µm of the bundle so as to block possible fluid flow. In the second type of experiment the fibres were pushed against the cuticular plate close to the base of the hair bundle. Although we observed changes in the bundle position in the latter case, no changes in the bundle spectra were detected in either type of experiment of this set.
Brownian motion of untreated bundles
At the beginning of each experiment, after the initial washout, two sets of spectra were recorded with an interval of 10–15 min between the sets to ensure the stability of the preparation. Brownian-motion spectra (Fig. 4) were similar in shape to those described earlier (Denk et al. 1989) for hair bundles of the bullfrog sacculus. The main components of the noise (Fig. 4, trace 4) emanate from off-focus bundles and tissue in the cone of laser light. The spectra recorded in HBSS varied from bundle to bundle in shape and amplitude but for any one bundle they remained stable during recording for up to 2
h (Fig. 4, inset). The slope of the spectra on a double-logarithmic plot and the magnitude of the Brownian motion at frequencies above 2–4 kHz did not change with time or after the application of BAPTA, subtilisin or concanavalin A. This frequency region of the spectra is controlled by viscous damping (Denk et al. 1989), thus in our experiments the viscous damping remained unchanged.
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After application of one or more of the agents, the Brownian-motion spectra changed. The ratio of spectra after and before the treatment (eqn (4)) was usually fitted well by the ratio of two Lorentzian functions. Taken over each set of experiments, coefficient a in eqn (6) was statistically close to 1, and ranged between 0.7 and 1.3 in individual experiments, thereby indicating that viscous damping had not been changed.
Concanavalin A. Previous experiments have shown that concanavalin A increases bundle stiffness by cross-linking glycoproteins on the stereocilia surfaces (Kössl et al. 1990). In our experiments, application of concanavalin A did not change the general shape of the spectrum, but reduced its amplitude (Fig. 6). Application of 0.3 µM of concanavalin A for 10 min resulted in a 1.8 ± 0.4-fold (mean ±S.D.) increase in stiffness (n= 19).
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(t) to a power function
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| (11) |
= 4.4 (Fig. 10). This value is an average number of the bonds that should be cleaved in a link to significantly change the link's stiffness. Here we assume the kinetics of the bond cleavage to be linear with the rate, p, equal to that of HCA loss.
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It is worth noting that there was no statistically significant stiffness change in the first 15 min of application, a period during which all of the ankle links disappear (Goodyear & Richardson, 1999).
Application of BAPTA after subtilisin or subtilisin after BAPTA
When BAPTA was applied after subtilisin or subtilisin was applied after BAPTA, the stiffness further decreased as a result of the application of the second solution (Fig. 11). Application of BAPTA after subtilisin caused the bundle to split into two or three parts in the first minute of the second treatment in seven out of nine preparations. In the two cases when the bundles remained intact, the stiffness reduction due to the BAPTA treatment was 38 ± 6% (n= 2), close to the value of 43% obtained with BAPTA alone. The subtilisin exposure time for BAPTA-treated bundles had to be reduced to 25 min to prevent bundle splitting. The stiffness decrease due to subtilisin was 32 ± 10% (n= 3), i.e. 1.5 times less than the 48% reduction in stiffness obtained for 40 min treatment with subtilisin alone, but very close to the 30% obtained as an estimate for a 25 min treatment from the kinetic data (Fig. 10). Data on sequential treatments show that the effects of BAPTA and subtilisin are additive, irrespective of the sequence of application.
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Anti-HCA labelling showed a significant reduction in the maculae treated with subtilisin for 5–10 min and declined to background values in the maculae treated for 30–40 min (Figs 10 and 12). No changes in the labelling were detected in the control maculae incubated in HBSS for 5–40 min. Immunofluorescence intensity during 50 mg l–1 subtilisin treatment is fitted well by a linear function with a slope of –0.035 ± 0.012 min–1 (Fig. 10).
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Discussion |
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Displacement density spectra of the chick vestibular hair bundles, when displayed on a double logarithmic plot, have slopes below the hair-bundle roll-off frequency ranging from –0.3 to –0.5, and slopes above it ranging from –0.7 to –1. These results are in a good agreement with the measurements of Denk et al. (1989) on bullfrog hair bundles. Our displacement density spectra, and indeed those measured from the bullfrog sacculus by Denk et al. (1989), are not fitted well by a single Lorentzian function. Thus, to find absolute stiffness we employed the equipartition theorem (Denk et al. 1989). However, motion at frequencies below 20 Hz, which we are not able to take into account, may contribute to the value of the integral in eqn (9). To find the relative stiffness change we calculated the ratio of spectra before and after treatments, which gave the full picture of the stiffness change across frequency. To obtain a single numeric measure of the relative stiffness we fitted the ratio of spectra by a ratio of two Lorentzian functions.
This approach tends to eliminate the effects of constant external and internal forces if they act on the bundle. It gives a simple analytical expression to calculate relative stiffness change at any frequency. It is not affected by variability in the corner frequencies of the bundles, and is therefore preferable over measurements made at a single frequency.
Disruption of tip links, ankle links and kinocilial links by the calcium chelator BAPTA causes a decrease in hair-bundle stiffness to 0.57 ± 0.18 of the original value. Our figure is in good agreement with the –4 dB change in the stiffness of chick cochlear hair bundles observed following the application of BAPTA (Pae & Saunders, 1994). The static stiffness of hair bundles in the bullfrog sacculus treated with BAPTA was also approximately half that of hair bundles perfused with 1 mM Ca2+ (Fig. 2 in Marquis & Hudspeth, 1997). Good agreement of the stiffness changes due to BAPTA treatment with earlier observations further validates our approach to stiffness measurements.
As a multivalent ligand that can bind to cell-surface glycoproteins, concanavalin A should cross-link the interstereocilial links and reduce their flexibility and/or mobility within the plane of the membrane. It may also form further molecular links between the membranes of adjacent stereocilia. The 1.8-fold increase in stiffness seen in this study following the application of concanavalin A shows that such additional cross-linking increases the overall stiffness of chick vestibular hair bundles. This value is half of the 3- to 5-fold increase reported by Kössl et al. (1990) for mouse cochlear hair bundles after the same treatment, possibly due to differences in the size, geometry and/or maturity of the hair bundles.
The effects of subtilisin reported here reveal that proteases that are used widely to remove overlying membranes and underlying stromal and neural tissue from hair cells could significantly change the mechanical properties of the bundles. However, when the utricles were exposed to 50 mg l–1 subtilisin for 15–20 min, which is the exposure time usually used to free hair cells from overlying and underlying tissue, hair-bundle stiffness changed by less than 10%. Longer application of subtilisin leads to a decrease in hair-bundle stiffness up to 48%. The most prominent changes in spectra were observed below 3 kHz, where Brownian motion is controlled by stiffness. No changes were detected in the range controlled by viscous damping of the external solution, suggesting negligible contribution of the viscoelasticity of the links in this range. Also, the links do not change significantly the viscous drag of the bundle, possibly because of their relatively small overall cross-section.
If a homogeneous population of links were uniformly digested by subtilisin without any spatial restrictions, one would expect the kinetics of link-loss to be close to linear, to observe direct proportionality between the loss of stiffness and immunofluorescence, and the normalized kinetics of link-loss and stiffness-decrease to be of equal slope. Although the loss of HCA is close to this prediction, the stiffness change is slower in the beginning and at the end of the treatment. To explain this discrepancy we employed two models, where link disruption results from either random cleavage of a number of chemical bonds per link, or sequential cleavage of several bonds. The sequence of cleavage could be determined by the chemical structure of the link, or spatial restrictions on the accessibility of particular bonds to subtilisin. The first model leads to the average value of 4.4 bonds per individual link. The final part of the kinetic curve does not fit, possibly, because the kinetics of bond cleavage deviate from linear, or a contribution from structures other than shaft connectors becomes noticeable. The second model gives an average time to break a link of 24 ± 8 min. Although the models are rough and reflect two extreme cases, both of them require a link to be an ordered structure rather then a random element of a network or a gel.
The other possibility is that the subtilisin-sensitive connectors are not uniform, different volumes or pools exhibiting different resistance or accessibility to subtilisin and different contribution to overall stiffness. Probably, only part of them is associated with HCA. There may be a pool of anti-HCA mAb immunoreactive hemi-shaft connectors between the stereocilia that do not contribute to hair-bundle stiffness (Goodyear & Richardson, 2003), but are more accessible to subtilisin than interstereociliary shaft connectors. This would slow down the initial segment of the stiffness-loss curve. Loss of the HCA may not reflect physical breakage of the shaft connector (i.e. the epitope may be located distal to the site of molecular interaction). Furthermore, the presence of cell-surface molecules, possibly charged components of the glycocalyx that do not appear as interstereociliary links but contribute to hair-bundle stiffness through electrostatic interactions (Dolgobrodov et al. 2000a,b), cannot be discounted.
Sequential application of BAPTA and subtilisin led to a further decrease in stiffness up to 30% of the original stiffness. Although stronger effects were not observed due to physical splitting of the bundles, the experiments show that the effects of subtilisin and BAPTA are additive in the measurable range of stiffness. If the additivity is still applicable for the treatments causing bundles to split, deletion of both subtilisin-sensitive and BAPTA-sensitive structures would result in 91% loss of stiffness. The rest of the bundle structure consists of stereocilia pivots at the base of the bundle and horizontal top connectors, and must contribute to the remaining 9% of the initial bundle stiffness.
Immunofluorescence data (Goodyear & Richardson, 2003) show that ankle links completely disappear within 15 min of subtilisin treatment, whereas our data do not show any stiffness changes at this time point. Thus, ankle link deletion does not appear to reduce the bundle stiffness. This finding is not surprising considering that the links are predominantly localized around the base of the bundle (Goodyear & Richardson, 1999) and do not confer significant mechanical advantage to influence rotational stiffness.
The results of this study reveal the relative contribution of subtilisin- and BAPTA-sensitive structures to the mechanical properties of the sensory hair bundle. They suggest that shaft connectors or other subtilisin-sensitive structures contribute 48% to stiffness, whereas BAPTA-sensitive structures, tip links, kinocilial links and ankle links, contribute 43% to the overall stiffness.
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References |
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