Brix, Jutta

Comis, S. D.

Gleich, Otto

Köppl, Christiane

Manley, Geoffrey A.

Osborne, M. P.

Pickles, J. O.

English

University of Regensburg Publication Server

Tip-Link organization in relation to the structure and orientation of stereovillar bundles

Cochlear Mechanisms Structure, Function, and Models Edi ted by J . P. Wi lson Universi ty of Keele Keele, Staf fordshi re, United K ingdom and D. T. Kemp Institute of Oto logy and Laryngology London, Uni ted K ingdom Plenum Press New York and London Published in Cooperation with NATO Scientific Affairs Division TIP-LINK ORGANIZATION IN RELATION TO THE STRUCTURE AND ORIENTATION OF STEREOVILLAR BUNDLES J . O . Pickles, M.P. Osborne, S.D. Comis, C. Köppl*, O. Gleich*, J . Brix*, G . A . Manley* Department of Physiology University of Birmingham, Birmingham B15 2TJ, UK * Institut für Zoologie, Technische Universität München D-8046 München, FDR Introduction The linkages between stereovilli (stereocilia) of hair cells have been implicated both in the mechanical properties of the bundles and in the transmission of s t imulus- induced movements to the ind iv idua l mechanotransducer Channels (e.g. Flock and Strelioff, 1984; Pickles et al . , 1984; Hudspeth, 1985). Here, we describe new observations on the fine structure of the tip links between stereovill i . We also show that the spatial Organization of the tip l inks in bundles of different conformations in a variety of species is appropriate for a role in transduction. Methods For transmission electron microscopy, temporal bones were extracted from anaesthetized guinea pigs. The bones were perfused wi th f ixat ive as described previously (Pickles et al . , 1987). Specimens were f ixed for 0.5 h at 4° C in 2.5% glutaraldehyde containing 2% tannic acid and buffered wi th 0.05 M B E S (NN-bis [2-Hydroxyethyl ] -2-aminoethane sulfonic acid), adjusted to p H 7.4 with N a O H . Af ter f ixat ion tissues were postfixed in 1% OsO in the same buffer for 5 min , followed by soaking in 2% tannic acid i n 4 dis t i l led H 2 0 , wi th the p H adjusted to 7.0 wi th N a O H . Dehydrat ion was accomplished with ethanol and the material stained en bloc wi th uranyl acetate and phosphotungstic acid before embedding in an Epon Ara ld i te resin mixture (Mollenhauer, 1964). Sections were stained in methanolic uranyl acetate and lead citrate before examination in a J E O L 120 C X I I transmission microscope operated at 80 k V . For scanning electron microscopy in guinea pigs, the temporal bones were extracted as described above and fixed with 2.5% glutaraldehyde in 0.05 M B E S buffer. For scanning electron microscopy in European lfzards (Podarcis mural is and P. sicula), chicks (Gallus gallus), pigeons (Columba livia) and starling (Sturnus vulgaris), the animals were anaesthetized and perfused either transcardially or directly through the oval or round Windows with f ixative (pigeon: 2.5% glutaraldehyde in 0.1 M phosphate buffer; ch ick , starling and l izard 1% or 2.5% glutaraldehyde and 15% saturated p icr ic acid in 0.1 M phosphate buffer). Specimens were stored in 2% glutaraldehyde in 0.05 or 0.1 M phosphate buffer unt i l further treatment. They were then dehydrated in acetone, dr ied by the c r i t i ca l -po in t technique wi th l iqu id C 0 2 , and sputter-coated wi th plat inum to a nominal depth of 25 nm. Specimens were examined in a J E O L 120 C X I I microscope wi th a scanning attachment at 40 k V , and images were observed by a secondary-electron detector. Results T H E FINE S T R U C T U R E OF TIP-LINKS IN GUINEA PIG HAIR C E L L S A fine, straight filament was visible in the centre of each tip l ink (F ig . 1). In many cases the fine central filament was surrounded by amorphous material , which had a variable conformation (F ig . 2). Similar material was also visible on the surface membranes of the stereovil l i (F ig . 2). The fine central filament often appeared more l ight ly-s ta in ing than the surrounding material. Its mean diameter measured in sections taken at a r änge of or iginal magnifications of 29,000 to 100,000 had a value of 5.5 nm (n=16), the same as the diameter of the actin filaments in the central core of the stereovil l i measured in the same sections. In sections cut perpendicular to the cuticular plate and perpendicular to the rows of stereovill i on an outer hair ce l l , the bundles had a t r i angu l ä r shape in lateral v iew, the stereovilli being closely apposed at the tips. The central filaments of the tip links running between the stereovil l i of the three rows were nearly coaxial wi th each other, and also nearly coaxial wi th the shortest of the three stereovill i in the cross section (F ig . 3). In such sections, therefore, the two tip links and the shortest stereovillus lay along a nearly straight line. Moreover , in those sections where the rootlets were vis ible , each rootlet continued along the axis of the stereovillus (F ig . 4). In addit ion to tip l inks, a broad band of l inks was visible running laterally between the rows of stereovil l i , part icularly visible between the tallest and second-tallest stereovill i in the b ü n d l e (arrowheads, Figs 3 and 4). A t its point of attachment on the taller of two stereovil l i , the central filament of the tip l ink ran to the centre of an electron-dense area, which formed a bridge between the external plasma membrane and the central actin filaments of the stereovillus (arrowheads, Figs 1 and 2). A t its lower end, the central filament met the plasma membrane at the tip of the shorter stereovillus, which was often pulled out into a point. This point overlay a clear area, wi th a denser cap over the ends of the actin filaments immediately underneath, visible in sections cut in a suitable plane. In some sections, fine filaments could be seen connecting the plasma membrane to the underlying density. The dense cap was only visible at the ends of the actin filaments immediately under lying the tip l ink: the other filaments ended without any obvious association wi th an electron-dense area. This was particularly apparent in the tips of stereovill i of the second tallest row on inner hair cells, which ended wi th a wider tip. TIP-LINK O R G A N I Z A T I O N IN R E L A T I O N T O T H E H A I R - C E L L AXIS We have previously suggested that tip links on guinea pig inner and outer hair cells are organized such that the horizontal component in their orientation runs parallel to the hair cell axis of bilateral symmetry (Pickles et a l . , 1984; Comis et al . , 1985). This axis defines the axis for excitatory and inhibi tory deflections of the stereovilli (Flock, 1965). We have further investigated the point in bird and lizard basilar papillae, where the hair bundles show a quite different conformation and, sometimes, a systematical ly-varying orientation with respect to the papi l lär edge ( M i l l e r , 1980; G l e i c h and Manley , 1988). In micrographs of bundles viewed perpendicular to the cuticular plate, the stereovill i were hexagonally packed (Fig . 5). The tip l inks were oriented along a 1,0 axis of the hexagon, parallel to the axis of bilateral symmetry, and at right angles to the long axis of the bünd l e as a whole (arrows: F i g . 5). The stereovilli were therefore organized into columns, running parallel to the hai r -ce l l axis of bilateral symmetry. These observations were made consistently in bundles from lizards and all birds. In micrographs of bundles viewed at an acute angle to the cuticular plate, the Organization of stereovilli into columns, connected by tip l inks , was also obvious (F ig . 6). In some bundles, wi th in the general gradation in height of the stereovill i from shortest on one side of the bünd l e to tallest on the opposite side, all the stereovilli in one column were a little taller, or a little shorter, than the corresponding stereovilli in adjacent columns (e.g. arrows, F i g . 6). Examples of this behaviour were found in all ears examined and in all regions of the papillae. Where bundles had separated, perhaps due to mechanical trauma during preparation of the specimen, the stereovilli separated most commonly into columns running parallel to the axis of bilateral symmetry of the hair cell (F ig . 7). These are the columns defined in the previous paragraphs on the basis of the t ip - l ink connections. When this occurred, there was no obvious debris on the upper ends of the separated, lateral, surfaces of the stereovill i in the gap (Fig . 7). Discussion The present observations on the fine structure of tip l inks suggests that the links have a fine central filamentous core, surrounded by an amorphous coat. We do not have Information on the nature of the central filament, although it may well be protein. L i k e actin, the central filament reacts poorly wi th positive stains. Moreover , its diameter is similar to that of a Single actin filament. On the other hand, the amorphous surrounding coat may well be an extension of the coat on the surface of the ind iv idua l s tereovil l i , and may therefore consist of glycocongugates (Santi and Anderson , 1987). One or both ends of the tip links are possible sites for the transducer Channels, on the hypothesis that Stretch of the tip l inks opens the transducer Channels (Pickles et al . , 1984). The densities associated wi th the ends of the filament probably represent anchorage points at which the forces are coupled to the internal cytoskeleton. A t its lower end, the tip l ink joins to a conical extension of the membrane at the tip of the shorter Fig. 1. Tip link on guinea pig outer hair cell, showing central filament. Arrowhead: upper dense point of attachment. Scale bar: 100 nm. Fig. 2. Tip link on guinea pig outer hair cell, showing variable thickness of amorphous material attached to central filament of tip link. Arrowhead: upper dense point of attachment. Arrow: site of lower density under the surface membrane of the stereovillus. Scale bar: 100 nm. Fig. 3. Tip links on guinea pig outer hair cell, showing that the tip links between the different rows are nearly coaxial, and coaxial with the shortest stereovillus on the bündle . Arrows: tip links. Arrowhead: lateral (inter-row) links. Scale bar: 200 nm. Fig. 4. Guinea pig outer hair cell, showing that the rootlets are coaxial with the individual stereovilli, and that the stereovilli slope towards each other at their tips. Arrowhead: sideways (inter-row) links. Scale bar: 200 nm. Fig. 5. Hair cell from neural edge of starling basilar papilla, viewed nearly perpendicular to the cuticular plate. Arrows indicate the axes of columns formed by the tip links. The tallest stereovilli in the bündle are at the top of the bündle . Arrowheads: extraneous material, perhaps from tectorial membrane, lying over stereovilli. Scale bar: 500 nm. Fig. 6. Hair cell of starling basilar papilla. Arrows indicate two columns of stereovilli, joined by tip links, the stereovilli in these columns are a little taller than those in the adjacent columns. Neural edge of papilla. Scale bar: Ifim. Fig. 7. Hair cell of starling basilar papilla, showing Separation of bündle along the columns defined by the tip links. No debris is visible on the lateral, separated, walls of the stereovilli (arrows). Scale bar: 500 nm. stereovillus. Fine Strands connect the membrane to the under lying dense cap over the ends of the actin filaments. The conical extension of the membrane is one possible site for the transducer channel(s). It is interesting that the conical extension of the membrane at the tips of shorter s tereovi l l i , w i th the underlying density, was first noted by F lock (1965). The association of densities wi th attachment sites at both ends of the tip l inks was first noted by ourselves (Pickles, Osborne and Comis , Society for Exper imenta l Biology, 5 January, 1984). The geometrical arrangement shown in Figs 1 and 2 would be expected to produce a Stretch of the tip l inks , in response to an angular deflection of the s tereovil l i . Indeed, the arrangement shown would tend to couple the deflections into the tip l inks wi th min imal Stretch or compression of the other l inks , for instance of the lateral l inks wh ich in guinea pigs j o i n the stereovil l i of the different rows just below their tips. This arrangement would therefore be efficient at coupling Stimulus energy to the tip l inks , as opposed to other structures of the hair b ü n d l e . The fine fi lament wi th in each tip l ink is ideal for transmitting the s t imulus- induced forces to a minute area of membrane. This would be mandatory i f energy is to be coupled efficiently to the small number of transducer Channels, perhaps between one and four, thought to exist on each stereovillus (Russell , 1983; Hol ton and Hudspeth, 1986). Calculat ions f rom the effective collecting area of the ear (Rosowski et al . , 1986), the number of hair cells per mm of cochlear duct (Ulehlova et al . , 1987), our own counts of the number of tip l inks on human inner and outer hair cells, and the cr i t ica l bandwidth expressed as a length of cochlear duct (Greenwood, 1961), lead us to conclude that the forces collected from the acoustic wavefront would be concentrated onto the central filaments of tip l inks wh ich have a total cross-sectional area which is smaller by a factor of 1 0 8 times. The orientation of the tip l inks parallel to the axis of bilateral symmetry of the hair ce l l , and so parallel to the exci ta tory- inhib i tory axis (F lock , 1965), is part icularly obvious in the compact, hexagonally-packed bundles of birds and lizards. In addit ion, the Observation of strict orientation of the tip l inks along the axis of bilateral symmetry of the ind iv idua l b ü n d l e , irrespective of the bundle's orientation wi th in the epi thel ium, is consistent wi th the hypothesis that the tip l inks are associated wi th the transduction process. This association of the direct ion of the tip l inks wi th the axis of bilateral symmetry of the hair cell was found even though in ^irds all orientations of hair cell axis could be seen, ranging f rom 0 to 90 with respect to the neural edge, depending upon Position in the papil la . The fact that stereovil l i tend to separate into columns connected by tip l inks suggests that any sideways connections between different columns are weaker than the connections running wi th in each column. Moreover , when the stereovil l i had separated into columns, no debris was visible on the lateral surfaces of stereovill i near their upper ends. This suggests that there are no, or only sparse, lateral l inks connecting the stereovill i of the different columns in this region. It is l ike ly therefore that the upper end of the b ü n d l e is relatively weakly braced against deflections which are oblique to the axis of bilateral symmetry. A g a i n , this would be appropriate i f the main mechanical input was parallel to the axis of bilateral symmetry, i.e. parallel to the exci ta tory- inhibi tory axis. A t the moment, we do not know i f the apparently stronger in t ra -co lumn connections result only from the tip l inks, or whether there is a contr ibution from lateral l inks running below the tips of the stereovil l i . On the other hand, where it was possible to look deep into the gaps where stereovil l i had separated, it was possible to see remains of lateral l inks at the lower ends of the stereovill i , consistent wi th the rieh lateral network of l inks in this region, as previously reported by Csukas et a l . (1987) for the l izard . Sometimes the stereovill i wi th in one column were, wi th in the general gradation in height of the stereovil l i , all a little taller, or a little shorter, than the stereovil l i in adjacent columns. This suggests that a factor wh ich governs the heights of the stereovilli during development has a component which is expressed along each column, the column being as defined above on the basis of the tip l inks connections. It appears therefore that the heights of the stereovill i and tip l ink orientation are closely related by common factors in development. Acknowledgements The skilled technical assistance of T . L . Hayward and L . M . Tomkins is gratefully acknowledged. This research was supported by the Medical Research Council of the U K , and the Deutsche Forschungsgemeinschaft within the programme of the SFB 204 "Gehör". References Comis, S.D., Pickles, J .O. and Osborne, M.P . 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Tip-Link organization in relation to the structure and orientation of stereovillar bundles

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