Brix, Jutta

Comis, S. D.

Gleich, Otto

Köppl, Christiane

Manley, Geoffrey A.

Osborne, M. P.

Pickles, J. O.

English

University of Regensburg Publication Server

Basic Issues in Hearing Proceedings of the 8th International Symposium on Hearing Paterswolde, Netherlands, April 5 -9 , 1988 Edi ted by H. Duifhuis Biophysics Department University of Groningen The Netherlands J. W. Horst and H. P. Wit Institute of Audiology University Hospital Groningen The Netherlands 1988 Academic Press Harcourt Brace Jovanovich, Publishers London San Diego New York Berkeley Boston Sydney Tokyo Toronto The fine structure and organization of tip links on hair cell stereovilli J . O . Pickles, J . Br ix* , O . G l e i ch * , C . Kftppl*, G . A . Manley* , M .P . Osborne and S .D . Comis Department of Physiology, University of Birmingham, Birmingham, B15 2TJ, UK, and *Institut fur Zoologie, Technische Universitdt Munchen, D-8046 Garching, FRG. Introduction We have recently described a set of l inks between the stereovi l l i o f hair cells in the mammal ian cochlea. The l inks emerge f rom the tips of the shorter stereovi l l i in the bundle, and extend upwards to j o in the s ide-wa l l o f the adjacent taller stereovillus (e.g. Pickles et al., 1984; Comis et al., 1985). The tip l inks may be part icular ly interesting, because the evidence suggests that they are l ike ly to be involved in sensory transduction, coupl ing the s t imulus-induced movement to the transducer channels, perhaps situated at one or both o f their points of insertion into the stereovi l l i (e.g. Hudspeth 1985; P ickles, 1985). In the present experiments, we have further investigated the f ine structure o f the t ip l inks , in order to see how the structure might be related to a possible role in transduction. We have also investigated the organization of the l inks in stereovi l l i f rom a variety of species, since i f they are involved in transduct ion, we might expect them to be universal ly present on mechano-receptor hair cells o f acousticolateral or ig in and to be oriented parallel to the exc i ta tory- inhib i tory axis of the cel l . Methods For transmission electron microscopy, guinea pigs were anaesthetised, the bullae extracted, and perfused wi th 2.5% glutaraldehyde and 2% tannic acid in 0.05 M B E S buf fer , adjusted to p H 7.4 wi th N a O H (method modi f i ed f rom L i t t l e and Neugebauer, 1985). A f t e r extraction of the mod io l i , the specimens were f i xed for 0.5 h in the same buffer but inc lud ing in addit ion 2% tannic ac id , fo l lowed by postf ixation in 1% O s 0 4 in the same buffer for 5 m i n , and soaking in 2% tannic acid in dist i l led H 2 0 , at p H 7.0. Dehydrat ion was accomplished wi th ethanol and the material stained en bloc w i th urany l acetate and phosphotungstic ac id , before embedding in an Epon Ara ld i te epoxy resin mixture. Sections were stained in methanolic uranyl acetate and lead citrate before examination. For scanning electron microscopy in l izards, pigeons and starlings, the subjects were anaesthetised and were perfused either intravenously or directly through the oval or round windows w i th f ixat ive (pigeon: 2.5% glutaraldehyde in 0.1 M phosphate buffer ; starl ing and l i zard: 1% g lutar-aldehyde and 15% saturated p ic r i c acid in 0.1 M phosphate buffer) . Spec i -mens were stored in 2% glutaraldehyde in 0.05 M or 0.1 M phosphate buf fer unti l further treatment. They were then dehydrated in acetone, dr ied by the cr i t ical point technique w i th l i qu id C 0 2 , and sputter-coated w i th p la t inum to a nominal depth of 25 nm. Specimens were examined in a J E O L 120 C X I I microscope w i th a scanning attachment, and images were observed by a secondary electron detector. In order to measure the electrophysiological responses of the ch ick basilar Papillae, chicks ( 1 - 1 4 days old) were anaesthetised, and the basilar papil lae removed v ia an intracranial approach. The papillae were mounted in b i rd ringer or buffered saline ( pH 7.4, 285 mOsm) in a recording chamber, w i th electrodes on either side of the sensory epi the l ium. The tectorial membrane was moved direct ly by means of a piezoelectric pusher (Burle igh) , usually at 200 H z and a peak to peak amplitude of 70 nm , the microphonic being detected w i th the help of a spectrum analyzer (Hewlet t -Packard 3580A) . Responses were tested every 5 m in ; after responses had been shown to be stable for 20 m i n , enzyme solution was introduced into the chamber, and responses were monitored at 10 min intervals for a further 40 - 60 m in . The Papillae were then f ixed in 2.5% glutaraldehyde and processed for scanning electron microscopy as above. For enzyme experiments in the guinea pig , temporal bones were extracted as above, the cochleae perfused w i th 2.5 % glutaraldehyde, and the cochleae opened under f ixat ive , g iv ing a total time in f ixat ive of 5 m in . The opened modio l i were immersed in enzyme solution at 4°C for 10 or 20 m i n , and then Processed for scanning electron microscopy as above. Results The fine structure of tip l inks In sections stained wi th uranyl acetate and lead citrate, a f ine relatively straight f i lament was visible in the centre of the tip l ink (F ig . 1). The central f i lament was surrounded by dark ly -s ta in ing material w i th a variable appear-ance. In some sections, the central f i lament appeared to be negatively stained. The central f i lament, inc lud ing the heavily dark ly -s ta in ing material i m m e d i -ately surrounding it , was measured as having a diameter of 5.5 nm ± 0.4 nm (sem, n = 16). A t its upper end, the central f i lament ran to the centre of a density on the sidewall o f the taller stereovillus, the density forming a bridge between the external membrane of the stereovillus and the internal act in paracrystal. A t its lower end, the f i lament ended on what usually appeared as a conical extension of the membrane of the stereovillus (F ig . 2). Below this, there was a clear area, and below that, there was a dense cap over the ends of the f i laments of the actin paracrystal (F ig . 2). In some cases it was just possible to see fi laments Figure 1. Tip link with central filament (arrow), and upper dense point of attachment (arrowhead). Insert: same, more densely printed. Guinea pig outer hair cell. Scale bar: 100 nm. Figure 2. Dense cap (arrow) over actin filaments just under tip link. Filaments (arrowhead) are just visible, connecting density to surface membrane just under tip link. Guinea pig inner hair cell. Scale bar: 100 nm. Figure 3. Chondroitinase-digested inner hair cell, showing some tip links remaining, but thinner than in control cochleae. Insert: detail. Guinea pig. Scale bars: 200 nm and 50 nm. Figure 4. Hair bundle from the lizard Podarcis sicula. showing columns of stereocilia joined by tip links (arrows). Scale bar: 500 nm. running between the dense cap and the over ly ing membrane near the point of insertion of the central f i lament of the tip l ink. The material of variable appearance surrounding the central f i lament of ^ S ^ r e 5. /fair fcwirf/e m starling, showing tip links, with stereocilia organized into columns running at right angles to the rows (i.e. along the axis of bilateral symmetry). Basal end of papilla, near abneural edge. Scale bar: 500 nm. Figure 6. Tip link organization in pigeon. Abneural edge, middle of papilla. Scale bar: 500 nm. Figure 7. Anomalous bundle in pigeon, with tip links over left and some of right of bundle oriented towards the right (arrows), and with those on the extreme right running towards the left (double-headed arrows). This hair cell »as oriented 30 degrees differently from its neighbours. The latter were oriented such that their rows of stereovilli ran at right angles to the arrows on the left of the figure, with their tip links running parallel to the arrows. The targe structure at the top is a bleb, probably associated with the hole in the cuticular plate. Middle of papilla, neural edge. Scale bar: 200 nm. the t ip l ink appeared to be continuous wi th the variable material surrounding the external membranes of the stereovillus. It may therefore consist of glycocongugates. This hypothesis was tested by enzyme digestion in the guinea pig. Opened cochleae were incubated in chondroitinase (Sigma C -3509, 1 unit/ml) for 10 min at 4°C. While some of the tip l inks were missing, those that d i d survive were usually f iner than in control cochleae (F ig . 3), suggesting that a surrounding coat had been removed, and that it was susceptible to chondroit inase. O n the other hand, incubat ion in crude protease (Sigma Type I P-4630, 200 /ig/ml for 10 min) or trypsin (Sigma Type III-S, T -2395, 1000 72 /ig/ml for 20 min) removed the l inks as soon as it produced other observable changes on the surface of the stereovi l l i . The l inks therefore may wel l contain a protein component, perhaps in the central f i lament, although we have no further in format ion on the composit ion of the f i lament. A common procedure for the isolation of viable hair cells f rom the m a m -mal ian cochlea involves incubat ion in collagenase (e.g. Za j i c and Schacht, 1987). Digest ion o f the isolated ch ick basilar papi l la in collagenase (Sigma type IV C-5138 , 1 mg/ml) for up to 1 hr at 20°C had little effect on the evoked microphonic current, and was associated wi th surv iva l o f the t ip l inks , although changes in the membrane texture sometimes made them d i f f i cu l t to see. Th is suggests that the l inks may not be composed o f collagen. T h e spatial organization of tip l inks We have previously reported that in the guinea p ig , t ip l inks have a horizontal component in their orientation wh i ch is parallel to the axis of bi lateral symmetry of the hair ce l l , or in other words paral le l to the exc i ta tory- inhib i tory axis (Pickles et al.y 1984; Comis et al., 1985). Th is was true in both inner and outer hair cells, i.e. in bundles of very di f ferent conformat ion. The presence and spatial organization o f t ip l inks was further investigated in hair cells of the b i rd and l izard basilar papil lae, wh i ch have conformations quite di f ferent f rom that o f the mammal ian cochlea. In the lizards Podarcis sicula and Podarcis muralis the tip l inks ran parallel to the axis o f bilateral symmetry, i.e. in the direct ion f rom the shortest stereovi l l i to the tallest (F ig . 4). The stereovi l l i and their associated t ip l inks in fact formed columns, running parallel to the axis of symmetry, sometimes clearly separating out into groups (F ig . 4). The same columnar organization was found in the starl ing (F ig . 5), pigeon (F ig . 6) and ch ick basilar papil lae. Where anomalous tip l ink orientations were found, they were generally associated w i th the fo l lowing: (i) the stereovi l l i had been obviously d i s -organized in preparation, so that the l inks had been distorted, and (ii) there were other abnormalit ies in the bundle. In some cases moreover extraneous material lay over the surface o f the bundle, making it d i f f i cu l t to identi fy the t ip l inks . In only a few hair cells (an average wel l under 1%) was it possible to f i nd anomalously-or iented t ip l inks in an otherwise normal bundle. Most ly there were other abnormalit ies as wel l . In the case i l lustrated in F i g . 7, the t ip l inks on the whole of the left half and on part of the right hal f ran towards the r ight; those on the extreme right of the bundle ran towards the left. Th is bundle was also anomalous in that the bundle as a whole was ti lted w i th respect to its neighbours, w i th the orientation of the rows as a whole being some 30° di f ferent f rom those on either side. The orientation was such that the t ip l inks over most of the bundle (i.e. over the left hal f and most of the right half) ran in a d irect ion nearly parallel to the t ip l inks on the adjacent hair cells. Discussion The results show that tip l inks have two components, namely a f ine central filament, and a variable surrounding coat. We do not have in format ion on the nature of the f i lament, although it is presumably protein. It has the same diameter as act in f i laments w i th in the paracrystal o f the stereovi l l i i n our material. L i k e act in , the f i lament is negat ively-staining, and is susceptible to osmium tetroxide (Comis et al„ 1985). A f ine f i lament would be ideal for concentrating the s t imulus- induced movements onto a small area of m e m -brane, suitable for opening the 1 - 4 transducer channels associated w i th each stereovillus (Russel l , 1983; Ho l ton and Hudspeth, 1986). We do not know at wh i ch end of the l ink the transducer channels might be situated. The density at the upper point of attachment, or the conica l extension of the stereovillar membrane at the lower point o f attachment, are both possible sites, under the hypothesis that the t ip l inks are invo lved in transduction (Pickles et at., 1984). We note that the membrane around the lower point of insert ion, at the tip of the shorter stereovil lus, is anchored by filaments onto the dense cap on the under ly ing act in paracrystal, so that s t imulus- induced shear can be concentrated on the membrane at that point. The f ind ing that t ip l inks are present in a wide range of species supports the notion that they have an important functional role. They have so far been reported for the mammalian cochlea and vestibular system, inc lud ing the guinea p ig , rat and man (Pickles et al., 1984; Rhys -Evans et al.% 1985) and the fish vestibular system (Lit t le and Neugebauer, 1985). Here, the observations have been extended to include birds (chick, starl ing and pigeon) and reptiles (lizards). Investigation of these species had advantages, in that the hair bundles have a compact f o rm, w i th the stereovi l l i being t ightly packed into a hexagonal array. In such bundles, the orientation of the t ip l inks paral lel to the hair cel l axis of symmetry is part icular ly obvious. The fact that the stereovil l i tend to split into columns wh ich run parallel to the axis of bi lateral symmetry (i.e. along the exc i tatory- inhib i tory axis) suggests that the mechanical connections along this axis are stronger than those in the other directions. Th is again emphasises the possible importance of the axis i n hair cell funct ion. The laying down of the axis of the hair cel l dur ing ontogeny, and the development of the tip l inks and their organization (Neugebauer, 1986), are Part icular ly interesting issues. Anomalous ly-or iented t ip l inks may give suggestive evidence here. We have as yet only pre l iminary evidence on this Point. We have previously reported in a group of guinea pigs where hair cells were found w i th anomalous axes, that tip l inks nevertheless tended to run at right angles to the long axis of the ind iv idua l rows of stereovi l l i (Pickles et al.% 1986). This suggests that tip l ink organization is more closely related to the hair cel l itself rather than to its surroundings. Here, however, we report a case where anomalous organization of the t ip l inks was associated w i th an anomalous orientation of the bundle as a whole. This suggests that the orientation o f the t ip l inks can be affected by the surroundings of the ce l l , as Hair cell stereovilli fine structure wel l as by factors internal to the cel l . Summary T i p l inks on hair cells of the guinea pig cochlea have a f ine (6-nm) central core, surrounded by a variable outer coat. The f ine central f i lament is related to densities at its points of attachment on the stereovi l l i (stereocilia). We report here t ip l inks for a wider range of systems, inc lud ing birds (starling, pigeon and chick ) and l izards. With very few exceptions, the tip l inks run paral lel to the axis of bi lateral symmetry of the hair ce l l , and the stereovi l l i are organized into columns parallel to this axis. When the organization o f the t ip l inks does not f i t into this scheme, there are generally other anomalies in the hair bundle. Acknowledgements The work reported here was supported by the Med i ca l Research C o u n c i l ( U K ) , and the Deutsche Forschungsgemeinschaft w i th in the programme of the S F B 204 "Gehflr". The expert technical assistance of L . M . Tompk ins and T . L . Hay ward is gratefully acknowledged. References Comis , S.D., P ickles , J .O . and Osborne, M .P . (1985). "Osm ium tetroxide post f ixat ion in relation to the crosslinkage and spatial organization of stereocil ia in the guinea pig cochlea, " J .Neurocyto l . 14, 113-130. Ho l t on , T . and Hudspeth, A . J . (1986). a T h e transduction channel of hair cells f r om the bu l l - f r og characterized by noise analysis, " J .Phys io l . 375, 195-227. Hudspeth , A . J . (1985). a T h e cel lular basis of hearing: the biophysics of hair ce l l s , " Science 230, 745-752. L i t t l e , K . F . and Neugebauer, D . C h . (1985). "Interconnections between the stereovi l l i o f the f ish inner ear II," Ce l l Tissue Res. 242, 427-432. Neugebauer, D . C h . (1986). "Interconnections between the stereovi l l i o f the f ish inner ear. I l l , " Ce l l Tissue Res. 246, 447-453. P ick les , J .O . (1985). "Recent advances in cochlear physio logy, " Prog. Neurob io l . 24, 1-42. P ick les , J .O . , Comis , S.D. and Osborne, M .P . (1984). "Cross - l inks between stereocil ia in the guinea pig organ of C o r t i , and their possible relation to sensory t ransduct ion, " Hear ing Res. 15, 103-112. P ickles , J .O . , Comis , S.D., Osborne, M .P . and Pepper, C . (1986). "Gu inea pigs w i th backwards outer hair ce l ls , " Abstracts, 23rd Meet ing Inner Ear Bio logy , p. 13, Be r l i n , D D R . R h y s - E v a n s , P .H . , Comis , S.D., Osborne, M.P . , P ickles, J .O . and Jef fr ies, D .J .R . (1985). "Cross - l inks between stereocil ia in the human organ of C o r t i , " J .Laryngo l .Oto l . 99 , 11-19. Russe l l , I .J. (1983). "Or i g in of the receptor potential in inner hair cells of the mammal ian cochlea: evidence for Dav is ' theory, " Nature 301 , 334-336. Za j ic , G . and Schacht, J . (1987). "Compar ison of isolated outer hair cells f rom five mammal ian species," Hear ing Res. 26, 249-256. Comments Slepecky: In your diagram you show the t ip l inks parallel to the d i rect ion o f bi lateral symmetry, wh i ch is also parallel to the direct ion of hair cel l excitat ion in the radial d i rect ion. Th is arrangement o f stereocil ia may be true for the basal region of the cochlea, but at the apical region the axes o f bi lateral symmetry of the stereocil ia bundle is not exactly radia l , and the f ibers of the tectorial membrane curve. Does the orientation of the tip l inks vary w i th the shape of the stereocil ia bundle, along the length of the cochlea? Reply by Pickles: There is almost certainly a longi tudinal component to the travel l ing wave in the cochlear duct , so it should not be surpis ing that the axis o f bi lateral symmetry o f the hair cells does not always run in a d irect ion wh ich is radial across the duct. There are problems in analyzing the three-dimensional organization of stereovillar bundles in the apex of the mammal ian cochlea, since the stereovil l i are rather long and are easily disturbed dur ing the preparation. However , it should be noted that in species such as b irds, the angle o f the hair cell axis changes in a regular and systematic manner across the w id th o f the Papi l la , undergoing a change in orientation of up to 90°. In these cases, the columns formed by the tip l inks , and the t ip l inks themselves, nevertheless continue to run parallel to the axis of bilateral symmetry o f the bundle, i n spite o f the change in orientation. 

The fine structure and organization of tip links on hair
cell stereovilli

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The fine structure and organization of tip links on hair cell stereovilli

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