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Fodrin is a constituent of the cortical lattice in outer hair cells of the guinea pig cochlea: Immunocytochemical evidence
lfeuri~fg Research, 65 (1993) 274-280 Q 1993 Elsevier Science fubiishers B.V. All rights reserved 0378-595S/93/$06.~
274
HEARES 01873
Fodrin is a constituent of the cortical lattice in outer hair cells of the guinea pig cochlea: ~mmunocytoc~emical evidence Yoshinao Nishida a,b,Toyoshi Fujimoto b, Akira Takagi ‘, Iwao Honjo a and Kazuo Ogawa ’ department
of a Otola~ngolo~
and b Anatomy, Facufty of Medicine, Kyoto Uniuersity, Kyoto, Japan
(Received 26 June 1992; Revision received 30 September 1992; Accepted 8 October 1992)
Locahzation of fodrin, a membrane skeletai protein, in the outer hair cell of the guinea pig cochlea was examined by immunocytochemical techniques. By immunofluorescence microscopy, fodrin was observed in the cuticular plate, in the infracuticular network and along the lateral wall. By immunoelectron microscopy of ultrathin cryosections, labeling for fodrin along the lateral wall was localized between the cell membrane and the outermost layer of the subsurface cisternae. Furthermore, pre-embedding immunoelectron microscopy of permeabilized specimens showed that most immunogolds for fodrin were on the thin cross-linking ~arn~nent of the cortical lattice. The results indicate that fodrin is a constituent of the cortical lattice which is thought to play an important role in outer hair cell motility. Fodrin; Cortical lattice; Outer hair cell; Cochlea; Cytoskeleton
Introduction The outer hair cehs (OHCs) isolated from the guinea pig cochlea have been observed to show two types of motility. The slow motility is initiated by various stimuli and depends upon ATP and calcium (Zenner et al., 1985; Zenner, 1986; Flock et al., 1986; Slepecky, 1989; Dulon et al., 19901, whereas the fast motility is induced by electrical stimulation (Brownell et al., 1985; Kachar et al., 1986; Ashmore, 1987; Evans et al., 1991). The motility of OHC is supposed to be related to the active process of hearing, but the detailed mechanism is not known. However, recent studies have shown that the structure in the lateral wall of OHC plays an important role in the motility (Holley and Ashmore, 1988a; Slepecky, 1989). A network of cytoskeletal filaments called the cortical lattice which exists between the cell membrane and the outermost layer of subsurface cisternae has been studied extensively, but its moiecular composition has not been clarified (Bannister et al., 1988; Holley and Ashmore, 1988b, 1990a,b; Lim et al., 1989; Arnold et al., 1990; Arima et al., 1991). In the present study, we investigated whether a membrane skeletal protein, fodrin, is a component of the lattice. Fodrin is a double stranded rod-shaped protein, which is analogous to erythrocyte spectrin and distributed beneath the cell membrane in various cells. The pro-
tein is thought to construct a membrane skeleton, which is involved in maintenance of cell shape, anchoring of cell membrane proteins to cytoskeleton and so on (Bennett et al., 1988; Coleman et al., 1989). Examination of the ultrastructural localization of fodrin is therefore important for understanding the highly differentiated cytoskeleton of OHC in relation to the well-delineated cytoskeletal structures of other cell types. The presence of fodrin in mammalian OHC has been reported already, but how it is related to the cortical lattice has not been demonstrated (Drenckhahn et al., 1985; Ylikoski et al., 1990; Holley and Ashmore, 1990b; Slepecky et al., 1992). In this paper, we studied the localization of fodrin with two immunoelectron microscopic methods and found that fodrin is a constituent of the cortical lattice in OHC.
Materials and Methods Adult Hartley guinea pigs (250-300 g) were anesthetized with pentobarbital and decapitated. After the temporal bone was removed, the bulla was opened in Hank’s balanced salt solution and the modiolus was cut into several pieces. Immunojluorescence
Correspondence
to: Yoshinao Nishida, Department of Otolaryngology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606 Japan. Fax: 81 (075) 751-7225.
microscopy
The specimens containing the organ of Corti were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4, for 1 h at room temperature and washed with 0.1 M PB
275
three times. For sectioning the third turn of the cochlea was used. The tissue pieces were treated with NaBH, (1 mg/ml) in 0.1 M PB for 30 min, washed with 0.1 M PB three times, immersed in 10% gelatin in 0.1 M PB for 1 h at 37°C and cooled in ice for solidification. They were then infused with 2.3 M sucrose in 0.1 M PB overnight at 4°C and rapidly frozen in liquid nitrogen. Semi-thin cryosections, OS-l.0 pm in thickness, were cut in a Reichert-Jung Ultracut E ultramicrotome equipped with a cryoattachment FC4. Sections were cut parallel to the axis of the modiolus starting from the periphery. By this method, OHCs always appeared before inner hair cells; the order was monitored by examining every one of several consecutive sections. Sections were collected with a semi-frozen droplet of saturated sucrose and mounted on glass slides. They were washed with phosphate buffered saline containing 10 mM glycine (PBSG), blocked with 2% gelatin in PBSG for 10 min and washed with PBSG for 10 min. They were then incubated with affinity-purified rabbit anti-rat brain fodrin antibody (lo-20 pg/mll (Fujimoto and Ogawa, 1989) for 30 min at 37°C washed with PBSG (2 X 5 min), and treated with fluorescein isothiocyanate (FIT0coupled goat anti-rabbit IgG antibody (Cappel), diluted 1: 25 with PBSG for 30 min at 37°C. Specimens were washed with PBSG, mounted in Citifluor glycerol/PBS solution and observed under an Olympus Vanox photomicroscope equipped with epifluorescent illumination and a phase contrast device. Immunoelectron
microscopy of cryosections
The specimens were fixed and treated similarly as above, but without NaBH, treatment. Ultrathin cryosections, 50-100 nm in thickness, were cut and mounted on formvar coated grids. Following a similar pretreatment as semi-thin sections, they were treated with the anti-fodrin antibody for 30 min at 37°C. After rinses with PBSG, they were incubated with 5 nm colloidal gold-conjugated goat anti-rabbit IgG antibody (Arnersham), and diluted 1: 20 in PBSG containing 1% bovine serum albumin (BSA), for 30 min at 37°C. They were washed with PBS and fixed with 2% glutaraldehyde in PBS for 10 min. After rinses with distilled water, they were stained with 2% neutral uranyl acetate and embedded in a mixture of 0.2% methylcellulose, 2% carbowax and 0.02% uranyl acetate. They were observed in a JEOL 1OOCor a 1200EX electron microscope. Pre-embedding immunoelectron
microscopy
The excised cochlea was extracted with 0.5% Triton X-100 in cytoskeletal buffer (CSK buffer; 100 mM NaCl, 300 mM sucrose, 10 mM Pipes, 3 mM MgCl, and 1.2 mM phenylmethylsulphonyl fluoride, pH 6.8) for 10 min at 4°C. They were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in CSK
buffer for 30 min at 4°C washed with CSK buffer once, with PBSG three times and treated with 1% BSA and 5% normal goat serum in PBSG for 30 min at 25°C. They were then incubated with the antibody to fodrin for 1 h at 25°C washed with PBSG (4 X 5 min), treated with the gold-conjugated secondary antibody, diluted 1:20 in 0.2% BSA and 1% normal goat serum in PBSG for 1 h at 25°C and washed with PBS (4 x 5 min). The samples were fixed with 2% glutaraldehyde and 0.2% tannic acid in 0.1 M cacodylate buffer (CB), pH 7.4, for 30 min at room temperature, washed with CB (5 X 2 min), and post-fixed in 1% osmium tetroxide in CB for 30 min at 4°C. After en bloc staining in 0.5% uranyl acetate in distilled water for 30 min at 4°C they were dehydrated in ethanol and embedded in Epon 812. Ultrathin sections were counterstained with lead citrate and observed.
Results Immunojluorescence
microscopy
Sections were cut almost parallel to the axis of the modiolus so that several OHCs in the same row could be observed simultaneously. Length of OHC appeared short because sections were cut obliquely to its long axis. Relatively weak fixation as well as thinness of the sections (0.5-1.0 pm1 made preservation of the original tissue architecture difficult, and thus the fluid-filled space appeared collapsed. In OHCs, labeling for fodrin was observed most intensely in the cuticular plate. In some OHCs, labeling of the infracuticular network was seen extending downward from. the cuticular plate (Ylikoski et al., 1990; Holley and Ashmore, 1990b). The lateral wall of OHC was also labeled positively. In contrast, the stereocilia were not labeled for fodrin (Fig. 1). These findings are mostly consistent with the previous reports about distribution of fodrin and spectrin (Drenckhahn et al., 1985; Ylikoski et al., 1990; Holley and Ashmore, 1990b; Slepecky et al., 1992). Immunoelectron
microscopy of cryosections
By immunoelectron microscopy of ultrathin cryosections, the presence of fodrin in the cuticular plate as well as its absence in the stereocilia was confirmed (Fig. 2a). Along the lateral wall, immunogold particles were consistently observed between the cell membrane and the outermost layer of the subsurface cisternae. There was scarcely any labeling between neighboring stacks of the subsurface cisternae. The localization of the labeling seemed to correspond to that of the cortical lattice, but its detailed structure could not be observed in cryosections (Fig. 2b). Pre-embedding immunoelectron microscopy
In order to localize fodrin in direct correlation to the cortical lattice structure, a pre-embedding im-
276
Fig. 1. Immunofluorescence microscopy of semi-thin cryosections of the guinea pig cochlea. Sections were cut almost parallel to the axis of the modioius so that several OHCs in the same row could be observed simultaneously. Since OHCs are cut obliquely, only the upper portion is observed in most ceils; on the other hand, ceils marked with arrows exhibit only their lower half, and thus the cuticuiar plate is not seen. Intense labeling for fodrin is observed in the cuticular plate and moderate labeling along the lateral wail. In some OHCs, labeling of the infracuticular network was seen extending downward from the cuticuiar plate (arrowheads). The stereocilia are not labeled positively. The fluid-filled space is marked by an asterisk. Scale bar: IO pm.
munocytochemical technique was employed. By treating the cochlea with Triton X-100, the membrane was permeabihzed, but the cortical lattice remained and could be labeled by antibodies. Ultrathin sections which cut the cell wall tangentially gave en face views of the
cortical lattice, thus enabling identification of the circumferential and cross-finking fiiaments. In the specimen, immunogoId particles for fodrin were observed more frequently on the thinner cross-linking filaments than on the thicker circumferential filaments (Fig. 3).
Fig. 2. Immunoeiectron microscopy of ultrathin cryosection of the OHC labeled with anti-fodrin and S-nm-immunogoid particles. (a) Apical cytoplasm. The presence of fodrin in the cuticular plate and its absence in the stereocilia is shown. S: stereocilia, CP: cuticular plate. Scale bar: 500 nm. (b) Lateral wail. Immunogoid particles for fodrin are observed between the cell membrane and the outermost layer of the subsurface cisternae. SSC: subsurface cisternae, M: mitochondria, Arrowheads: ceil membrane. Scale bar: IO0 nm.
Pig. 3. Pre-embedding immunoelectron microscopy of the detergent-treated OHC. Immunola~ling for fodrin can be observed in relation to the tort ical lattice, (a) Control. Circumferential (large arrows> as well as cross-linking (small arrows) are preserved in the preparation. Scarcely any im munolabeling is seen. (b-h) Immunogold particles for fodrin are observed mostly on cross-linking filaments (arrowheads). Scale bar: 200 nm.
278
By counting the number of gold particles, 58.7% of them were on the cross-links, 16.0% on the circumferential filaments and 25.3% on the intersections of the two kinds of filaments: more than 80% of them were related to the cross-links. The quantitative analysis indicates that fodrin is a component of the cross-links. But because the cortical lattice ultrastructure was not well preserved due to the weak fixation, the possibility remains that the protein may also be involved for the circumferential filaments.
Discussion The properties of fodrin
Fodrin, also referred to as calspectin and nonerythroid spectrin, belongs to the spectrin family. The protein is rod-shaped and is believed to exist mostly as heterodimers in vivo. Fodrin dimers are about 100 nm in length and about 2-4 nm in diameter. Fodrin is localized beneath the cell membrane in a variety of differentiated cells. It was shown to bind many cytoskeletal as well as membrane proteins in vitro. Although its precise function in vivo is not clear at present, it is likely that the protein mediates linkage between the cell membrane and the cytoskeleton, and is thus involved in various cell functions: maintenance of cell shape, cell surface receptor mobility, endocytosis, exocytosis, cell motility and so on (Byers et al., 1985; Shen et al., 1986; Sobue et al., 1987; Bennett et al., 1988; Coleman et al., 1989). Fodrin in outer hair cells
Fodrin has been demonstrated to exist in mammalian OHCs by immunofluorescence microscopy (Drenckhahn et al., 1985; Ylikoski et al., 1990; Holley and Ashmore, 1990b; Slepecky et al., 1992). The labeling for fodrin has been observed in the cuticular plate, in the infracuticular network which extends from the cuticular plate toward the nucleus, and along the lateral wall; the stereocilia were not labeled for fodrin. Our present results by immunofluorescence microscopy confirmed the previous reports. In spite of the above observations, the ultrastructural localization of fodrin has not been shown to date. The distribution of the protein in the cuticular plate may be analogous to that in the terminal web of the intestinal epithelium: fodrin is likely to cross-link actin filaments in the rootlet of neighboring microvilli (Hirokawa et al., 1983). On the other hand, its localization in the lateral wall of OHC is interesting because a special macromolecular structure has been known to be present between the cell membrane and the outermost layer of the subsurface cisternae. Various models have been proposed and there may be some species differences, but
basically the structure is thought to consist of a filamentous lattice which is parallel to the cell membrane, and pillars vertical to it (Raphael et al., 1986; Flock et al., 1986; Bannister et al., 1988; Holley and Ashmore, 1988b, 1990a,b; Lim et al., 1989; Arnold et al., 1990; Arima et al., 1991). For example, by electron microscopy of the guinea pig cochlea, Holley and Ashmore observed a cortical lattice composed of two types of filaments: the circumferential filaments 5-8 nm in diameter and the cross-links 2-3 nm in diameter and 40-50 nm in length. Based on the filament’s size as well as the immunofluorescence results, they suggested that the circumferential filaments and cross-links are composed of actin and spectrin (fodrin), respectively. In the present study, by immunoelectron microscopy of cryosections, we observed that immunogold particles for fodrin were localized between the cell membrane and the outermost layer of the subsurface cisternae. Furthermore, by pre-embedding immunoelectron microscopy, we demonstrated that a majority of immunogold particles for fodrin were localized on the crosslinks of the cortical lattice. The result is in agreement with the assumption of Holley and Ashmore and presents the first direct evidence that the cortical lattice in OHC contains fodrin. The length of the cross-link which was observed by conventional thin section electron microscopy is shorter than the size of isolated fodrin molecule (Fishkind et al., 1987), 100 nm in dimer and 200 nm in tetramer. Several interpretations are possible to explain the discrepancy. For example, each fodrin molecule may span the circumferential filament two filaments away instead of the very next filament. It is also possible that the cross-links may be heterogeneous in length and in composition and only some of them may consist of fodrin. However, the most plausible explanation can be given by assuming high elasticity for fodrin. Erythrocyte spectrin in tetramer has been shown to change its length from 50 nm to 200 nm according to ionic strength (Byers et al., 1985; Shen et al., 1986; Vertessy et al., 1989). Functions of fodrin in the lateral wall have been postulated in various ways. Firstly, as in many other nonerythroid cells, fodrin may maintain cell shape as a membrane skeletal protein. In OHC, the mechanical strength of the lateral wall may be important because the intracellular pressure should be maintained positively for the fast motility (Holley and Ashmore, 1990a). Secondly, fodrin may be linked to stretch-activated ion channels which sense membrane tension and regulate the intracellular pressure (Sachs, 1988; Ding et al., 1991). Thirdly, fodrin may be involved in the fast motility of OHC. As proposed by Holley (19911, elastic force induced by the ionic environmental alteration and electrostatic charge affected by membrane potential may cause changes in the molecular length of fodrin and, eventually, cell length. The existence of
fodrin as cross-links in the regularly organized cortical lattice of OHC in contrast to forming random networks in other cells (Fujimoto et al., 1991) may be important in performing above functions. The cortical lattice of the guinea pig is assumed to play important roles in the motility of OHCs, but there are some species, e.g. humans and mole-rats, which are known to devoid of it (Arnold et al., 1990; Raphael et al., 1986). In the chinchilla also, the structure beneath the lateral cell membrane is likely to be different from that of the guinea pig (Lim et al., 1989). In those species, the role of the cortical lattice in the guinea pig should be fulfilled by other structures. In this regard, it would be interesting to see where fodrin is localized in OHCs of other species, because a structure functionally analogous to the cortical lattice may also contain the protein. The identity of the two other main components of the lateral wall, the circumferential filament and the pillar, has not been determined by the present study. Actin has been the most likely candidate for the circumferential filament because of its diameter. In fact, several immunofluorescence as well as immunoelectron microscopic studies reported that actin exists along the lateral wall (Flock et al., 1986; Zenner, 1986; Thorne et al., 1987; Slepecky et al., 1988; Slepecky, 1989; Dulon et al., 1990). However, a recent study utilizing a combination of laser scanning confocal microscopy and rhodamine-phalloidin failed to detect a significant amount of F-actin along the lateral wall (Altschuler and Raphael, 1991). Since fodrin can interact with other cytoskeletal components as well as actin, the present result does not preclude either of the possibilities. Neither was the molecular composition of the pillar indicated, but an intriguing possibility is that proteins known to interact with fodrin in other cells, such as ankyrin, protein 4.1, band 3 and so on, may be related to the structure. In conclusion, the current study presented a direct demonstration that a component of the cortical lattice is made of fodrin. The physiological function of the cortical lattice will only be amenable to detailed analysis by identifying other constituent molecules in the future.
References Altschuler, R.A. and Raphael, Y. (1991) Laser scanning confocal microscopy of actin in hair cells: Evidence against F-actin in outer hair cell lateral walls. Abstr. Assoc. Res. Otolaryngol. 14, 12. Arima, T., Kuraoka, A., Toriya, R., Shibata, Y. and Uemura, T. (1991) Quick-freeze, deep-etch visualization of the cytoskeletal spring of cochlear outer hair cells. Cell Tissue Res. 263, 91-97. Arnold, W. and Anniko, M. (1990) Structurally based new functional interpretations of the subsurface cisternal network in human outer hair cells. Acta. Otolaryngol. (Stockh) 109, 213-220.
Ashmore, J.F. (1987) A fast motile response in guinea-pig outer hair ceils: The cellular basis of the cochlear amplifier. J. Physiol. 388,323-347. Bannister, L.H., Dodson, H.C., Astbury, A.R. and Douek, E.E. (1988) The cortical lattice: A highly ordered system of subsurface filaments in guinea pig cochlear outer hair cells. Prog. Brain Res. 74, 213-219. Bennett, V., Steiner, J. and Davis, J. (1988) Diversity in protein associations of the spectrin-based membrane skeleton of nonerythroid cells. Protoplasma 145, 89-94. Brownell, W.E., Bader, C.R., Bertrand, D. and de Ribaupierre, Y. (1985) Evoked mechanical responses of isolated cochlear outer hair cells. Science 227, 194-196. Byers, T.J. and Branton D. (1985) Visualization of the protein associations in the erythrocyte membrane skeleton. Proc. Natl. Acad. Sci. USA 82, 6153-6157. Coleman, T.R., Fishkind, D.J., Mooseker, M.S. and Morrow, J.S. (1989) Functional diversity among spectrin isoforms. Cell Motil. Cytoskeleton 12, 225-247. Ding, J.P., Salvi, R.J. and Sachs, F. (1991) Stretch-activated ion channels in guinea pig outer hair cells. Hear. Res. 56, 19-28. Drenckhahn, D., Schafer, T. and Prinz, M. (1985) Actin, myosin, and associated proteins in the vertebrate auditory and vestibular organs: Immunocytochemical and biochemical studies. In: D.G. Drescher (Ed.), Auditory Biochemistry, Charles C Thomas, Springfield, IL, pp. 317-335. Dulon, D., Zajic, G. and Schacht, J. (1990) Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells. J. Neurosci. 10, 1388-1397. Evans, B.N., Hallworth, R. and Dallas, P. (1991) Outer hair cell electromotility: The sensitivity and vulnerability of the DC component. Hear. Res. 52, 288-304. Fishkind, D.J., Bonder, E.M. and Begg, D.A. (1987) Isolation and characterization of sea urchin egg spectrin: Calcium modulation of the spectrin-actin inte’raction. Cell Motil. Cytoskeleton 7, 304314. Flock, A., Flock, B. and Ulfendahl, M. (1986) Mechanisms of movement in outer hair cells and a possible structural basis. Arch. Otorhinolaryngol. 243, 83-90. Fujimoto, T. and Ogawa, K. (1989) Immunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium. J. Histochem. Cytochem. 37, 1345-1352. Fujimoto, T., Lee, K., Miwa, S. and Ogawa, K. (1991) Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro. J. Histochem. L%ochem. 39, 1485-1493. Hirokawa, N., Cheney, R.E. a td Willard, M. (1983) Location of a protein of the fodrin-spectrinTW260/240 family in the mouse intestinal brush border. Cell 32, 953-965. Holley, M.C. and Ashmore, J.F. (1988a) On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea. Proc. R. Sot. Lond. B. 232, 413-429. Holley, M.C. and Ashmore, J.F. (1988b) A cytoskeletal spring in cochlear outer hair cells. Nature 335, 635-637. Holley, M.C. and Ashmore, J.F. (199Oa) A cytoskeletal spring for the control of cell shape in outer hair cells isolated from the guinea pig cochlea. Eur. Arch. Otorhinolaryngol. 247, 4-7. Holley, M.C. and Ashmore, J.F. (199Ob) Spectrin, actin and the structure of the cortical lattice in mammalian cochlear outer hair cells. J. Cell Sci. 96, 283-291. Holley, M.C. (1991) High frequency force generation in outer hair cells from the mammalian ear. BioEssays. 13, 115-120. Kachar, B., Brownell, W.E., Altschuler, R. and Fex, J. (1986) Electrokinetic shape changes of cochlear outer hair cells. Nature 322,365-368. Lim, D.J., Hanamure, Y. and Ohashi, Y. (1989) Structural organization of the outer hair cell wall. Acta. Otolaryngol. (Stockh) 107, 398-405.
Raphael, Y. and Wroblewski, R. (1986) Linkage of sub-membranecisterns with the cytoskeleton and the plasma membrane in cochlear outer hair cells. J. Submicrosc. Cytol. 18, 731-737. Sachs, F. (1988) Mechanical transduction in biological systems. CRC Crit. Rev. Biomed. Eng. 16, 141-169. Shen, B.W., Josephs, R. and Steck, T.L. (1986) Ultrastructure of the intact skeleton of the human erythrocyte membrane. J. Cell Biol. 102, 997-1006. Slepecky, N., Ulfendahl, M. and Flock, A. (1988) Effects of caffeine and tetracaine on outer hair cell shortening suggest intracellular calcium involvement. Hear. Res. 32, 11-22. Slepecky, N. (1989) Cytoplasmic actin and cochlear outer hair cell motility. Cell Tissue Res. 257, 69-75. Slepecky, N. and Ulfendahl, M. (1992) Actin-binding and microtubule-associated proteins in the organ of Gxti. Hear. Res. 57, 201-21s. Sobue, K., Okabe, T., Itoh, K., Kadowaki, K., Tanaka, T., Kanda, K., Ueki, N. and Fujio, Y. (1987) The Ca*+-dependent regulation of the submembranous cytoskeleton in nonerythroid cells: The pos-
sible involvement of calspectin (nonerythroid spectrin or fodrin) and its interacting proteins. Biomed. Res. 8, sup. 13-22. Thorne, P.R., Carlisle, L., Zajic, G., Schacht, J. and Altschuler, R.A. (1987) Differences in the distribution of F-actin in outer hair cells along the organ of Corti. Hear. Res. 30, 253-266. Vertessy, B.G. and Steck, T.L. (1989) Elasticity of the human red cell membrane skeleton: Effects of temperature and denaturants. Biophys. J. 55255-262. Ylikoski, J., Pirvola, U., Narvanen, 0. and Virtanen, I. (1990) Nonerythroid spectrin (fodrin) is a prominent component of the cochlear hair cells. Hear. Res. 43, 199-204. Zenner, H.P., Zimmermann, U. and Schmitt, U. (1985) Reversible contraction of isolated mammalian cochlear hair cells. Hear. Res. 18, 127-133. Zenner, H.P. (1986) Motile responses in outer hair cells. Hear. Res. 22, 83-90. Zenner, H.P. (1988) Motility of outer hair cells as an active, actinmediated process. Acta. Otolaryngol. (Stockh) 105, 39-44.
274
HEARES 01873
Fodrin is a constituent of the cortical lattice in outer hair cells of the guinea pig cochlea: ~mmunocytoc~emical evidence Yoshinao Nishida a,b,Toyoshi Fujimoto b, Akira Takagi ‘, Iwao Honjo a and Kazuo Ogawa ’ department
of a Otola~ngolo~
and b Anatomy, Facufty of Medicine, Kyoto Uniuersity, Kyoto, Japan
(Received 26 June 1992; Revision received 30 September 1992; Accepted 8 October 1992)
Locahzation of fodrin, a membrane skeletai protein, in the outer hair cell of the guinea pig cochlea was examined by immunocytochemical techniques. By immunofluorescence microscopy, fodrin was observed in the cuticular plate, in the infracuticular network and along the lateral wall. By immunoelectron microscopy of ultrathin cryosections, labeling for fodrin along the lateral wall was localized between the cell membrane and the outermost layer of the subsurface cisternae. Furthermore, pre-embedding immunoelectron microscopy of permeabilized specimens showed that most immunogolds for fodrin were on the thin cross-linking ~arn~nent of the cortical lattice. The results indicate that fodrin is a constituent of the cortical lattice which is thought to play an important role in outer hair cell motility. Fodrin; Cortical lattice; Outer hair cell; Cochlea; Cytoskeleton
Introduction The outer hair cehs (OHCs) isolated from the guinea pig cochlea have been observed to show two types of motility. The slow motility is initiated by various stimuli and depends upon ATP and calcium (Zenner et al., 1985; Zenner, 1986; Flock et al., 1986; Slepecky, 1989; Dulon et al., 19901, whereas the fast motility is induced by electrical stimulation (Brownell et al., 1985; Kachar et al., 1986; Ashmore, 1987; Evans et al., 1991). The motility of OHC is supposed to be related to the active process of hearing, but the detailed mechanism is not known. However, recent studies have shown that the structure in the lateral wall of OHC plays an important role in the motility (Holley and Ashmore, 1988a; Slepecky, 1989). A network of cytoskeletal filaments called the cortical lattice which exists between the cell membrane and the outermost layer of subsurface cisternae has been studied extensively, but its moiecular composition has not been clarified (Bannister et al., 1988; Holley and Ashmore, 1988b, 1990a,b; Lim et al., 1989; Arnold et al., 1990; Arima et al., 1991). In the present study, we investigated whether a membrane skeletal protein, fodrin, is a component of the lattice. Fodrin is a double stranded rod-shaped protein, which is analogous to erythrocyte spectrin and distributed beneath the cell membrane in various cells. The pro-
tein is thought to construct a membrane skeleton, which is involved in maintenance of cell shape, anchoring of cell membrane proteins to cytoskeleton and so on (Bennett et al., 1988; Coleman et al., 1989). Examination of the ultrastructural localization of fodrin is therefore important for understanding the highly differentiated cytoskeleton of OHC in relation to the well-delineated cytoskeletal structures of other cell types. The presence of fodrin in mammalian OHC has been reported already, but how it is related to the cortical lattice has not been demonstrated (Drenckhahn et al., 1985; Ylikoski et al., 1990; Holley and Ashmore, 1990b; Slepecky et al., 1992). In this paper, we studied the localization of fodrin with two immunoelectron microscopic methods and found that fodrin is a constituent of the cortical lattice in OHC.
Materials and Methods Adult Hartley guinea pigs (250-300 g) were anesthetized with pentobarbital and decapitated. After the temporal bone was removed, the bulla was opened in Hank’s balanced salt solution and the modiolus was cut into several pieces. Immunojluorescence
Correspondence
to: Yoshinao Nishida, Department of Otolaryngology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606 Japan. Fax: 81 (075) 751-7225.
microscopy
The specimens containing the organ of Corti were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4, for 1 h at room temperature and washed with 0.1 M PB
275
three times. For sectioning the third turn of the cochlea was used. The tissue pieces were treated with NaBH, (1 mg/ml) in 0.1 M PB for 30 min, washed with 0.1 M PB three times, immersed in 10% gelatin in 0.1 M PB for 1 h at 37°C and cooled in ice for solidification. They were then infused with 2.3 M sucrose in 0.1 M PB overnight at 4°C and rapidly frozen in liquid nitrogen. Semi-thin cryosections, OS-l.0 pm in thickness, were cut in a Reichert-Jung Ultracut E ultramicrotome equipped with a cryoattachment FC4. Sections were cut parallel to the axis of the modiolus starting from the periphery. By this method, OHCs always appeared before inner hair cells; the order was monitored by examining every one of several consecutive sections. Sections were collected with a semi-frozen droplet of saturated sucrose and mounted on glass slides. They were washed with phosphate buffered saline containing 10 mM glycine (PBSG), blocked with 2% gelatin in PBSG for 10 min and washed with PBSG for 10 min. They were then incubated with affinity-purified rabbit anti-rat brain fodrin antibody (lo-20 pg/mll (Fujimoto and Ogawa, 1989) for 30 min at 37°C washed with PBSG (2 X 5 min), and treated with fluorescein isothiocyanate (FIT0coupled goat anti-rabbit IgG antibody (Cappel), diluted 1: 25 with PBSG for 30 min at 37°C. Specimens were washed with PBSG, mounted in Citifluor glycerol/PBS solution and observed under an Olympus Vanox photomicroscope equipped with epifluorescent illumination and a phase contrast device. Immunoelectron
microscopy of cryosections
The specimens were fixed and treated similarly as above, but without NaBH, treatment. Ultrathin cryosections, 50-100 nm in thickness, were cut and mounted on formvar coated grids. Following a similar pretreatment as semi-thin sections, they were treated with the anti-fodrin antibody for 30 min at 37°C. After rinses with PBSG, they were incubated with 5 nm colloidal gold-conjugated goat anti-rabbit IgG antibody (Arnersham), and diluted 1: 20 in PBSG containing 1% bovine serum albumin (BSA), for 30 min at 37°C. They were washed with PBS and fixed with 2% glutaraldehyde in PBS for 10 min. After rinses with distilled water, they were stained with 2% neutral uranyl acetate and embedded in a mixture of 0.2% methylcellulose, 2% carbowax and 0.02% uranyl acetate. They were observed in a JEOL 1OOCor a 1200EX electron microscope. Pre-embedding immunoelectron
microscopy
The excised cochlea was extracted with 0.5% Triton X-100 in cytoskeletal buffer (CSK buffer; 100 mM NaCl, 300 mM sucrose, 10 mM Pipes, 3 mM MgCl, and 1.2 mM phenylmethylsulphonyl fluoride, pH 6.8) for 10 min at 4°C. They were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in CSK
buffer for 30 min at 4°C washed with CSK buffer once, with PBSG three times and treated with 1% BSA and 5% normal goat serum in PBSG for 30 min at 25°C. They were then incubated with the antibody to fodrin for 1 h at 25°C washed with PBSG (4 X 5 min), treated with the gold-conjugated secondary antibody, diluted 1:20 in 0.2% BSA and 1% normal goat serum in PBSG for 1 h at 25°C and washed with PBS (4 x 5 min). The samples were fixed with 2% glutaraldehyde and 0.2% tannic acid in 0.1 M cacodylate buffer (CB), pH 7.4, for 30 min at room temperature, washed with CB (5 X 2 min), and post-fixed in 1% osmium tetroxide in CB for 30 min at 4°C. After en bloc staining in 0.5% uranyl acetate in distilled water for 30 min at 4°C they were dehydrated in ethanol and embedded in Epon 812. Ultrathin sections were counterstained with lead citrate and observed.
Results Immunojluorescence
microscopy
Sections were cut almost parallel to the axis of the modiolus so that several OHCs in the same row could be observed simultaneously. Length of OHC appeared short because sections were cut obliquely to its long axis. Relatively weak fixation as well as thinness of the sections (0.5-1.0 pm1 made preservation of the original tissue architecture difficult, and thus the fluid-filled space appeared collapsed. In OHCs, labeling for fodrin was observed most intensely in the cuticular plate. In some OHCs, labeling of the infracuticular network was seen extending downward from. the cuticular plate (Ylikoski et al., 1990; Holley and Ashmore, 1990b). The lateral wall of OHC was also labeled positively. In contrast, the stereocilia were not labeled for fodrin (Fig. 1). These findings are mostly consistent with the previous reports about distribution of fodrin and spectrin (Drenckhahn et al., 1985; Ylikoski et al., 1990; Holley and Ashmore, 1990b; Slepecky et al., 1992). Immunoelectron
microscopy of cryosections
By immunoelectron microscopy of ultrathin cryosections, the presence of fodrin in the cuticular plate as well as its absence in the stereocilia was confirmed (Fig. 2a). Along the lateral wall, immunogold particles were consistently observed between the cell membrane and the outermost layer of the subsurface cisternae. There was scarcely any labeling between neighboring stacks of the subsurface cisternae. The localization of the labeling seemed to correspond to that of the cortical lattice, but its detailed structure could not be observed in cryosections (Fig. 2b). Pre-embedding immunoelectron microscopy
In order to localize fodrin in direct correlation to the cortical lattice structure, a pre-embedding im-
276
Fig. 1. Immunofluorescence microscopy of semi-thin cryosections of the guinea pig cochlea. Sections were cut almost parallel to the axis of the modioius so that several OHCs in the same row could be observed simultaneously. Since OHCs are cut obliquely, only the upper portion is observed in most ceils; on the other hand, ceils marked with arrows exhibit only their lower half, and thus the cuticuiar plate is not seen. Intense labeling for fodrin is observed in the cuticular plate and moderate labeling along the lateral wail. In some OHCs, labeling of the infracuticular network was seen extending downward from the cuticuiar plate (arrowheads). The stereocilia are not labeled positively. The fluid-filled space is marked by an asterisk. Scale bar: IO pm.
munocytochemical technique was employed. By treating the cochlea with Triton X-100, the membrane was permeabihzed, but the cortical lattice remained and could be labeled by antibodies. Ultrathin sections which cut the cell wall tangentially gave en face views of the
cortical lattice, thus enabling identification of the circumferential and cross-finking fiiaments. In the specimen, immunogoId particles for fodrin were observed more frequently on the thinner cross-linking filaments than on the thicker circumferential filaments (Fig. 3).
Fig. 2. Immunoeiectron microscopy of ultrathin cryosection of the OHC labeled with anti-fodrin and S-nm-immunogoid particles. (a) Apical cytoplasm. The presence of fodrin in the cuticular plate and its absence in the stereocilia is shown. S: stereocilia, CP: cuticular plate. Scale bar: 500 nm. (b) Lateral wail. Immunogoid particles for fodrin are observed between the cell membrane and the outermost layer of the subsurface cisternae. SSC: subsurface cisternae, M: mitochondria, Arrowheads: ceil membrane. Scale bar: IO0 nm.
Pig. 3. Pre-embedding immunoelectron microscopy of the detergent-treated OHC. Immunola~ling for fodrin can be observed in relation to the tort ical lattice, (a) Control. Circumferential (large arrows> as well as cross-linking (small arrows) are preserved in the preparation. Scarcely any im munolabeling is seen. (b-h) Immunogold particles for fodrin are observed mostly on cross-linking filaments (arrowheads). Scale bar: 200 nm.
278
By counting the number of gold particles, 58.7% of them were on the cross-links, 16.0% on the circumferential filaments and 25.3% on the intersections of the two kinds of filaments: more than 80% of them were related to the cross-links. The quantitative analysis indicates that fodrin is a component of the cross-links. But because the cortical lattice ultrastructure was not well preserved due to the weak fixation, the possibility remains that the protein may also be involved for the circumferential filaments.
Discussion The properties of fodrin
Fodrin, also referred to as calspectin and nonerythroid spectrin, belongs to the spectrin family. The protein is rod-shaped and is believed to exist mostly as heterodimers in vivo. Fodrin dimers are about 100 nm in length and about 2-4 nm in diameter. Fodrin is localized beneath the cell membrane in a variety of differentiated cells. It was shown to bind many cytoskeletal as well as membrane proteins in vitro. Although its precise function in vivo is not clear at present, it is likely that the protein mediates linkage between the cell membrane and the cytoskeleton, and is thus involved in various cell functions: maintenance of cell shape, cell surface receptor mobility, endocytosis, exocytosis, cell motility and so on (Byers et al., 1985; Shen et al., 1986; Sobue et al., 1987; Bennett et al., 1988; Coleman et al., 1989). Fodrin in outer hair cells
Fodrin has been demonstrated to exist in mammalian OHCs by immunofluorescence microscopy (Drenckhahn et al., 1985; Ylikoski et al., 1990; Holley and Ashmore, 1990b; Slepecky et al., 1992). The labeling for fodrin has been observed in the cuticular plate, in the infracuticular network which extends from the cuticular plate toward the nucleus, and along the lateral wall; the stereocilia were not labeled for fodrin. Our present results by immunofluorescence microscopy confirmed the previous reports. In spite of the above observations, the ultrastructural localization of fodrin has not been shown to date. The distribution of the protein in the cuticular plate may be analogous to that in the terminal web of the intestinal epithelium: fodrin is likely to cross-link actin filaments in the rootlet of neighboring microvilli (Hirokawa et al., 1983). On the other hand, its localization in the lateral wall of OHC is interesting because a special macromolecular structure has been known to be present between the cell membrane and the outermost layer of the subsurface cisternae. Various models have been proposed and there may be some species differences, but
basically the structure is thought to consist of a filamentous lattice which is parallel to the cell membrane, and pillars vertical to it (Raphael et al., 1986; Flock et al., 1986; Bannister et al., 1988; Holley and Ashmore, 1988b, 1990a,b; Lim et al., 1989; Arnold et al., 1990; Arima et al., 1991). For example, by electron microscopy of the guinea pig cochlea, Holley and Ashmore observed a cortical lattice composed of two types of filaments: the circumferential filaments 5-8 nm in diameter and the cross-links 2-3 nm in diameter and 40-50 nm in length. Based on the filament’s size as well as the immunofluorescence results, they suggested that the circumferential filaments and cross-links are composed of actin and spectrin (fodrin), respectively. In the present study, by immunoelectron microscopy of cryosections, we observed that immunogold particles for fodrin were localized between the cell membrane and the outermost layer of the subsurface cisternae. Furthermore, by pre-embedding immunoelectron microscopy, we demonstrated that a majority of immunogold particles for fodrin were localized on the crosslinks of the cortical lattice. The result is in agreement with the assumption of Holley and Ashmore and presents the first direct evidence that the cortical lattice in OHC contains fodrin. The length of the cross-link which was observed by conventional thin section electron microscopy is shorter than the size of isolated fodrin molecule (Fishkind et al., 1987), 100 nm in dimer and 200 nm in tetramer. Several interpretations are possible to explain the discrepancy. For example, each fodrin molecule may span the circumferential filament two filaments away instead of the very next filament. It is also possible that the cross-links may be heterogeneous in length and in composition and only some of them may consist of fodrin. However, the most plausible explanation can be given by assuming high elasticity for fodrin. Erythrocyte spectrin in tetramer has been shown to change its length from 50 nm to 200 nm according to ionic strength (Byers et al., 1985; Shen et al., 1986; Vertessy et al., 1989). Functions of fodrin in the lateral wall have been postulated in various ways. Firstly, as in many other nonerythroid cells, fodrin may maintain cell shape as a membrane skeletal protein. In OHC, the mechanical strength of the lateral wall may be important because the intracellular pressure should be maintained positively for the fast motility (Holley and Ashmore, 1990a). Secondly, fodrin may be linked to stretch-activated ion channels which sense membrane tension and regulate the intracellular pressure (Sachs, 1988; Ding et al., 1991). Thirdly, fodrin may be involved in the fast motility of OHC. As proposed by Holley (19911, elastic force induced by the ionic environmental alteration and electrostatic charge affected by membrane potential may cause changes in the molecular length of fodrin and, eventually, cell length. The existence of
fodrin as cross-links in the regularly organized cortical lattice of OHC in contrast to forming random networks in other cells (Fujimoto et al., 1991) may be important in performing above functions. The cortical lattice of the guinea pig is assumed to play important roles in the motility of OHCs, but there are some species, e.g. humans and mole-rats, which are known to devoid of it (Arnold et al., 1990; Raphael et al., 1986). In the chinchilla also, the structure beneath the lateral cell membrane is likely to be different from that of the guinea pig (Lim et al., 1989). In those species, the role of the cortical lattice in the guinea pig should be fulfilled by other structures. In this regard, it would be interesting to see where fodrin is localized in OHCs of other species, because a structure functionally analogous to the cortical lattice may also contain the protein. The identity of the two other main components of the lateral wall, the circumferential filament and the pillar, has not been determined by the present study. Actin has been the most likely candidate for the circumferential filament because of its diameter. In fact, several immunofluorescence as well as immunoelectron microscopic studies reported that actin exists along the lateral wall (Flock et al., 1986; Zenner, 1986; Thorne et al., 1987; Slepecky et al., 1988; Slepecky, 1989; Dulon et al., 1990). However, a recent study utilizing a combination of laser scanning confocal microscopy and rhodamine-phalloidin failed to detect a significant amount of F-actin along the lateral wall (Altschuler and Raphael, 1991). Since fodrin can interact with other cytoskeletal components as well as actin, the present result does not preclude either of the possibilities. Neither was the molecular composition of the pillar indicated, but an intriguing possibility is that proteins known to interact with fodrin in other cells, such as ankyrin, protein 4.1, band 3 and so on, may be related to the structure. In conclusion, the current study presented a direct demonstration that a component of the cortical lattice is made of fodrin. The physiological function of the cortical lattice will only be amenable to detailed analysis by identifying other constituent molecules in the future.
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