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The incorporation and turnover of radiolabelled amino acids in developing stereocilia of the chick cochlea
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Hearing Research 101 (1996) 45-54
The incorporation and turnover of radiolabelled amino acids in developing stereocilia of the chick cochlea J.O.
Pickles *, D.A. Billieux-Hawkins, G.W. Rouse
a
Vision Touch and Hearing Research Centre, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Qld. 4072, Australia Received 20 March 1996; revised 1 July 1996; accepted 21 July 1996
Abstract
Hair cell stereocilia are composed of packed actin filaments, oriented such that the preferred end for the addition of actin monomers is at the tips of the stereocilia. It has therefore been suggested that when stereocilia grow, they do so from their tips (Tilney and DeRosier, 1986, Dev. Biol. 116, 119 129). In order to test the hypothesis, radiolabelled amino acids were applied to the air-sac of chicken eggs at day 17 of incubation, i.e., at the beginning of a phase in which the stereocilia have achieved their mature width, but are growing rapidly in length. Incorporation of radiolabel was studied autoradiographically, followed by image analysis and averaging grain counts over many hair cells. In contrast to the position expected from the above hypothesis, there was no sign of preferential incorporation of label in the upper part of the stereociliary bundle. The greatest density of labelling was found in the lower part of the bundle, while the upper part of the bundle was under-represented in the autoradiographic averages. The turnover time (to fall to l/e) was significantly greater in the bundle (16 days) than in the cuticular plate or in the rest of the cell (9 days). The results (i) give no support for the hypothesis that stereocilia grow from the tips, and (ii) suggest that during development at least some components of the stereocilia turn over with a relatively short time course.
Keywords." Stereocilia; Protein; Turnover; Actin; Chick; Development; Hair cell
1. Introduction
Hair cells have an array of 50-150 stereocilia on their apical surface, deflection of which initiates mechanotransduction (Hudspeth, 1989; Pickles and Corey, 1992). The stereocilia are composed of actin filaments, tightly packed into what is known as a paracrystalline array (Tilney et al., 1980; Flock et al., 1981; Zenner, 1981). The actin paracrystal gives the stereocilia their strength and rigidity, so that when stereocilia are deflected, they bend only at their base and do not flex (Flock et al., 1977). This dense, tight and large-scale organization of actin filaments in such a paracrystal is unique to acousticolateral hair cells. The high degree of organization of the stereociliar paracrystal means that it * Corresponding author. Tel.: +61-7-3365-4125; Fax: +61-7-3365-4522; E-mail: [email protected].
a Present address." Department of Zoology, University of New South Wales, Sydney, NSW 2006, Australia.
is likely to form a relatively simple model for protein organization. It is also likely that the high degree of organization, close packing, and large number of inter-molecular cross-links of the actin filaments of the paracrystal impose severe restraints on the way that the filaments can be laid down or added to during development and turnover. As suggested by Tilney and DeRosier (1986), this makes it likely that the addition of m o n o m e r s occurs only at the borders of the paracrystal. Tilney and DeRosier therefore suggested that when stereocilia grow in length, but not in width, that they do so primarily by addition of actin monomers to one end or the other of the actin paracrystal. The pattern of decoration of the actin filaments with the S1 fragment of myosin shows that the preferred end for the addition of actin m o n o m e r s is at the tips of the stereocilia, and this suggested to Tilney and DeRosier that when stereocilia elongate, they do so by the addition of actin m o n o m e r s at their upper ends. There is no direct evidence of the way that stereocilia
0378-5955/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 5 9 5 5 ( 9 6 ) 0 0 129-3
46
J.O. Pickles et al./Hearing Research 101 (1996) 45 5 4
elongate. The suggestion that stereocilia elongate by addition of actin at their tips is uncertain, because it is also possible for monomers to be added at the nonpreferred end of actin filaments, although this requires a higher concentration of monomers (Woodrum et al., 1975). A related unresolved question concerns the turnover and replacement of stereociliar proteins. Once stereocilia are established, do they remain for a long period, such as years, or are they replaced in a matter of weeks or days? Actin turnover time in intestinal microvilli is several hours (Stidwill et al., 1984), while that of cultured myotubes has been measured as several days (Rubinstein et al., 1976). In addition, membrane-associated proteins commonly show a high degree of turnover. Given the substantial involvement of stereociliar damage in hair-cell pathology, knowledge of the rate of turnover of proteins in stereocilia would have important implications for our understanding of repair after cochlear damage. In order to test whether stereocilia grow by elongation at the tips, chick hair cells were incubated with radiolabelled amino acids, during the late phase in development, when the stereocilia are growing rapidly in length, but not in width (Tilney and DeRosier, 1986). After different waiting times, the distribution of the label was assessed by autoradiography of the hair bundles. Fig. 1 shows the expected sites of radiolabelling under the most direct interpretations of the two possible hypotheses. If stereocilia grow by the addition of new protein to the tips of the stereocilia, the concentration of radiolabel should be highest in the upper part of the bundle (Fig. 1B). If, however, stereocilia grow by the addition of new protein at the base, the concentration of label should be greatest in the lower part of the bundle (Fig. 1C). In addition, decreases in labelling with increasing times between injection and fixing will give an indication of protein turnover. To the extent that protein incorporating radiolabel is replaced by new, unlabelled protein, an indication of the rate of turnover will be obtained by measuring the rate of decay of radiolabel with time.
2. Methods Chicken eggs (white Leghorn) were incubated in a forced-draught incubator at 37°C. At 17 days of incubation, control embryos were staged by the criteria described by H a m b u r g e r and Hamilton (1951). At this age in experimental embryos, 0.5 mCi of a cocktail of 3Hlabelled amino acids (Amersham TRK550: leucine, lysine, phenylalanine, proline, tyrosine, 4.7 nmoles total) in 0.1 ml H 2 0 was applied to the air-sac of the eggs. After different periods of survival (2-13 days), the embryos or hatched chickens were decapitated, the head bisected, and the bony covering of the basilar papilla
l A
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Fig. 1. A: profile of a stereociliary bundle from the chick basilar papilla, seen from the side, before the late phase of elongation of the stereocilia (embryonic age 17). Tall stereocilia are to the left of the bundle, and short to the right. B: if stereocilia grow by addition of actin monomers at the tips of the stereocilia, the new protein will be laid down in the upper part of the bundle. C: if stereocilia grow by addition of actin monomers at the base of the stereocilia, the new protein will be laid down in the lower part of the bundle.
opened. The half-heads were immersed in fixative (2.5% paraformaldehyde in 0.1 M phosphate buffer) for at least 24 h. The basilar papilla was opened widely, and the tegmentum vasculosum removed. The tectorial membrane was removed with fine forceps, and the remaining papilla was rinsed, dehydrated in ethanol and embedded in Spurr's resin. One g m sections were collected starting at a point 5% from the apical (i.e., distal) end of the papilla, and continuing for another 5% of the distance along the papilla, i.e., to a point 10% of the distance from the apical end of the papilla. Sections were cut orthogonal to the long axis of the papilla. Slides were dipped in autoradiographic emulsion (Ilford LM-1), and exposed at 4°C for 15 months. After autoradiographic development, the sections were lightly stained with toluidine blue. Sections were photographed under uniform conditions with a 100 × oil-immersion objective, and selected hair cells analysed by N I H - i m a g e software. The great majority of bundles were upright in the papilla and showed a compact bundle without splaying of stereocilia, i.e., they did not show any signs of disturbance from removal of the tectorial membrane. Criteria for selection of a bundle for further analysis were: (i) the vertical axis of bundle was orthogonal to cuticular plate, (2) the bundle showed a clear gradient in heights of the stereocilia, with a clear sharp taper to the tallest stereocilia which were towards the abneural side of the papilla, and (3) the bundle was compact, without splaying of the stereocilia. After scanning of the printed images by Macintosh OneScanner (256 grey levels, resolution equivalent to 94 nm/pixel; see Fig. 3A), each image was aligned so that the tip of the bundle was situated on the centre line of the frame, the line running along the centre of the bundle was aligned vertically, and the border of the cuticular plate was situated in the centre of the frame. Each image had a fixed value subtracted, adjusted
47
J.O. Pickles et al./Hearing Research 101 (1996) 45-54
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Fig. 2. Dark-field micrographs of labelled (A) and control (B) cochleae, 2 days between injection and fixation. A shows heavy labelling in the papilla, but a low density of grains in the space above the papilla. B shows a low density of grains in all parts of the specimen. C and D: bright-field images of sections in A and B. C is very lightly stained, the dark regions showing autoradiographic grains. D is more heavily stained, to show the positions of the hair cells in the papilla. Scale bar: 20 gm (applies to all subfigures).
for each image, so that only pixels lying within autoradiographic grains had values above zero ("thresholding"; Fig. 3B). F o r the whole image, pixel values were then multiplied by the m a x i m u m value (255). This means that all pixels within grains had equal values (set at the maximum, 255), while the pixels outside grains had zero values. This produced an image in which all the grains were represented uniformly black, while all areas outside grains were uniformly white (Fig. 3C). Images of all cells in the group were then superimposed and then averaged. Fig. 3D shows an example of an intermediate stage of the analysis, in which six images such as in Fig. 3C have been averaged. Grain size was measured by counting the number of non-zero pixels contained within the images of individual grains.
As discussed later, it was possible that differences in autoradiographic grain density in different parts of the image might be a result of differences in the amount of tissue in different parts of the bundle. In order to test this possibility, the value of each pixel in the image of autoradiographic grain density (e.g. Fig. 4B) was divided by the value of the corresponding pixel in the raw image, i.e., before removal of toluidine blue stain and thresholding. The raw image has a full representation of the stain in the image, in addition to images of the autoradiographic grains. In this way, total staining could be compared with grain density. For a constant density of autoradiographic grains, the calculated ratio of (density grains after thresholding)/(density toluidine blue stain+density grains) will be low where the level of
48
J.O. Pickles et al./ Hearing Research 101 (1996) 45-54
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Fig. 3. Image processing. A: lightly stained raw image of hair cell. B: image after subtraction of constant level, to leave pixel values above zero only in the autoradiographic grains. C: image after the pixel values in all autoradiographic grains have been brought up to the same, maximum, value. D: intermediate stage of averaging, showing average image formed by superimposing and averaging 6 images such as are shown in Fig. 3C. Scale bar: 5 gm.
stain is relatively high, i.e., where there is relatively more toluidine blue staining material in the average. In this way, the calculation will provide a scaling for the amount of tissue which contributed to the average. The scaling will be most accurate where the density of grains is low relative to the amount of stain, i.e., when the divisor in the ratio will be most equal to the density of stain alone. The calculated ratio will be too small, where the density of grains is high relative to the amount of stain. The profiles of calculated ratios (Fig. 6) will therefore show less variation, i.e., will be flatter than would have been calculated using the ideal ratio (density grains/density stain). Nevertheless, differences in the calculated ratio will indeed reflect real differences in the ratio (density grains/density stain), although the differences will be understated. Chicks were put into one of five groups, depending on delay between injection and fixation: group 1, 2 days delay, 3 chicks; group 2, 5.5 days delay, 2 chicks;
group 3, 8 days delay, 1 chick; group 4, 10 days delay, 1 chick; group 5, 13 days delay, 2 chicks. Overall, 155 cells were averaged for group 1, 320 for group 2, 106 for group 3, 217 for group 4, and 241 for group 5. The differing numbers of cells in each group reflected the numbers of cells found conforming to the criteria listed above. Differences in grain density (measured over an area corresponding to the whole cellular area below the apical surface of the hair cells in Fig. 4A-E) for the different chicks within each group above showed that the standard deviation of grain density, and hence for uptake and incorporation of radiolabel in an individual animal, was 17% (df= 4).
3. Results
3.1. Growth of bundle in neural and abneural hair cells Bundle heights were measured in control cochleae
Fig. 4. A schematic outline of hair cell, corresponding to the hair cells in parts B F. b, bundle; cp, cuticular plate; n, nucleus. B-F: mean density of autoradiographic grains after different waiting times: (B) 2 days, (C) 5.5 days, (D) 8 days, (E) 10 days, (F) 13 days. The horizontal arrows point to the tip of the bundle, averaged over all hair cells in the group. Vertical arrow in F: point at which the vertical profile in Fig. 8 was obtained. Scale bar: 5 ~am.
J.O. Pickles et al./Hearing Research 101 (1996) 45-54
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F). fixed at 17 days of incubation (staged to Hamburger and Hamilton stages 43-44), i.e., at the stage at which radiolabel was applied in experimental chicks. The heights were measured in plastic-embedded sections over the region situated between 5 and 10% of the distance from the cochlear apex. The heights averaged 3.32 gm + 0.57 lam (S.D. ; n = 112) for bundles situated in the neural 50% of the papilla, and 3.63+0.80 gm (S.D.; n = 7 8 ) for cells situated in the abneural 50% of the papilla. The average height of bundles measured in the experimental chicks after autoradiographic processing was 6.17 g m + 0 . 4 3 gm (S.D.; n - 4 1 5 ) in the neural 50% of the papilla, and 6.01+0.71 gm (S.D.; n = 369) in the abneural 50%. The mean increase in bundle height over the period of the experiment was therefore 2.85 gm (86%) for neurally situated hair cells, and 2.38 gm (66%) for abneurally situated hair cells. Most of this growth (mean of 2.25 ~tm for neural and abneural cells) occurred in the 2 days after labelling, with a further 0.32 gm occurring between 2 and 5.5 days after labelling. There was no significant growth in the period 5.5-13 days after labelling, the regression line of height as a function of time in this period having a mean slope of 0.024 + 0.07 gm/day, not significantly different from zero. Because the neural and abneural groups of cells did not show any systematic differences in the pattern of incorporation of radiolabel, the later figures show data pooled from the two groups of cells.
3.2. Distribution of radiolabel and image processing Fig. 2A shows an autoradiograph of the basilar papilla, photographed in dark-field illumination, with 2 days delay between labelling and fixation. The sensory epithelium is heavily labelled. The hair cell layer shows a greater density of label, although the brightness of the image means that individual hair cells cannot be distinguished. Fig. 2C shows a bright field image of the same area, showing how the hair cells are marked by a greater density of grains (this section was only very lightly stained with toluidine blue, so that the dark material in the image represents only autoradiographic grains). In both images, the space above the sensory epithelium, from which the tectorial membrane has been removed, shows only a very low density of grains. Fig. 2B shows a control cochlea, following saline injection, but exposed for autoradiography with the same regime. The density of grains in all parts of the image is very low. These results show that the density of background labelling, even with 15 months of autoradiographic development, is very low. Hair cells, selected according to the criteria described in Section 2, were photographed at higher magnification, and the images scanned for further processing. Fig. 3A shows a typical hair cell after scanning, while Fig. 3B shows the same image after subtracting a constant value chosen so as to remove all stain, leaving only autoradiographic grains. Fig. 3C shows the same
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Fig. 6. Density of grains in bundle, as a ratio to density of toluidine blue staining material in image (see Section 2). The upper part of the bundle incorporates a relatively low a m o u n t of radiolabel in proportion to the toluidine blue staining material in the bundle. Data are means for 2-8 days, or for 10 13 days, delay between injection and fixation. The ratios were measured in a narrow (1 g m wide) vertical strip along the centre of the stereociliary bundle. The average ratios were constant across the width of the strip.
image once all non-zero pixels have been brought to the same, 100% black, value. This image, which represents only autoradiographic grains, was used for further analysis. As it appears in the stage of analysis shown in Fig. 3C, averaged over all animals used in the experiment and for autoradiographic grains situated within the hair cell body, the mean size of a single autoradiographic grain was 0.23l gm 2. For grains situated within the stereociliary bundle the mean size of a single autoradiographic grain was 8% smaller, at 0.213 ~tm2. The above figures show that only small errors are introduced by this variation in the measured grain size, leading to an under-representation of the autoradiographic signal in the bundle by only 8%, compared with that in the cell body. The above mean value for the grain size in the hair bundle (0.213 gm 2) was used for the calibration of later density plots. Images such as in Fig. 3C were averaged over all hair cells which had bundles conforming to the selection criteria to produce density plots of autoradiographic grains as in Fig. 4. In making the averages, the images of the tallest part of the bundles were accurately superimposed (see Section 2), so that the resulting images would be an accurate reflection of the mean density in the hair bundle. Because the registration of the bundles was most accurate in the centre of the bundle, and because of slight variation in the widths and shapes of
the bundles, the density of grain was greatest in the centre of the averaged image. Raw (unprocessed) images, composed of grains plus toluidine blue stain, were also averaged. For a 2 gm wide strip along the centre of the bundle, the density of the averaged raw images was uniform in a lateral direction across the width of the strip, over the basal 80% of the bundle height, thus indicating accurate registration of the images over this region. For a 1 ~tm wide strip the density was uniform in a lateral direction over the basal 90% of the bundle height. The lack of uniformity in the very tip region was due to the bundle tapering towards its tip. Fig. 4B shows the averaged grain density for 2 days delay between injection of radiolabel and fixing. In this image, the outline of the cell body and bundle is clearly visible, following the diagram in Fig. 4A. Parts of the adjacent hair cells are also visible on the left and right edges of the sub-figure. The region of the nucleus and cuticular plate is most heavily labelled. In the bundle, the density of label is greatest in the lower, rather than in the upper, part of the bundle. Fig. 4C F show the density of radiolabel, measured 5.5, 8, 10 and 13 days after labelling. The density of label falls with increasing delay between injection and fixation. However, the decline appears to be greater in the cell body than in the bundle (see below), so that with the greatest delays (Fig. 4F), the cell body and the bundle have nearly the same density of labelling. Changes in density were measured for each group of hair cells in a vertical 2 ~tm wide strip centred on the bundle (Fig. 5). Fig. 5 confirms that the density of label is greatest in the lower part of the hair bundle. It also shows that with increasing delays between injection and fixation, the density of label proportionately fell most in the cell body, and less in the bundle. Fig. 5 also shows a dip in the density of label just above the cuticular plate, 5-10% of the way up the bundle, which may in part arise because the stereocilia become thinner towards their rootlets, just before they enter the cuticular plate. It might be supposed that the relatively low level of incorporation in the upper part of the hair bundle is due to the small amount of tissue present in that region, as a result of the taper of the bundle, possibly with a small contribution from variation in the heights of the bundles (the S.D. of bundle height was 0.57 gm). In order to test this possibility, the value of each pixel in the grain density plot was divided by the value of the corresponding pixel in the average of the raw images (consisting of toluidine blue stain plus grains) used to make the plots. As described in Section 2, this provides scaling for the amount of toluidine blue staining material in the image. A high value of the resulting ratio reflects a high value of the ratio (grain density)/(toluidine blue stain density) and therefore reflects a high level of radiolabel incorporation relative to the amount
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Fig. 7. A: grain density in cuticular plate, nucleus, whole cellular area, and supporting cells, as a function of delay between injection and fixation. The fitted slopes are not significantly different at the P < 0.05 level. B: grain density in upper and lower 50% of bundle, as a function of delay between injection and fixation. The slope for the mean bundle is significantly shallower than that for the whole cell in part A, while slopes in the upper and lower halves of the bundle are not significantly different.
of tissue in the section. Particularly near the top of the bundle, the calculation is noise-prone, because the divisor in the calculation becomes small. Therefore, values were averaged over all cells analysed for the experiment before calculation of the ratio. The cells were divided into two groups, one for delays between injection and fixation of 2-8 days, and the other for delays of 10-13 days. As shown in Fig. 6, both groups of cells show a lower ratio of density in the upper part of the bundle than in the lower part, showing that the upper part of the bundle incorporates less radiolabel, relative to the amount of material in the section. The change in the ratio between the two groups of cells is approximately similar in all parts of the bundle. The mean decline in ratio is 39%, almost exactly equal to the decline in the grain density (36%) measured before the ratio calculation had been made, suggesting that the average density of toluidine blue staining material was comparable in the two groups of cells.
3.3. Turnover times A change in grain density with increasing delays between labelling and fixing presumably represents replacement of labelled amino acids by unlabelled ones and therefore represents turnover of the constituent proteins (by contrast, the half-life of 3H is 12.3 years). Grain density was measured in the averaged images in (1) a rectangle 2 gm wide covering the upper 50% of the bundle, (2) a rectangle 2 gm wide covering the lower
50% of the bundle, (3) a rectangle 3 gm wide and 1 gm high placed on the most densely labelled part of the cuticular plate, (4) a square 4 gm wide placed over the central densely labelled part of the hair cell, identified as the nucleus, (5) the whole image areas of Fig. 4 which lie below the apical surface of the hair cell, and (6) a square 2 p.m wide placed in the bottom left corner of the image, i.e., in the supporting cell area. Fig. 7A shows how grain density fell in the different regions of the hair cell body, in the supporting cells, and in the whole region below the apical surface of the epithelium. On a logarithmic vertical axis, all curves are well fitted by straight lines, showing single exponential decay. The time constants (to fall to l/e) ranged from 8.9 to 10.5 days for the different structures, with no significant differences. Fig. 7B shows curves for the two parts of the stereociliary bundle, and for the mean bundle. The time constant for the mean bundle was significantly greater than the values for the whole cell area ( P < 0 . 0 1 ; t = 6.70, df= 3). The time constants for the two parts of the bundle were not significantly different (t = 1.23, d f = 3 , P = 0.3).
3.4. Spread of radiolabel In order to determine the spread of radiolabelled grains around the site of radioactive disintegration, the profile of labelling was measured at a sharp edge in the tissue, in a mean image of grain density formed by averaging Fig. 4B-F. The density of label was meas-
J.O. Pickles et al./ Hearing Research 101 (1996) 45 -54
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Fig. 8. Density of grain along a 1 ~tm wide vertical strip, the density being averaged over all sub-figures of Fig. 4B F. The strip was centred on the point on the mean image indicated by the open arrow in Fig. 4F. The slope is the derivative of the density curve and shows the spread of radiolabel around a line source situated along the edge of the cuticular plate. 70°/,, of autoradiographic grains lie within 0.5~tm of the source, and 90% within 0.8 ~tm of the source. ured in a 1 ~m wide strip running vertically at a point to the left of the bundle in the images, at a point at which there was minimal labelled material in the space above the hair cells (shown by vertically pointing arrow in Fig. 4F). The profile of labelling is shown in Fig. 8. In the case of an infinitely sharp edge of material, the derivative of the density is the spread of labelling around a line source situated along the edge. The slope function in Fig. 8 has its peak situated precisely at the apical surface of the basilar papilla, as expected. The curve falls to 70% of its m a x i m u m value within +0.5 ~tm, indicating that at least 70% of grains will be localised to within 0.5 ~tm of the source. The true localisation will be more precise than this, because the averaged edge of material at the apical boundary of the hair and supporting cells is not infinitely sharp, due to for instance the presence of microvilli, attached fragments of the tectorial membrane, and variation in tissue position from one section to another, due to irregular curvature of the papillar surface.
3.5. Fixation of free amino acids Tissue in the main series in the experiment was fixed with paraformaldehyde, which has a p o o r ability to fix free amino acids, compared with glutaraldehyde (e.g. Sharp, 1976). Radiolabelling was compared in one chick (2 days delay between injection and fixation), where one ear had been fixed with 2.5% glutaraldehyde and the other with 2.5% paraformaldehyde. Mean grain density was compared in 100 hair cells from the glutaraldehyde-fixed ear, and 236 hair cells from the paraformaldehyde-fixed ear. The mean grain density in the glutaraldehyde-fixed ear was 1.7%+2.4% (S.E.M.) greater than in the paraformaldehyde-fixed ear, the small difference suggesting, if glutaraldehyde indeed
The low level of background labelling, even with 15 months autoradiographic development, and the good spatial localisation of the label (at least 70% of grains within 0.5 ~tm of the source) suggest that the radiolabelling technique is suitable for the measurement of the incorporation and turnover of amino acids, and hence presumably proteins, in hair cells. As shown in Fig. 4, the hair cells labelled more heavily than the surrounding supporting cells and, within the hair cells, the cuticular plate and the nucleus labelled most heavily. The reason for the differences in the patterns of labelling is not clear, but are presumably related to the local density of protein in the tissue, and the rate of laying down of protein soon after the application of the radiolabel. In Fig. 4B, within the stereociliary bundle, the pattern of labelling is closer to that shown in Fig. 1C than to that in Fig. lB. Therefore the most direct interpretation of the results is that the new amino acids, and therefore proteins, are preferentially added at the base of the stereocilia. The density of radiolabel is greater in the bottom part of the bundle, even once the density has been scaled by the amount of material in each part of the bundle (Fig. 6). These results imply that, contrary to the hypothesis of Tilney and DeRosier (1986), stereocilia do not grow from their tips. They may instead grow from the base, being pushed up from the cell body, or possibly even grow at all points along their length. The presumably greater availability of actin monomers in the cell body, compared to the tips of the stereocilia, could allow the actin monomers to be added at the non-preferred end. Tilney and DeRosier suggested that addition of actin monomers at the rootlet would be unlikely, because the filaments that were growing would have to detach from and reattach to the actin paracrystal as they moved up through the stereocilium. However, if monomers were added to the filaments in the rootlet proper (i.e., the filaments that continue into the cuticular plate) at the same rate as they were added to the filaments that end in association with the membrane in the taper region of the stereociliary ankle, then the whole paracrystal could be pushed up together, and no disassembly and reassembly of the paracrystal would have to take place during growth. The labelling seen in all points in the bundle in Fig. 4A suggests that there is some incorporation of label in previously established regions of the stereocilia. Therefore the simple model does not entirely explain the data. The labelling seen at all points on the stereocilia may arise from incorporation in rapidly turning over components of the stereocilia such as the membrane, from
J.O. Pickles et al./Hear&g Research 101 (1996) 45-54
labelling of unincorporated protein, from some growth of the stereocilia at all points along their length, or from some redistribution of radiolabelled material within the stereocilia after laying down of the protein. Because the decline of radiolabel occurs at the same rate in all parts of the bundle (Fig. 7B), it appears that mechanisms requiring different rates of turnover in different parts of the bundle, as in the first two of these explanations, cannot be the major ones. The results presented in the present paper give information on the average rate of turnover of proteins in hair cells. They suggest an overall turnover time of 16 days for the bundle, and 9 days for the rest of the cell. It is recognised that the times calculated here will be upper limits, because of the likelihood of some re-use of label during the period of turnover. Therefore the proteins may in fact be replaced more rapidly. The total amount of available label must be constant in the egg until hatching, except for amounts lost due to respiration. After hatching, the concentration of radiolabel will be reduced by excretion, and by the overall growth of the chick. However, any label which re-enters the general pool will be diluted as a result of the massive, several-fold, increase in the body weight of the chickens during the period of the experiment. Therefore we would expect the availability of label to fall with time, so that protein re-synthesised during the experiment will contain a lower level of radiolabel. It should also be noted that these values were obtained over the period from 1 day before to 12 days after hatching, and that turnover rates may be different in older chicks. It is likely that the different proteins within the tissue turn over at different rates. While attempts were made to analyse the labelled proteins on gels, unfortunately insufficient radiolabel was incorporated in extracted isolated sensory epithelia to permit autoradiography of the gels. However, the major proteins in stereocilia are known to include actin, its linking proteins, and membrane-associated proteins (Gillespie and Hudspeth, 1991), and it is likely that at least some of these were labelled. There is evidence that in microvilli membrane proteins turn over faster than do proteins associated with the cytoskeletal core (Stidwill et al., 1984). In developing muscle cells there is evidence for asynchronous turnover of the different actin-associated proteins (Funabiki and Cassens, 1973), although in microvilli the proteins associated with actin bundling turn over at about the same rate as actin itself (Stidwill et al., 1984). It is possible therefore that at least some of the change in labelling in the stereociliary bundle is due to turnover of membrane proteins and other proteins not associated with the more permanent components of the actin paracrystal. Superimposed on the hypothesised new growth of stereocilia from the base, turnover of the less permanent components could explain the incorporation and later decline of radiolabelling in older
53
parts of the stereocilia, i.e., in those parts which had developed before the radiolabel was applied. Because we do not know the proportion of radiolabel incorporated in the presumably more permanent components of the stereocilium, such as the actin filaments of the paracrystal, we do not know the extent to which the results are able to tell us the pattern of turnover in the paracrystal alone. The radiolabel in the bundle fell by 49% between days 2 and 13 after the injection. It would be surprising if a decline of this magnitude could occur without some measurable decrease in labelling in the paracrystal, given the relative proportion of the stereocilium occupied by the actin paracrystal as judged from ultramicrographs. However, it is also possible that different components of the paracrystal turn over asynchronously, so for instance the actin bundling proteins may be replaced while the Factin remains intact. Therefore, it cannot be guaranteed that the present experiments give information on turnover of the most permanent components of the stereocilium, and so tell us the degree of replacement of the stereocilium as a whole. Stereocilia are readily damaged by cochlear insult, including acoustic trauma, and ototoxic drugs. The changes include fracture and depolymerisation of the actin paracrystal (e.g. Tilney et al., 1982; Lim, 1986; Thorne et al., 1986; Liberman, 1987; Liberman and Dodds, 1987; Avinash et al., 1993), and may be responsible for some of the loss of stiffness seen after overstimulation (Szymko et al., 1995). The site of fracture forms a point of weakness at which the stereocilia flex further. At the moment we have no information as to the degree of repair possible in the paracrystal. The data of Liberman (1987) and Liberman and Dodds (1987) suggested that very little repair is possible, except perhaps to the rootlet. Nevertheless, it is likely that a gradual turnover and replacement of the actin paracrystal of the stereocilium over time would be an effective way of renewing the structural integrity of the stereocilium after accumulated trauma. The direction of replacement of the paracrystal would be critical for the effectiveness of the repair. If stereocilia are replaced by being pushed up from the cell body, damaged regions of the paracrystal would eventually be removed at the upper ends of the stereocilia. If replacement occurred from the upper ends downwards, it is possible that as a region of damage reached the rootlet, the attachment of the stereocilium would be so weakened that the whole stereocilium would be shed. It would also be interesting to know whether the turnover process could be artificially enhanced, thus possibly reversing the gradual increase in stereociliar pathologies found in older ears.
Acknowledgments This research was supported by the Australian Re-
54
J.o. Pickles et al./Hearing Research 101 (1996) 45-54
s e a r c h C o u n c i l . T h e s k i l l e d t e c h n i c a l a s s i s t a n c e o f P. R o g e r s a n d E. O b a r s k i is g r a t e f u l l y a c k n o w l e d g e d , and the advice of Alan Cody on image processing was strongly appreciated.
References Avinash, G.B., Nuttall, A.L. and Raphael, Y. (1993) 3-D analysis of F-actin in stereocilia of cochlear hair cells after loud noise exposure. Hear. Res. 67, 139-146. Flock, A., Flock, B. and Murray, E. (1977) Studies on the sensory hairs of receptor cells in the inner ear. Acta Otolaryngol. (Stockh.) 83, 85 91. Flock, A., Cheung, H.C., Flock, B. and Utter, G. (1981) Three sets of actin filaments in sensory cells of the inner ear. Identification and functional orientation determined by gel electrophoresis, immunofluorescence and electron microscopy. J. Neurocytol. 10, 133 147. Funabiki, R. and Cassens, R.G. (1973) Asynchronous turnover of the thin filament proteins, actin, tropomyosin and troponin by a continuous double isotope method. J. Nutr. Sci. Vitaminol. 19, 361368. Gillespie, P.G. and Hudspeth, A.J. (1991) High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. J. Cell Biol. 112, 625-640. Hamburger, V. and Hamilton, H.L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49 92. Hudspeth, A.J. (1989) How the ear's works work. Nature 341, 397 404. Liberman, M.C. (1987) Chronic ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates. Hear. Res. 26, 65 88. Liberman, M.C. and Dodds, L.W. (1987) Acute ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates. Hear. Res. 26, 45-64.
Lim, D.J. (1986) Effects of noise and ototoxic drugs at the cellular level in the cochlea: a review. Am. J. Otolaryngol. 7, 73 99. Pickles, J.O. and Corey, D.P. (1992) Mechanoelectrical transduction by hair cells. Trends Neurosci. 15, 254-259. Rubinstein, N., Chi, J. and Holtzer, H. (1976) Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells. Exp. Cell Res. 97, 387-393, Sharp, F.R. (1976) Activity-related increases of glucose utilization associated with reduced incorporation of glucose into its derivatives. Brain Res. 107, 663 666. Stidwill, R.P., Wysolmerski, T. and Burgess, D.R. (1984) The brush border is not static: in vivo turnover of proteins. J. Cell Biol. 98, 641-645. Szymko, Y.M., Nelson-Adesokan, P.M. and Saunders, J.C. (1995) Stiffness changes in chick hair bundles following in vitro overstimulation. J. Comp. Physiol. A 176, 727 735. Thorne, P.R., Duncan, C.E. and Gavin, J.B. (1986) The pathogenesis of stereocilia abnormalities in acoustic trauma. Hear. Res. 21, 4l 49. Tilney, L.G. and DeRosier, D.J. (1986) Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate. Dev. Biol. 116, 119-129. Tilney, L.G., DeRosier, D.J. and Mulroy, M.J. (1980) The organization of actin filaments in the stereocilia of cochlear hair cells. J. Cell Biol. 86, 244-259. Tilney, L.G., Saunders, J.C., Egelman, E. and DeRosier, D.J. (1982) Changes in the organization of actin filaments in the stereocilia of noise-damaged lizard cochleae. Hear. Res. 7, 181 197. Woodrum, D.T., Rich, S. and Pollard, T.D, (1975) Evidence for the biased bidirectional polymerisation of actin using heavy meromyosin produced by an improved method. J. Cell Biol. 67, 231 237. Zenner, H.P. (1981) Cytoskeletal and muscle-like elements in cochlear hair cells. Arch. Otorhinolaryngol. 230, 81 92.
Hearing Research 101 (1996) 45-54
The incorporation and turnover of radiolabelled amino acids in developing stereocilia of the chick cochlea J.O.
Pickles *, D.A. Billieux-Hawkins, G.W. Rouse
a
Vision Touch and Hearing Research Centre, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Qld. 4072, Australia Received 20 March 1996; revised 1 July 1996; accepted 21 July 1996
Abstract
Hair cell stereocilia are composed of packed actin filaments, oriented such that the preferred end for the addition of actin monomers is at the tips of the stereocilia. It has therefore been suggested that when stereocilia grow, they do so from their tips (Tilney and DeRosier, 1986, Dev. Biol. 116, 119 129). In order to test the hypothesis, radiolabelled amino acids were applied to the air-sac of chicken eggs at day 17 of incubation, i.e., at the beginning of a phase in which the stereocilia have achieved their mature width, but are growing rapidly in length. Incorporation of radiolabel was studied autoradiographically, followed by image analysis and averaging grain counts over many hair cells. In contrast to the position expected from the above hypothesis, there was no sign of preferential incorporation of label in the upper part of the stereociliary bundle. The greatest density of labelling was found in the lower part of the bundle, while the upper part of the bundle was under-represented in the autoradiographic averages. The turnover time (to fall to l/e) was significantly greater in the bundle (16 days) than in the cuticular plate or in the rest of the cell (9 days). The results (i) give no support for the hypothesis that stereocilia grow from the tips, and (ii) suggest that during development at least some components of the stereocilia turn over with a relatively short time course.
Keywords." Stereocilia; Protein; Turnover; Actin; Chick; Development; Hair cell
1. Introduction
Hair cells have an array of 50-150 stereocilia on their apical surface, deflection of which initiates mechanotransduction (Hudspeth, 1989; Pickles and Corey, 1992). The stereocilia are composed of actin filaments, tightly packed into what is known as a paracrystalline array (Tilney et al., 1980; Flock et al., 1981; Zenner, 1981). The actin paracrystal gives the stereocilia their strength and rigidity, so that when stereocilia are deflected, they bend only at their base and do not flex (Flock et al., 1977). This dense, tight and large-scale organization of actin filaments in such a paracrystal is unique to acousticolateral hair cells. The high degree of organization of the stereociliar paracrystal means that it * Corresponding author. Tel.: +61-7-3365-4125; Fax: +61-7-3365-4522; E-mail: [email protected].
a Present address." Department of Zoology, University of New South Wales, Sydney, NSW 2006, Australia.
is likely to form a relatively simple model for protein organization. It is also likely that the high degree of organization, close packing, and large number of inter-molecular cross-links of the actin filaments of the paracrystal impose severe restraints on the way that the filaments can be laid down or added to during development and turnover. As suggested by Tilney and DeRosier (1986), this makes it likely that the addition of m o n o m e r s occurs only at the borders of the paracrystal. Tilney and DeRosier therefore suggested that when stereocilia grow in length, but not in width, that they do so primarily by addition of actin monomers to one end or the other of the actin paracrystal. The pattern of decoration of the actin filaments with the S1 fragment of myosin shows that the preferred end for the addition of actin m o n o m e r s is at the tips of the stereocilia, and this suggested to Tilney and DeRosier that when stereocilia elongate, they do so by the addition of actin m o n o m e r s at their upper ends. There is no direct evidence of the way that stereocilia
0378-5955/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 5 9 5 5 ( 9 6 ) 0 0 129-3
46
J.O. Pickles et al./Hearing Research 101 (1996) 45 5 4
elongate. The suggestion that stereocilia elongate by addition of actin at their tips is uncertain, because it is also possible for monomers to be added at the nonpreferred end of actin filaments, although this requires a higher concentration of monomers (Woodrum et al., 1975). A related unresolved question concerns the turnover and replacement of stereociliar proteins. Once stereocilia are established, do they remain for a long period, such as years, or are they replaced in a matter of weeks or days? Actin turnover time in intestinal microvilli is several hours (Stidwill et al., 1984), while that of cultured myotubes has been measured as several days (Rubinstein et al., 1976). In addition, membrane-associated proteins commonly show a high degree of turnover. Given the substantial involvement of stereociliar damage in hair-cell pathology, knowledge of the rate of turnover of proteins in stereocilia would have important implications for our understanding of repair after cochlear damage. In order to test whether stereocilia grow by elongation at the tips, chick hair cells were incubated with radiolabelled amino acids, during the late phase in development, when the stereocilia are growing rapidly in length, but not in width (Tilney and DeRosier, 1986). After different waiting times, the distribution of the label was assessed by autoradiography of the hair bundles. Fig. 1 shows the expected sites of radiolabelling under the most direct interpretations of the two possible hypotheses. If stereocilia grow by the addition of new protein to the tips of the stereocilia, the concentration of radiolabel should be highest in the upper part of the bundle (Fig. 1B). If, however, stereocilia grow by the addition of new protein at the base, the concentration of label should be greatest in the lower part of the bundle (Fig. 1C). In addition, decreases in labelling with increasing times between injection and fixing will give an indication of protein turnover. To the extent that protein incorporating radiolabel is replaced by new, unlabelled protein, an indication of the rate of turnover will be obtained by measuring the rate of decay of radiolabel with time.
2. Methods Chicken eggs (white Leghorn) were incubated in a forced-draught incubator at 37°C. At 17 days of incubation, control embryos were staged by the criteria described by H a m b u r g e r and Hamilton (1951). At this age in experimental embryos, 0.5 mCi of a cocktail of 3Hlabelled amino acids (Amersham TRK550: leucine, lysine, phenylalanine, proline, tyrosine, 4.7 nmoles total) in 0.1 ml H 2 0 was applied to the air-sac of the eggs. After different periods of survival (2-13 days), the embryos or hatched chickens were decapitated, the head bisected, and the bony covering of the basilar papilla
l A
D
Newprotein ,-,
Fig. 1. A: profile of a stereociliary bundle from the chick basilar papilla, seen from the side, before the late phase of elongation of the stereocilia (embryonic age 17). Tall stereocilia are to the left of the bundle, and short to the right. B: if stereocilia grow by addition of actin monomers at the tips of the stereocilia, the new protein will be laid down in the upper part of the bundle. C: if stereocilia grow by addition of actin monomers at the base of the stereocilia, the new protein will be laid down in the lower part of the bundle.
opened. The half-heads were immersed in fixative (2.5% paraformaldehyde in 0.1 M phosphate buffer) for at least 24 h. The basilar papilla was opened widely, and the tegmentum vasculosum removed. The tectorial membrane was removed with fine forceps, and the remaining papilla was rinsed, dehydrated in ethanol and embedded in Spurr's resin. One g m sections were collected starting at a point 5% from the apical (i.e., distal) end of the papilla, and continuing for another 5% of the distance along the papilla, i.e., to a point 10% of the distance from the apical end of the papilla. Sections were cut orthogonal to the long axis of the papilla. Slides were dipped in autoradiographic emulsion (Ilford LM-1), and exposed at 4°C for 15 months. After autoradiographic development, the sections were lightly stained with toluidine blue. Sections were photographed under uniform conditions with a 100 × oil-immersion objective, and selected hair cells analysed by N I H - i m a g e software. The great majority of bundles were upright in the papilla and showed a compact bundle without splaying of stereocilia, i.e., they did not show any signs of disturbance from removal of the tectorial membrane. Criteria for selection of a bundle for further analysis were: (i) the vertical axis of bundle was orthogonal to cuticular plate, (2) the bundle showed a clear gradient in heights of the stereocilia, with a clear sharp taper to the tallest stereocilia which were towards the abneural side of the papilla, and (3) the bundle was compact, without splaying of the stereocilia. After scanning of the printed images by Macintosh OneScanner (256 grey levels, resolution equivalent to 94 nm/pixel; see Fig. 3A), each image was aligned so that the tip of the bundle was situated on the centre line of the frame, the line running along the centre of the bundle was aligned vertically, and the border of the cuticular plate was situated in the centre of the frame. Each image had a fixed value subtracted, adjusted
47
J.O. Pickles et al./Hearing Research 101 (1996) 45-54
C
.
[~
,
"7
Fig. 2. Dark-field micrographs of labelled (A) and control (B) cochleae, 2 days between injection and fixation. A shows heavy labelling in the papilla, but a low density of grains in the space above the papilla. B shows a low density of grains in all parts of the specimen. C and D: bright-field images of sections in A and B. C is very lightly stained, the dark regions showing autoradiographic grains. D is more heavily stained, to show the positions of the hair cells in the papilla. Scale bar: 20 gm (applies to all subfigures).
for each image, so that only pixels lying within autoradiographic grains had values above zero ("thresholding"; Fig. 3B). F o r the whole image, pixel values were then multiplied by the m a x i m u m value (255). This means that all pixels within grains had equal values (set at the maximum, 255), while the pixels outside grains had zero values. This produced an image in which all the grains were represented uniformly black, while all areas outside grains were uniformly white (Fig. 3C). Images of all cells in the group were then superimposed and then averaged. Fig. 3D shows an example of an intermediate stage of the analysis, in which six images such as in Fig. 3C have been averaged. Grain size was measured by counting the number of non-zero pixels contained within the images of individual grains.
As discussed later, it was possible that differences in autoradiographic grain density in different parts of the image might be a result of differences in the amount of tissue in different parts of the bundle. In order to test this possibility, the value of each pixel in the image of autoradiographic grain density (e.g. Fig. 4B) was divided by the value of the corresponding pixel in the raw image, i.e., before removal of toluidine blue stain and thresholding. The raw image has a full representation of the stain in the image, in addition to images of the autoradiographic grains. In this way, total staining could be compared with grain density. For a constant density of autoradiographic grains, the calculated ratio of (density grains after thresholding)/(density toluidine blue stain+density grains) will be low where the level of
48
J.O. Pickles et al./ Hearing Research 101 (1996) 45-54
13
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gl e
%
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otlt
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Fig. 3. Image processing. A: lightly stained raw image of hair cell. B: image after subtraction of constant level, to leave pixel values above zero only in the autoradiographic grains. C: image after the pixel values in all autoradiographic grains have been brought up to the same, maximum, value. D: intermediate stage of averaging, showing average image formed by superimposing and averaging 6 images such as are shown in Fig. 3C. Scale bar: 5 gm.
stain is relatively high, i.e., where there is relatively more toluidine blue staining material in the average. In this way, the calculation will provide a scaling for the amount of tissue which contributed to the average. The scaling will be most accurate where the density of grains is low relative to the amount of stain, i.e., when the divisor in the ratio will be most equal to the density of stain alone. The calculated ratio will be too small, where the density of grains is high relative to the amount of stain. The profiles of calculated ratios (Fig. 6) will therefore show less variation, i.e., will be flatter than would have been calculated using the ideal ratio (density grains/density stain). Nevertheless, differences in the calculated ratio will indeed reflect real differences in the ratio (density grains/density stain), although the differences will be understated. Chicks were put into one of five groups, depending on delay between injection and fixation: group 1, 2 days delay, 3 chicks; group 2, 5.5 days delay, 2 chicks;
group 3, 8 days delay, 1 chick; group 4, 10 days delay, 1 chick; group 5, 13 days delay, 2 chicks. Overall, 155 cells were averaged for group 1, 320 for group 2, 106 for group 3, 217 for group 4, and 241 for group 5. The differing numbers of cells in each group reflected the numbers of cells found conforming to the criteria listed above. Differences in grain density (measured over an area corresponding to the whole cellular area below the apical surface of the hair cells in Fig. 4A-E) for the different chicks within each group above showed that the standard deviation of grain density, and hence for uptake and incorporation of radiolabel in an individual animal, was 17% (df= 4).
3. Results
3.1. Growth of bundle in neural and abneural hair cells Bundle heights were measured in control cochleae
Fig. 4. A schematic outline of hair cell, corresponding to the hair cells in parts B F. b, bundle; cp, cuticular plate; n, nucleus. B-F: mean density of autoradiographic grains after different waiting times: (B) 2 days, (C) 5.5 days, (D) 8 days, (E) 10 days, (F) 13 days. The horizontal arrows point to the tip of the bundle, averaged over all hair cells in the group. Vertical arrow in F: point at which the vertical profile in Fig. 8 was obtained. Scale bar: 5 ~am.
J.O. Pickles et al./Hearing Research 101 (1996) 45-54
Bundle =
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Fig. 5. Grain densities in a strip 2 ~ m wide, centred on the hair bundle, for different delays between injection and fixation (data from Fig. 4 B -
F). fixed at 17 days of incubation (staged to Hamburger and Hamilton stages 43-44), i.e., at the stage at which radiolabel was applied in experimental chicks. The heights were measured in plastic-embedded sections over the region situated between 5 and 10% of the distance from the cochlear apex. The heights averaged 3.32 gm + 0.57 lam (S.D. ; n = 112) for bundles situated in the neural 50% of the papilla, and 3.63+0.80 gm (S.D.; n = 7 8 ) for cells situated in the abneural 50% of the papilla. The average height of bundles measured in the experimental chicks after autoradiographic processing was 6.17 g m + 0 . 4 3 gm (S.D.; n - 4 1 5 ) in the neural 50% of the papilla, and 6.01+0.71 gm (S.D.; n = 369) in the abneural 50%. The mean increase in bundle height over the period of the experiment was therefore 2.85 gm (86%) for neurally situated hair cells, and 2.38 gm (66%) for abneurally situated hair cells. Most of this growth (mean of 2.25 ~tm for neural and abneural cells) occurred in the 2 days after labelling, with a further 0.32 gm occurring between 2 and 5.5 days after labelling. There was no significant growth in the period 5.5-13 days after labelling, the regression line of height as a function of time in this period having a mean slope of 0.024 + 0.07 gm/day, not significantly different from zero. Because the neural and abneural groups of cells did not show any systematic differences in the pattern of incorporation of radiolabel, the later figures show data pooled from the two groups of cells.
3.2. Distribution of radiolabel and image processing Fig. 2A shows an autoradiograph of the basilar papilla, photographed in dark-field illumination, with 2 days delay between labelling and fixation. The sensory epithelium is heavily labelled. The hair cell layer shows a greater density of label, although the brightness of the image means that individual hair cells cannot be distinguished. Fig. 2C shows a bright field image of the same area, showing how the hair cells are marked by a greater density of grains (this section was only very lightly stained with toluidine blue, so that the dark material in the image represents only autoradiographic grains). In both images, the space above the sensory epithelium, from which the tectorial membrane has been removed, shows only a very low density of grains. Fig. 2B shows a control cochlea, following saline injection, but exposed for autoradiography with the same regime. The density of grains in all parts of the image is very low. These results show that the density of background labelling, even with 15 months of autoradiographic development, is very low. Hair cells, selected according to the criteria described in Section 2, were photographed at higher magnification, and the images scanned for further processing. Fig. 3A shows a typical hair cell after scanning, while Fig. 3B shows the same image after subtracting a constant value chosen so as to remove all stain, leaving only autoradiographic grains. Fig. 3C shows the same
50
ZO. Pickles et aL /Hearing Research 101 (1996) 45-54
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80 60 40 20 Percentagebundle height
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Fig. 6. Density of grains in bundle, as a ratio to density of toluidine blue staining material in image (see Section 2). The upper part of the bundle incorporates a relatively low a m o u n t of radiolabel in proportion to the toluidine blue staining material in the bundle. Data are means for 2-8 days, or for 10 13 days, delay between injection and fixation. The ratios were measured in a narrow (1 g m wide) vertical strip along the centre of the stereociliary bundle. The average ratios were constant across the width of the strip.
image once all non-zero pixels have been brought to the same, 100% black, value. This image, which represents only autoradiographic grains, was used for further analysis. As it appears in the stage of analysis shown in Fig. 3C, averaged over all animals used in the experiment and for autoradiographic grains situated within the hair cell body, the mean size of a single autoradiographic grain was 0.23l gm 2. For grains situated within the stereociliary bundle the mean size of a single autoradiographic grain was 8% smaller, at 0.213 ~tm2. The above figures show that only small errors are introduced by this variation in the measured grain size, leading to an under-representation of the autoradiographic signal in the bundle by only 8%, compared with that in the cell body. The above mean value for the grain size in the hair bundle (0.213 gm 2) was used for the calibration of later density plots. Images such as in Fig. 3C were averaged over all hair cells which had bundles conforming to the selection criteria to produce density plots of autoradiographic grains as in Fig. 4. In making the averages, the images of the tallest part of the bundles were accurately superimposed (see Section 2), so that the resulting images would be an accurate reflection of the mean density in the hair bundle. Because the registration of the bundles was most accurate in the centre of the bundle, and because of slight variation in the widths and shapes of
the bundles, the density of grain was greatest in the centre of the averaged image. Raw (unprocessed) images, composed of grains plus toluidine blue stain, were also averaged. For a 2 gm wide strip along the centre of the bundle, the density of the averaged raw images was uniform in a lateral direction across the width of the strip, over the basal 80% of the bundle height, thus indicating accurate registration of the images over this region. For a 1 ~tm wide strip the density was uniform in a lateral direction over the basal 90% of the bundle height. The lack of uniformity in the very tip region was due to the bundle tapering towards its tip. Fig. 4B shows the averaged grain density for 2 days delay between injection of radiolabel and fixing. In this image, the outline of the cell body and bundle is clearly visible, following the diagram in Fig. 4A. Parts of the adjacent hair cells are also visible on the left and right edges of the sub-figure. The region of the nucleus and cuticular plate is most heavily labelled. In the bundle, the density of label is greatest in the lower, rather than in the upper, part of the bundle. Fig. 4C F show the density of radiolabel, measured 5.5, 8, 10 and 13 days after labelling. The density of label falls with increasing delay between injection and fixation. However, the decline appears to be greater in the cell body than in the bundle (see below), so that with the greatest delays (Fig. 4F), the cell body and the bundle have nearly the same density of labelling. Changes in density were measured for each group of hair cells in a vertical 2 ~tm wide strip centred on the bundle (Fig. 5). Fig. 5 confirms that the density of label is greatest in the lower part of the hair bundle. It also shows that with increasing delays between injection and fixation, the density of label proportionately fell most in the cell body, and less in the bundle. Fig. 5 also shows a dip in the density of label just above the cuticular plate, 5-10% of the way up the bundle, which may in part arise because the stereocilia become thinner towards their rootlets, just before they enter the cuticular plate. It might be supposed that the relatively low level of incorporation in the upper part of the hair bundle is due to the small amount of tissue present in that region, as a result of the taper of the bundle, possibly with a small contribution from variation in the heights of the bundles (the S.D. of bundle height was 0.57 gm). In order to test this possibility, the value of each pixel in the grain density plot was divided by the value of the corresponding pixel in the average of the raw images (consisting of toluidine blue stain plus grains) used to make the plots. As described in Section 2, this provides scaling for the amount of toluidine blue staining material in the image. A high value of the resulting ratio reflects a high value of the ratio (grain density)/(toluidine blue stain density) and therefore reflects a high level of radiolabel incorporation relative to the amount
51
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Fig. 7. A: grain density in cuticular plate, nucleus, whole cellular area, and supporting cells, as a function of delay between injection and fixation. The fitted slopes are not significantly different at the P < 0.05 level. B: grain density in upper and lower 50% of bundle, as a function of delay between injection and fixation. The slope for the mean bundle is significantly shallower than that for the whole cell in part A, while slopes in the upper and lower halves of the bundle are not significantly different.
of tissue in the section. Particularly near the top of the bundle, the calculation is noise-prone, because the divisor in the calculation becomes small. Therefore, values were averaged over all cells analysed for the experiment before calculation of the ratio. The cells were divided into two groups, one for delays between injection and fixation of 2-8 days, and the other for delays of 10-13 days. As shown in Fig. 6, both groups of cells show a lower ratio of density in the upper part of the bundle than in the lower part, showing that the upper part of the bundle incorporates less radiolabel, relative to the amount of material in the section. The change in the ratio between the two groups of cells is approximately similar in all parts of the bundle. The mean decline in ratio is 39%, almost exactly equal to the decline in the grain density (36%) measured before the ratio calculation had been made, suggesting that the average density of toluidine blue staining material was comparable in the two groups of cells.
3.3. Turnover times A change in grain density with increasing delays between labelling and fixing presumably represents replacement of labelled amino acids by unlabelled ones and therefore represents turnover of the constituent proteins (by contrast, the half-life of 3H is 12.3 years). Grain density was measured in the averaged images in (1) a rectangle 2 gm wide covering the upper 50% of the bundle, (2) a rectangle 2 gm wide covering the lower
50% of the bundle, (3) a rectangle 3 gm wide and 1 gm high placed on the most densely labelled part of the cuticular plate, (4) a square 4 gm wide placed over the central densely labelled part of the hair cell, identified as the nucleus, (5) the whole image areas of Fig. 4 which lie below the apical surface of the hair cell, and (6) a square 2 p.m wide placed in the bottom left corner of the image, i.e., in the supporting cell area. Fig. 7A shows how grain density fell in the different regions of the hair cell body, in the supporting cells, and in the whole region below the apical surface of the epithelium. On a logarithmic vertical axis, all curves are well fitted by straight lines, showing single exponential decay. The time constants (to fall to l/e) ranged from 8.9 to 10.5 days for the different structures, with no significant differences. Fig. 7B shows curves for the two parts of the stereociliary bundle, and for the mean bundle. The time constant for the mean bundle was significantly greater than the values for the whole cell area ( P < 0 . 0 1 ; t = 6.70, df= 3). The time constants for the two parts of the bundle were not significantly different (t = 1.23, d f = 3 , P = 0.3).
3.4. Spread of radiolabel In order to determine the spread of radiolabelled grains around the site of radioactive disintegration, the profile of labelling was measured at a sharp edge in the tissue, in a mean image of grain density formed by averaging Fig. 4B-F. The density of label was meas-
J.O. Pickles et al./ Hearing Research 101 (1996) 45 -54
52 1 O0
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Fig. 8. Density of grain along a 1 ~tm wide vertical strip, the density being averaged over all sub-figures of Fig. 4B F. The strip was centred on the point on the mean image indicated by the open arrow in Fig. 4F. The slope is the derivative of the density curve and shows the spread of radiolabel around a line source situated along the edge of the cuticular plate. 70°/,, of autoradiographic grains lie within 0.5~tm of the source, and 90% within 0.8 ~tm of the source. ured in a 1 ~m wide strip running vertically at a point to the left of the bundle in the images, at a point at which there was minimal labelled material in the space above the hair cells (shown by vertically pointing arrow in Fig. 4F). The profile of labelling is shown in Fig. 8. In the case of an infinitely sharp edge of material, the derivative of the density is the spread of labelling around a line source situated along the edge. The slope function in Fig. 8 has its peak situated precisely at the apical surface of the basilar papilla, as expected. The curve falls to 70% of its m a x i m u m value within +0.5 ~tm, indicating that at least 70% of grains will be localised to within 0.5 ~tm of the source. The true localisation will be more precise than this, because the averaged edge of material at the apical boundary of the hair and supporting cells is not infinitely sharp, due to for instance the presence of microvilli, attached fragments of the tectorial membrane, and variation in tissue position from one section to another, due to irregular curvature of the papillar surface.
3.5. Fixation of free amino acids Tissue in the main series in the experiment was fixed with paraformaldehyde, which has a p o o r ability to fix free amino acids, compared with glutaraldehyde (e.g. Sharp, 1976). Radiolabelling was compared in one chick (2 days delay between injection and fixation), where one ear had been fixed with 2.5% glutaraldehyde and the other with 2.5% paraformaldehyde. Mean grain density was compared in 100 hair cells from the glutaraldehyde-fixed ear, and 236 hair cells from the paraformaldehyde-fixed ear. The mean grain density in the glutaraldehyde-fixed ear was 1.7%+2.4% (S.E.M.) greater than in the paraformaldehyde-fixed ear, the small difference suggesting, if glutaraldehyde indeed
The low level of background labelling, even with 15 months autoradiographic development, and the good spatial localisation of the label (at least 70% of grains within 0.5 ~tm of the source) suggest that the radiolabelling technique is suitable for the measurement of the incorporation and turnover of amino acids, and hence presumably proteins, in hair cells. As shown in Fig. 4, the hair cells labelled more heavily than the surrounding supporting cells and, within the hair cells, the cuticular plate and the nucleus labelled most heavily. The reason for the differences in the patterns of labelling is not clear, but are presumably related to the local density of protein in the tissue, and the rate of laying down of protein soon after the application of the radiolabel. In Fig. 4B, within the stereociliary bundle, the pattern of labelling is closer to that shown in Fig. 1C than to that in Fig. lB. Therefore the most direct interpretation of the results is that the new amino acids, and therefore proteins, are preferentially added at the base of the stereocilia. The density of radiolabel is greater in the bottom part of the bundle, even once the density has been scaled by the amount of material in each part of the bundle (Fig. 6). These results imply that, contrary to the hypothesis of Tilney and DeRosier (1986), stereocilia do not grow from their tips. They may instead grow from the base, being pushed up from the cell body, or possibly even grow at all points along their length. The presumably greater availability of actin monomers in the cell body, compared to the tips of the stereocilia, could allow the actin monomers to be added at the non-preferred end. Tilney and DeRosier suggested that addition of actin monomers at the rootlet would be unlikely, because the filaments that were growing would have to detach from and reattach to the actin paracrystal as they moved up through the stereocilium. However, if monomers were added to the filaments in the rootlet proper (i.e., the filaments that continue into the cuticular plate) at the same rate as they were added to the filaments that end in association with the membrane in the taper region of the stereociliary ankle, then the whole paracrystal could be pushed up together, and no disassembly and reassembly of the paracrystal would have to take place during growth. The labelling seen in all points in the bundle in Fig. 4A suggests that there is some incorporation of label in previously established regions of the stereocilia. Therefore the simple model does not entirely explain the data. The labelling seen at all points on the stereocilia may arise from incorporation in rapidly turning over components of the stereocilia such as the membrane, from
J.O. Pickles et al./Hear&g Research 101 (1996) 45-54
labelling of unincorporated protein, from some growth of the stereocilia at all points along their length, or from some redistribution of radiolabelled material within the stereocilia after laying down of the protein. Because the decline of radiolabel occurs at the same rate in all parts of the bundle (Fig. 7B), it appears that mechanisms requiring different rates of turnover in different parts of the bundle, as in the first two of these explanations, cannot be the major ones. The results presented in the present paper give information on the average rate of turnover of proteins in hair cells. They suggest an overall turnover time of 16 days for the bundle, and 9 days for the rest of the cell. It is recognised that the times calculated here will be upper limits, because of the likelihood of some re-use of label during the period of turnover. Therefore the proteins may in fact be replaced more rapidly. The total amount of available label must be constant in the egg until hatching, except for amounts lost due to respiration. After hatching, the concentration of radiolabel will be reduced by excretion, and by the overall growth of the chick. However, any label which re-enters the general pool will be diluted as a result of the massive, several-fold, increase in the body weight of the chickens during the period of the experiment. Therefore we would expect the availability of label to fall with time, so that protein re-synthesised during the experiment will contain a lower level of radiolabel. It should also be noted that these values were obtained over the period from 1 day before to 12 days after hatching, and that turnover rates may be different in older chicks. It is likely that the different proteins within the tissue turn over at different rates. While attempts were made to analyse the labelled proteins on gels, unfortunately insufficient radiolabel was incorporated in extracted isolated sensory epithelia to permit autoradiography of the gels. However, the major proteins in stereocilia are known to include actin, its linking proteins, and membrane-associated proteins (Gillespie and Hudspeth, 1991), and it is likely that at least some of these were labelled. There is evidence that in microvilli membrane proteins turn over faster than do proteins associated with the cytoskeletal core (Stidwill et al., 1984). In developing muscle cells there is evidence for asynchronous turnover of the different actin-associated proteins (Funabiki and Cassens, 1973), although in microvilli the proteins associated with actin bundling turn over at about the same rate as actin itself (Stidwill et al., 1984). It is possible therefore that at least some of the change in labelling in the stereociliary bundle is due to turnover of membrane proteins and other proteins not associated with the more permanent components of the actin paracrystal. Superimposed on the hypothesised new growth of stereocilia from the base, turnover of the less permanent components could explain the incorporation and later decline of radiolabelling in older
53
parts of the stereocilia, i.e., in those parts which had developed before the radiolabel was applied. Because we do not know the proportion of radiolabel incorporated in the presumably more permanent components of the stereocilium, such as the actin filaments of the paracrystal, we do not know the extent to which the results are able to tell us the pattern of turnover in the paracrystal alone. The radiolabel in the bundle fell by 49% between days 2 and 13 after the injection. It would be surprising if a decline of this magnitude could occur without some measurable decrease in labelling in the paracrystal, given the relative proportion of the stereocilium occupied by the actin paracrystal as judged from ultramicrographs. However, it is also possible that different components of the paracrystal turn over asynchronously, so for instance the actin bundling proteins may be replaced while the Factin remains intact. Therefore, it cannot be guaranteed that the present experiments give information on turnover of the most permanent components of the stereocilium, and so tell us the degree of replacement of the stereocilium as a whole. Stereocilia are readily damaged by cochlear insult, including acoustic trauma, and ototoxic drugs. The changes include fracture and depolymerisation of the actin paracrystal (e.g. Tilney et al., 1982; Lim, 1986; Thorne et al., 1986; Liberman, 1987; Liberman and Dodds, 1987; Avinash et al., 1993), and may be responsible for some of the loss of stiffness seen after overstimulation (Szymko et al., 1995). The site of fracture forms a point of weakness at which the stereocilia flex further. At the moment we have no information as to the degree of repair possible in the paracrystal. The data of Liberman (1987) and Liberman and Dodds (1987) suggested that very little repair is possible, except perhaps to the rootlet. Nevertheless, it is likely that a gradual turnover and replacement of the actin paracrystal of the stereocilium over time would be an effective way of renewing the structural integrity of the stereocilium after accumulated trauma. The direction of replacement of the paracrystal would be critical for the effectiveness of the repair. If stereocilia are replaced by being pushed up from the cell body, damaged regions of the paracrystal would eventually be removed at the upper ends of the stereocilia. If replacement occurred from the upper ends downwards, it is possible that as a region of damage reached the rootlet, the attachment of the stereocilium would be so weakened that the whole stereocilium would be shed. It would also be interesting to know whether the turnover process could be artificially enhanced, thus possibly reversing the gradual increase in stereociliar pathologies found in older ears.
Acknowledgments This research was supported by the Australian Re-
54
J.o. Pickles et al./Hearing Research 101 (1996) 45-54
s e a r c h C o u n c i l . T h e s k i l l e d t e c h n i c a l a s s i s t a n c e o f P. R o g e r s a n d E. O b a r s k i is g r a t e f u l l y a c k n o w l e d g e d , and the advice of Alan Cody on image processing was strongly appreciated.
References Avinash, G.B., Nuttall, A.L. and Raphael, Y. (1993) 3-D analysis of F-actin in stereocilia of cochlear hair cells after loud noise exposure. Hear. Res. 67, 139-146. Flock, A., Flock, B. and Murray, E. (1977) Studies on the sensory hairs of receptor cells in the inner ear. Acta Otolaryngol. (Stockh.) 83, 85 91. Flock, A., Cheung, H.C., Flock, B. and Utter, G. (1981) Three sets of actin filaments in sensory cells of the inner ear. Identification and functional orientation determined by gel electrophoresis, immunofluorescence and electron microscopy. J. Neurocytol. 10, 133 147. Funabiki, R. and Cassens, R.G. (1973) Asynchronous turnover of the thin filament proteins, actin, tropomyosin and troponin by a continuous double isotope method. J. Nutr. Sci. Vitaminol. 19, 361368. Gillespie, P.G. and Hudspeth, A.J. (1991) High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins. J. Cell Biol. 112, 625-640. Hamburger, V. and Hamilton, H.L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49 92. Hudspeth, A.J. (1989) How the ear's works work. Nature 341, 397 404. Liberman, M.C. (1987) Chronic ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates. Hear. Res. 26, 65 88. Liberman, M.C. and Dodds, L.W. (1987) Acute ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates. Hear. Res. 26, 45-64.
Lim, D.J. (1986) Effects of noise and ototoxic drugs at the cellular level in the cochlea: a review. Am. J. Otolaryngol. 7, 73 99. Pickles, J.O. and Corey, D.P. (1992) Mechanoelectrical transduction by hair cells. Trends Neurosci. 15, 254-259. Rubinstein, N., Chi, J. and Holtzer, H. (1976) Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells. Exp. Cell Res. 97, 387-393, Sharp, F.R. (1976) Activity-related increases of glucose utilization associated with reduced incorporation of glucose into its derivatives. Brain Res. 107, 663 666. Stidwill, R.P., Wysolmerski, T. and Burgess, D.R. (1984) The brush border is not static: in vivo turnover of proteins. J. Cell Biol. 98, 641-645. Szymko, Y.M., Nelson-Adesokan, P.M. and Saunders, J.C. (1995) Stiffness changes in chick hair bundles following in vitro overstimulation. J. Comp. Physiol. A 176, 727 735. Thorne, P.R., Duncan, C.E. and Gavin, J.B. (1986) The pathogenesis of stereocilia abnormalities in acoustic trauma. Hear. Res. 21, 4l 49. Tilney, L.G. and DeRosier, D.J. (1986) Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate. Dev. Biol. 116, 119-129. Tilney, L.G., DeRosier, D.J. and Mulroy, M.J. (1980) The organization of actin filaments in the stereocilia of cochlear hair cells. J. Cell Biol. 86, 244-259. Tilney, L.G., Saunders, J.C., Egelman, E. and DeRosier, D.J. (1982) Changes in the organization of actin filaments in the stereocilia of noise-damaged lizard cochleae. Hear. Res. 7, 181 197. Woodrum, D.T., Rich, S. and Pollard, T.D, (1975) Evidence for the biased bidirectional polymerisation of actin using heavy meromyosin produced by an improved method. J. Cell Biol. 67, 231 237. Zenner, H.P. (1981) Cytoskeletal and muscle-like elements in cochlear hair cells. Arch. Otorhinolaryngol. 230, 81 92.