Spectrin, act in and the structure of the cortical lattice in mammalian

cochlear outer hair cells

M. C. HOLLEY and J. F. ASHMOEE

Department of Physiology, The Medical School, University Walk, Bristol BS8 1TD, UK

Summary

Mammalian cochlear outer hair cells generate high-frequency forces in response to electrical stimu-lation. Force generation occurs in the lateral cortexof the cell, which includes the plasma membrane, atwo-dimensional 'cortical lattice' of filamentous pro-tein, and a multi-layered membrane system, the lat-eral cisternae. The cortical lattice is composed ofrelatively long filaments, 6.7 nm in diameter, whichare wound circumferentially about the cell. Thesefilaments are spaced about 42 nm apart and arecross-linked by a second type of filament 3.2 nm indiameter approximately aligned with the longitudi-nal axis of the cell. The cortical lattice is the onlycortical structure that remains after the cell is fullyextracted in the detergent Triton X-100 and high-saltsolution. It retains the original cylindrical shape ofthe cell and is reversibly deformable. Antibodies

raised against chicken gizzard actin, human bloodspectrin and pig brain spectrin all react positivelywith the extracted lattice viewed using immunofluor-escence. Three protein subunits identified in theorgan of Corti have approximate molecular weightsof 220, 235 and 240K (K=103Mr) and react with thespectrin antibodies. A structural model of the latticeis proposed in which the circumferential filamentsare composed of actin and the cross-links of spectrin.The model can account for the unusual cylindricalshape of outer hair cells and suggests a mechanism offorce generation based upon the elastic and electro-static properties of spectrin.

Key words: outer hair cells, cytoskeleton, spectrin, actin, cellmotility.

Introduction

This paper describes the structure and molecular com-ponents of the cortical cytoskeleton in mammalian outerhair cells, and proposes a model for the role of the skeletonin high-frequency cell-length changes. Hair cells arespecialised mechanoreceptors (Hudspeth, 1989), but mam-malian outer hair cells are also able to generate high-frequency forces along the cell body in response to electri-cal stimulation. These forces are visible in isolated cells aslength changes (Brownell et al. 1985; Kachar et al. 1986;Ashmore, 1987) and their function in vivo may be to refinethe tuning properties of the basilar membrane (Davis,1983; Kim, 1986; Mountain and Hubbard, 1989). Forcesare generated in the lateral cortex of the outer hair cell(Holley and Ashmore, 1988a), which includes the plasmamembrane, an underlying membrane system called thelateral cisternae (Angelborg and Engstrom, 1973;Ekstrom von Lubitz, 1981; Saito, 1983; Flock et al. 1986)and a two-dimensional cytoskeleton, the cortical lattice,which lies between the two (Bannister et al. 1988; Holleyand Ashmore, 19886).

The mechanism of high-frequency force generation dif-fers from that in most other motile systems because it isindependent of ATP and calcium (Kachar et al. 1986;Holley and Ashmore, 1988a). It has a latency of less than100 /is and can be driven reversibly at frequencies of atleast 8 kHz (Ashmore, 1987). Maximal displacements are5 % of cell length. Outer hair cells in the guinea pig areJournal of Cell Science 96, 283-291 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

uniformly about 10/an in diameter, but they increase inlength from about 30 /an at the basal, high-frequency endof the cochlea, to about 100 /.cm at the apical, low-frequencyend. The maximal length change in an apical cell is thusabout 5jum, readily visible in the light microscope.

Slower cell-length changes can be stimulated 'acousti-cally' (Canlon et al. 1988; Brundin et al. 1989), by applyingATP and calcium following permeabilisation (Flock et al.1986), or by increasing external potassium concentration(Zenner, 1986). The mechanism driving these responsesmay differ from that driving high-frequency responses andit is not discussed in this paper.

The cortical lattice is composed of at least two filamen-tous proteins (Holley and Ashmore, 19886), and three linesof evidence suggest that they are actin and some form ofspectrin. First, the two filaments are 5-8 nm and 2-3 nmin diameter, the approximate values for actin and spectrin,respectively. Second, antibodies to both actin (Flock et al.1986) and spectrin (Drenckhahn et al. 1985) bind to thelateral cortex. Third, demembranated cells are reversiblydeformable (Holley and Ashmore, 19886), a propertyshared with the spectrin skeleton in mammalian erythro-cytes (Steck, 1989). Spectrins are a class of elastic, fila-mentous proteins originally typified by mammalian eryth-rocyte spectrin (Bennett, 1985; Speicher, 1986; Goodman etal. 1988). Non-erythrocyte spectrins include fodrin, de-scribed primarily from brain tissue, and TW260/240 fromgut epithelial cells (Burridge et al. 1982; Glenney andGlenney, 1983).

283

In this paper we present evidence that the corticallattice is an ordered array of spectrin molecules alignedwith the longitudinal axis of the cell. The array isparticularly well adapted to facilitate the length changesobserved in outer hair cells and might form the basis of theforce-generating mechanism.

Materials and methods

Cell dissociationGuinea pigs were killed by cervical dislocation, the bullaeremoved and opened in L-15 cell culture medium (Leibovitz, L-16Gibco) to remove the cochlear spiral. The organ of Corti wasdissected from the spiral with a fine needle, dissociated mechan-ically by reflux through a pipette tip and then the cells wereallowed to settle in a 150 /il droplet of L-15 for lOmin on a glassslide. The slide was precoated with poly-L-lysine (Sigma14000Mr) by incubation in a 1.5mgml solution with 0.1%Triton X-100 for 10 min. It was then briefly washed and air dried.

Cell extraction buffersThe extraction buffers were those devised for biochemical frac-tionation by Fey et al. (1984). Outer hair cells were extracted incytoskeleton buffer (CSK: 100 mM NaCl, 300 mM sucrose, 10 milPipes, pH6.8, 3mM MgCl2, 0.5% Triton X-100 and 1.2minphenylmethylsulphonyl fluoride (PMSF)) for two periods of10 min on ice. The soluble fraction was removed and replaced by ahigh-salt buffer (250 mM ammonium sulphate, 300 mM sucrose,10 mM Pipes, pH 6.8, 3 mM MgCl2, 1.2 mM PMSF and 0.5 % TritonX-100) for lOminonice.

ImmunofluorescenceOuter hair cells were fixed on ice for 30 min in CSK buffercontaining 0.1 % glutaraldehyde and 2 % formaldehyde and pro-cessed as follows at 20°C: PBS (10 mM phosphate buffer, pH7.4,150mM NaCl, 2mM NaN3) with 0 . 1 M glycine (2x5min); PBS(2x5min); BSA-TBS (1% bovine serum albumin in PBS with0.05M Tris-HCl, pH7.6) (3x5min); primary antibody diluted1:1000 in BSA-TBS (overnight at 4°C); BSA-TBS (3x5min);FITC-conjugated secondary antibody diluted 1:500 in BSA-TBS(2hs); BSA-TBS (2x5 min); PBS (2x5 min). The cells were thenmounted and observed using a Nikon Diaphot equipped fordifferential interference contrast and epifluorescence.

Gel electrophoresis and immunoblottingSamples were run on discontinuous polyacrylamide mini-gels(Bio-Rad Mini Protean II) according to the method of Laemmli(1970) and then stained with silver (Sigma). For immunoblottingthey were unstained and transferred to nitrocellulose paper(Towbin et al. 1979) using a semi-dry blotter (LKB Novablot).Nitrocellulose papers were incubated in 4% bovine serum albu-min and 0.1 % gelatin in PBS, rinsed in PBS, and then incubatedovernight in primary antibody (1:100) in 'incubation buffer' (PBScontaining 0.8% bovine serum albumin, 0.1% gelatin and 1%normal goat serum). They were then washed in PBS three timesand incubated for 4 h in gold-conjugated second antibody (Auro-Probe One, Janssen Life Sciences Products) in incubation buffer.The signal was then enhanced with silver (Janssen IntensE).Whole protein patterns were obtained by staining nitrocellulosepapers with colloidal gold (Janssen Aurodye).

Antibodies and protein samplesThe three polyclonal antibodies used were raised in rabbitagainst: (1) human erythrocyte spectrin (a gift from Dr DavidShotton, University of Oxford); (2) pig brain spectrin (a gift fromDr Keith Burridge, University of N. Carolina at Chapel Hill); and(3) chicken gizzard actin (Biotechnologies Ltd). They were used ata dilution of 1:1000, as was the normal rabbit serum that wasused as a control.

Protein samples were prepared in a standard sample buffer

(SDS, Tris-HCl, pH 6.8, glycerol, 5 % /S-mercaptoethanol). Organsof Corti were dissected from the cochlear spiral and spun to thebottom of small Pyrex tubes in a bench centrifuge. Fractionatedtissue was washed in two 30-/il volumes of fractionation buffer perorgan of Corti. Each whole or fractionated organ of Corti wasprepared in 10 /il of sample buffer and boiled for 3 min. Sampleswere prepared fresh for each analysis.

Electron microscopyThe organ of Corti was left on the cochlear spiral and washed inL-15. It was then incubated in CSK and high-salt buffers asdescribed above, fixed in 2.5 % glutaraldehyde in either buffer for30 min on ice and rinsed in 0.1 M cacodylate. It was then incubatedin 1% osmium tetroxide for 10 min on ice, blocked stained inethanolic Uranyl acetate, dehydrated in ethanol and embedded inEpon. Thin sections were post-stained with uranyl acetate andlead citrate and observed in a Philips EM 300 electron microscope.Images were calibrated using a graticule of 2160 lines per mm andcatalase crystals with periodicity of 8.75 run (Agar Scientific).Filament diameters were measured from printed images at a totalmagnification of x 150 000.

Results

Outer hair cells

Isolated outer hair cells from the guinea pig are cylindricalwith a prominent nucleus at the base and a cuticular plateand stereociliary bundle at the apex (Fig. 1A). Followingextraction in CSK buffer these features were retainedalthough the membranes and much of the cytoplasmiccontent were lost (Fig. IB). The original cell shape wasaccurately defined by the Triton-insoluble cortical lattice.In some cells a fibrous structure, the infracuticularnetwork, projected intracellularly from the cuticularplate. Incubation in the high-salt buffer did not alter thecell morphology any further.

Fig. 1. An outer hair cell from the apical turn of the cochleabefore (A) and after (B) extraction in Triton X-100 in CSKbuffer, sc, stereocilia; cp, cuticular plate; n, nucleus; in,infracuticular network; cl, cortical lattice. Bar, 10 fan.

284 M. C. Holley and J. F. Ashmore

Fig. 2. Immunofluorescence of outer hair cells extracted inTriton X-100 in CSK buffer. A. Control using normal rabbitserum in place of primary antibody. B. Antibody raised to pigbrain spectrin. C. Antibody raised to human blood spectrin. Thecuticular plates, infracuticular networks and cortical latticebind both antibodies, but the stereocilia bind neither of them.Some reactivity between the brain spectrin antibody and thenucleus is observed. Bar, 10 /an.

ImmunofluorescenceBoth spectrin antibodies reacted positively with isolatedouter hair cells (OHCs) fixed and subsequently permeabi-lised with Triton X-100. They also reacted with cells fixedafter extraction with CSK buffer (Fig. 2) and high-saltbuffer. Strong fluorescence was observed from the cuticu-lar plate, the infracuticular network and the corticallattice. The stereocilia were not stained. Control cellsincubated with normal rabbit serum produced a weakfluorescence around the cuticular plate but were otherwisenegative (Fig. 2A).

Similar labelling was observed using the actin antibodyand the actin-binding toxin phalloidin, which was conju-gated with rhodamine. The major difference was thatthese two labels bound to the stereocilia but the spectrinantibodies did not.

Gel electrophoresis and immunoblottingProtein bands with the expected electrophoretic mobilityof spectrin subunits were identified by running samples of

Humanblood

GPblood

GPbrain

Organ ofCorti

Organ ofCorti

T-CSK

GPbrain

Humanblood

Fig. 3. A 5 % polyacrylamide gel to compare high molecular weight components of blood and brain, and of organ of Corti both beforeand after extraction with Triton X-100 in CSK buffer (T-CSK). Human blood was used as a marker for the 220K and 240K subunitsof spectrin. Blood samples were diluted in 170 volumes of sample buffer to provide approximately the same total proteinconcentration as one dissected organ of Corti. Brain samples were prepared with LGmgrnl"1 wet weight of brain tissue in samplebuffer. Bars indicate bands in the organ of Corti that have similar mobilities to the 220K, 235K and 240K subunits of spectrin inhuman blood and guinea pig brain. Guinea pig blood (GP) has no bands with mobilities comparable to those of these three subunits.

Cortical cytoskeleton in outer hair cells 285

human blood, guinea pig blood and guinea pig brainagainst the organ of Corti (Fig. 3).

Human erythrocyte spectrin is composed of two subunitswith molecular weights of 220K (K=103Mr) and 240K(Goodman et al. 1988). Guinea pig brain spectrin, althoughoriginally reported to be composed of 240K and 250Ksubunits (Levine and Willard, 1981), is composed of 235Kand 240K subunits (Goodman et al. 1988). Prominentbands of equivalent electrophoretic mobility to the 220K,235K and 240K aubunits were found in the organ of Cortiin approximately equal concentrations. Guinea pig bloodpossessed no bands with directly equivalent mobilities tothese three subunits, but instead had a pair of roughlyequal bands, one that migrated slightly slower than the220K band from human blood and the other slightly fasterthan the 235K band of guinea pig brain.

The identity of the spectrin bands was confirmed byimmunoblotting. The antibody raised against pig brainspectrin bound relatively strongly to the 240K band butonly weakly to the 235K band from guinea pig brain(Fig. 4). It bound only the 240K band from the organ ofCorti. There was very little cross-reactivity of the antibodywith other proteins from either sample.

The antibody raised against human erythrocyte spectrinbound to the two bands in guinea pig blood that migrated

GPbrain

Organof

Corti

Organof

Corti

GPblood

GPbrain

Controlorgan

ofCorti

-240K240K_235K-220K-

B B

close to the 220K and 235K bands (Fig. 5). It also bound tothe 235K and 240K bands from guinea pig brain and to allthree bands in the organ of Corti.

The results indicate that the organ of Corti in the guineapig contains at least three forms of spectrin, two of whichhave mobilities equivalent to those of guinea pig brainspectrin. Guinea pig blood spectrin, however, is composedof two subunits that differ from those in both the organ ofCorti and the brain. For clarity the subunits from theorgan of Corti will hereafter be called cochlear spectrin.

The 235K and 240K bands from the organ of Corti wereinsoluble in the CSK buffer, but the 220K band appearedfrom the gels to be at least partially soluble (Fig. 3).However, immunoblots revealed a soluble protein thatcomigrated with the band recognised by the antibody, andshowed that all three cochlear spectrin subunits wereinsoluble in Triton X-100 (Fig. 6). Most of the actin wasalso insoluble.

Electron microscopyThe undissociated organ of Corti retained its generalorganisation following extraction by Triton in CSK bufferand the cuticular plates of the hair cells remained at-tached to neighbouring supporting cells (Fig. 7). Hair cells

Organ of Corti

Insoluble Soluble

240K_2 3 5 K -2 2 0 K - ~

D B C D

Fig. 4. Western blot to illustrate specificity of the pig brain spectrin antibody against guinea pig brain and organ of Corti. Lane A isstained for total protein and lanes B and C are labelled with antibody. The antibody recognised the 240K subunit from brain and asimilar subunit from the organ of Corti.Fig. 5. Western blot to illustrate specificity of the human blood spectrin antibody against the guinea pig organ of Corti (A), blood (B)and brain (C). The antibody labels bands equivalent to the 220K, 235K and 240K subunits of spectrin. The control (D) was labelledwith normal rabbit serum instead of primary antibody. A weakly labelled band (arrow in lane A) may represent a degradationproduct of spectrin, particularly as it was not always evident (see Fig. 6).Fig. 6. Western blot of the insoluble and soluble fractions of the organ of Corti in Triton X-100 in CSK buffer. Each lane representsprotein from one dissected organ of Corti. Lanes A and C are stained for whole protein, and B and D are labelled with the antibodyto human blood spectrin. All three bands recognised by the antibody are insoluble. An additional protein that comigrates with the220K band is soluble (lane C) but it does not bind the antibody (arrow in lane D).

286 M. C. Holley and J. F. Ashmore

,cp

sc\•*+l

o \

Fig. 7. Electron micrograph of the apical region of an outer hair cell after extraction in Triton X-100 in CSK buffer, fixation,embedding and sectioning. The cortical lattice is indicated by filled arrowheads. A tangential section of the lattice is indicated by theopen arrowhead. Arrows indicate vesicles that were probably remnants of mitochondria and the lateral cisternae. The section passedthrough the cuticular plate and cut the lattice tangentially. This occurs because in some cells the cuticular plate is wider than thediameter of the cell body (see Fig. 1). The thicker filaments of the lattice can be distinguished from those of the infracuticularnetwork because they are orientated transversely with respect to the cell axis rather than longitudinally, sc, stereocilium; cp,cuticular plate. Bar, 1 /jm.Fig. 8. Section through the cortical lattice in the lower middle region of an outer hair cell. Bar, 0.5/nn.

contained very little cytoplasmic structure. Numerousvesicles located in the cortical region of the cell wereprobably the remains of mitochondria and the lateralcisternae.

The cortical lattice was present along the full length ofthe cell (Figs 7, 8) and was composed of relatively thickfilaments oriented circumferentially and cross-linked byshorter, thinner filaments (Fig. 9). The dimensions ofthese filaments were taken from electron micrographs at amagnification of x 150 000 (Figs 10, 11). Circumferentialfilaments had a mean diameter of 6.7±0.5nm (rc=20) andtheir cross-links a mean diameter of 3.2±1.0nm (n=17).The length of the cross-links, estimated from the spacingbetween neighbouring circumferential filaments, was42.4±6.2nm (/i=33). In all of these measurements themean is accompanied by a standard deviation and thenumber of measurements taken from electron micro-graphs.

The lattice filaments were integrated with the irregular,three-dimensional web of filaments in the cuticular plate

(Fig. 9). Individual circumferential filaments could betraced into the cuticular plate where they were cross-linked by thinner filaments similar to those in the lattice.The infracuticular network was composed of filamentswith similar dimensions (Fig. 12). In this network thethicker filaments were 6.8±0.6nm (n.=20) in diameter,and aligned roughly along the axis of the cell. They werecross-linked transversely by filaments 3.7±0.9nm (n=20)in diameter, in a three-dimensional array distinct fromthat in either the cuticular plate (Fig. 9) or the corticallattice.

The lateral cisternae and plasma membrane are nor-mally connected by a regular array of'pillars' about 30 nmlong and 6-8 nm in diameter (Flock et al. 1986; Fig. 13). Infully extracted cells they are absent (Figs 9, 10). Afterpartial extraction, however, they are clearly visible andassociated with the circumferential filaments (Fig. 14).The pillars thus appear to form an important mechanicallink between the lattice and the plasma membrane(Figs 15, 16).

Cortical cytoskeleton in outer hair cells 287

rp

Fig. 9. Enlarged image of the region indicated by the open arrowhead in Fig. 7. Circumferential filaments (cf) and crosslinks (cl) inthe cortical lattice are integrated with the more irregular array of filaments in the cuticular plate (cp). Bar, 200 nm.Fig. 10 A tangential section of the cortical lattice at higher magnification to illustrate circumferential filaments (cf) and cross-links(cl). Bar, 50 nm.Fig. 11. Section cutting circumferential filaments transversely (see Fig. 15). The section is 40-50 nm thick, so unless the filamentsare aligned perpendicular to the plane of the section they appear slightly thicker than in Fig. 10. Remnants of the plasma membrane(pm) and lateral cisternae (lc) are seen on either side of the lattice. A pillar (p) may also be visible. Bar, 50 nm.

Discussion

Structure of the cortical latticeThe cortical lattice in outer hair cells fully extracted inTriton X-100 is composed of relatively long circumfer-ential filaments about 6.7 nm thick, which are cross-linkedby thinner filaments 3.2 nm thick and at least 42 nm long.Circumferential filaments are pitched at a mean angle of15° to the transverse axis of the cell, and thus collectivelyresemble a coiled spring (Holley and Ashmore, 19886).Bannister et al. (1988) constructed a model of the latticebased upon 3nm filaments whose ends were attachedsolely to the pillars, with each pillar anchoring six fila-ments. The evidence for this was taken from thin sectionsof whole cells and cells lightly permeabilised in thedetergent saponin. Circumferential filaments are difficultto see in such preparations because they do not contrastwell, particularly against a background of plasma mem-brane or lateral cisternae (Fig. 14). Our fully extractedcells may have lost some of the lattice components andfurther experiments would be required to test this. In thefully extracted cells pillars were absent from both tangen-tial (Fig. 10) and transverse (Fig. 11) sections of thelattice, thus implying that they are not focal points of thelattice and that they are unnecessary for its integrity. Inour model the 3 nm filaments are connected directly to thecircumferential filaments. The most important structuralfunction of the pillars is probably to connect the lattice tothe plasma membrane.

The cortical lattice, cuticular plate and infracuticularnetworkThe ultrastructure and immunofluorescence suggest thatthese three networks are composed of the same two typesof filament. The cortical lattice and infracuticular network

may simply be specialised extensions of the cuticularplate, with their distinctive morphologies determined bythe nature of the cross-linking. The availability of bindingsites on the thicker filaments could dictate the cross-linking pattern. The thicker filaments must possess repe-titive binding sites for the cross-links along their length.Electron micrographs of the cortical lattice suggest eitherthat the intervals between these binding sites are irregu-lar, or that they are not all occupied. In all three networksthe cross-links must possess a single binding site for thethicker filament at each end. In contrast to the corticallattice both the cuticular plate and the infracuticularnetwork are cross-linked in three dimensions. The less-ordered array of thicker filaments in the cuticular platemay be a consequence of their shorter length.

An apical actin ring described from the edge of thecuticular plate in other hair cells (Flock et al. 1981;Hirokawa and Tilney, 1982) was not clear in this study,but it may correspond to the top end of the cortical latticeas illustrated in Fig. 9. The infracuticular lattice is notpresent in all cells and its function is unknown (Thome etal. 1987).

Actin and spectrin in the cortical latticeThe presence of actin and spectrin in the lattice issupported by immunofluorescence along the lateral cortexof cells extracted with Triton X-100 in both high-salt andlow-salt buffers. Electron microscopy reveals that thelattice is the only remaining structure in this region. Fewproteins are resistant to high-salt extraction in the pres-ence of Triton X-100, but they include spectrin. Erythro-cytes prepared in this way have a very simple compositionof spectrin, actin and two other proteins called bands 4.1and 4.9 (Bennett, 1985; Goodman et al. 1988). Evidence forspectrin has been reported from the cortex of unextracted

288 M. C. Holley and J. F. Ashmore

Fig. 12. Section through the infracuticular network that is formedfrom a parallel array of cross-linked filaments aligned with thelongitudinal axis of the cell, pi, parallel filaments; cl, cross-links.Bar, 100 nm.Fig. 13. Section of an unextracted outer hair cell. The cortex is cutin two planes due to deformation of the cell. The plasmamembrane (pm) is linked to the lateral cistemae (lc) by pillars (p).These structures are summarised in Fig. 16. Mitochondria (m)typically lie close to the inner surface of the lateral cistemae. Bar,0.5 \sm. Micrograph supplied by Dr Andrew Forge.Fig. 14. Tangential section of a partially extracted cell illustratingthe relationship between pillars (p) and circumferential filaments(cf). Circumferential filaments did not contrast well with thelateral cistemae. Bar, 100 nm.

rat outer hair cells (Drenckhahn et al. 1985). Actin hasbeen reported in the cortex of guinea pig outer hair cells(Flock et al. 1986).

Spectrin immunoreactivity has also been reported in thecuticular plates of vestibular hair cells (Scarfone et al.

1989). The network of cross-linked actin filaments in thecuticular plates of chick hair cells (Hirokawa and Tilney,1982; Hirokawa, 1986) is comparable with that of the'terminal webs' in the apices of both mammalian gutepithelial cells (Glenney et al. 1983) and ciliated epithelial

Cortical cytoskeleton in outer hair cells 289

15A B

Fig. 15. Drawing of the extracted cortical lattice in tangential(A) and transverse (B) section. Note the absence of pillars thatare visible in Fig. 14. cf, circumferential filaments; cl, cross-links.Fig. 16. Diagram illustrating the location of the lattice in thecortex of an outer hair cell, pm, plasma membrane; lc, lateralcisternae; p, pillars; cf, circumferential filaments.

cells (Koboyashi and Hirokawa, 1988). These terminalwebs are composed of actin filaments cross-linked by aspecific form of spectrin, and the cuticular plate of mam-malian hair cells may be very similar.

A model for the structure of the cortical latticeThe structure of the cortical lattice and the morphology ofthe two component filaments suggest that circumferentialfilaments are composed of actin and the cross-links ofspectrin.

Actin filaments are characteristically 5-8 run in diam-eter, similar to that of the circumferential filaments. Actincan polymerise to form filaments of varying length withrepetitive binding sites for a wide variety of proteins(Stossel et al. 1985). It could thus account for the structuralproperties of the circumferential filaments in the lattice,and the 6-7 nm filaments of the cuticular plate andinfracuticular network.

Spectrin molecules are about 2-4 nm in diameter (Shot-ton et al. 1979) and usually occur as tetramers formed fromtwo pairs of dimers (Bennett, 1985). They thus formfilaments that vary in length from 50 to 260 nm dependingupon the type of spectrin and its tertiary structure. Mostsignificantly, each filament has a binding site for actin ateach end. The cross-links are estimated to be about 42 nmlong, which is slightly short for the expected length ofspectrin filaments (Byers and Branton, 1985; Shen et al.1986). This value may be an underestimate because it wastaken from the spacing between circumferential fila-ments, assuming that the cross-links were straight. Spec-trin filament length is also susceptible to ionic environ-ment (see below), so the length of the cross-links shouldnot rule out the possibility that they are spectrin.

In the erythrocyte membrane spectrin forms an isotro-pic, two-dimensional network beneath the plasma mem-brane (Liu et al. 1987). The terminal regions of the spectrintetramers are linked by relatively short actin filaments(Bennett, 1985). The cortical lattice in outer hair cells isanisotropic, and the proposed actin filaments are muchlonger than those in the erythrocyte. It would thusrepresent an unusual network for actin and spectrin.

Mechanical properties of the cortical latticeThe orientation and mechanical properties of the circum-

ferential filaments and their cross-links should determinethe nature of the shape changes observed in isolated cells(Holley and Ashmore, 19886). Under positive intracellularpressure the forces generated in the cortex circumferen-tially should be twice the magnitude of those generatedlongitudinally, and the cell should tend to become spheri-cal (Holley and Ashmore, 1990). Actin filaments areflexible but can withstand relatively high tensile forcessuch as occur in muscle. Wound circumferentially aboutthe hair cell they are ideally located to maintain celldiameter, opposing the lines of greatest stress (Holley andAshmore, 1990). Spectrin is by contrast an elastic molecule(Vertessy and Steck, 1989) and an ideal partner for actin inthe lattice. The cross-links theoretically account for mostof the longitudinal stiffness of the lattice (Holley andAshmore, 19886), but their relatively low tensile strengthmay be the key to effecting changes in cell length ratherthan diameter.

Spectrin as a source of force generationSpectrin could generate the forces required to drive celllength changes. Spectrin tetramers in mammalian eryth-rocytes in vivo are about 70 nm long, but following incu-bation in high- or low-salt buffers they may shorten to50 nm or elongate to 200 nm, respectively (Byers andBranton, 1985; Shen et al. 1986). These changes arereflected in cell diameter (Vertessy and Steck, 1989). Thelength of the molecule is determined by the equilibriumbetween its inherent elasticity and the repulsion betweenintramolecular charges (Vertessy and Steck, 1989). Elec-trically induced length changes in outer hair cells wouldrequire no more than a 5 % length change, that is 2.5 nm ina cross-link 50 nm long. Charge-dependent length changesin the cross-links could drive length changes in the cell.How this mechanism might be coupled to the stimulus,that is to membrane potential (Ashmore, 1987), is notclear, but the model can be tested by direct experiments onthe isolated lattice.

ConclusionFurther ultrastructural work will be required to identifyspectrin and actin unequivocally in cross-links and cir-cumferential filaments, respectively. If the proposed modelis true, then the cortical lattice represents a novel combi-nation of the two proteins with spectrin molecules in aloosely parallel array. Mechanical measurements alongthe axes of extracted hair cells should provide informationabout the mechanical properties not only of hair cells butalso of spectrin molecules.

This work was supported by grants from the Medical ResearchCouncil and The Wellcome Trust. We thank Dr Andrew Forge forFigure 13, and Debbie Carter for assistance with electron mi-croscopy. M. C. Holley is a Beit Memorial Research Fellow.

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(Received 15 January 1990 - Accepted 27 February 1990)

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