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Fodrin immunocytochemical localization in the striated organelles of the rat vestibular hair cells
Hearing Research. 61 (1992) 155-160
155
© 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00 H E A R E S 01769
Fodrin immunocytochemical localization in the striated organelles of the rat vestibular hair cells D. Dem~mes and E. Scarfone INSERM U.254. Laboratoire de Neurophysiologie Sensorielle, Montpellier. France (Received 9 October 1991; Revision received 26 February 1992; Accepted 2 March 1992)
The immunocytochemical distribution of a spectrin-related protein, fodrin was studied at the electron microscopic level in the rat vestib."-lar hair cells. As previously demonstrated [Scarfone et al., Neurosci. Lett. 93, 13-18, 1988], an intense immunoreactivity was found in the cuticular plates. We demonstrate furthermore, here, for the first time the association of foddn immunoreactivity with the striated infracuticular structures called striated organelles (SO). Fodrin was found in striated structures clearly identified as SO in both Type I and Type 1I hair cells. SO were labelled regardless of their location, subcuticular or associated with the plasma membrane of the cells. We suggest that fodrin, as in the cuticular plate, could participate to the Ca -'+ dependent cross-linking of the actin filaments of the striated organelles and could play a role in their interaction with the submembraneous cytoskeleton. Fodrin; Vestibular hair cell; lmmunocytochemical; Distribution; Striated organelles
Introduction
Inner ear sensory cells of human and several animal species contain at the level of the cuticular plate and in the infracuticular region striated structures called striated organelles (SO) (Slepecky et al., 1981; Slepecky and Chamberlain, 1982; Sans, 1989). These SO were found to be structural components of normal hair cells from healthy vestibular and cochlear receptors despite having previously been associated with pathological or ageing phenomena (Friedman et al., 1963; Kimura, 1966; Spoendlin, 1966; Engtr0m et al., 1972). Slepecky and Chamberlain (1982) have demonstrated S-1 myosin decoration of actin filaments in the dense bands of SO situated at the base of the cuticular plate of hair cells in the cochlea of the chinchilla and suggested that SO coud be involved in the active processes of the cochlea. Motile processes associated with sensory transduction have also been proposed to occur in vestibular hair cells and SO, because of their structure and their location at the apex of the cells, have been implicated in them (Ross, 1982; Ross and Bourne, 1983; Sans, 1989) A spectrin-related protein, a fodrin, has recently been found in vestibular end organs by immunoblotting and localised in the cuticular plate of vestibular hair
Correspondance to: DanieHe DemC:mes, INSERM U.254, U.S.T.L.~ Laboratoire de Neurophysiologie Sensorielle, C.P. 089, Place E. Bataillon, 34095 Montpellier Cedex 5, France.
cells by immunofluorescence and immunoelectron microscopy (Scarfone et al., 1988). Cytoskeletal and contractile proteins such as actin (Flock et al., 1981), myosin and troporayosin (Sans et al., 1989) were found in the same area of the sensory cells. We have suggested that fodrin participates in the cuticular plate organisation by cross-linking actin filaments and by anchoring the cuticular plate to the apical membrane (Scarfone et al., 1988). The fact that SO are also partly constituted of actin filaments and are frequently closely associated with the plasma membrane may indicate that these SO may also contain fodfin. We have therefore carried out a thorough ultrastructural study of the immunolocalization of fodrin in the SO of vestibular hair cells,
Material and Methods
Young adult rats were transcardially perfused with freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The vestibular receptors were dissected out, postfixed for 1 h in the same fixative then rinsed in the phosphate buffer. They were immersed twice in acetone at -30°C and then thawed in phosphate buffer. Pre-embedding immunocytochemistry was carried out by incubating the receptors with affinity-purified anti-fodrin antibodies (Perrin and Aunis, 1985), diluted 3 - 2 0 / z g / m l , for 15 h at 4°C. The receptors were then transferred to horseradish peroxidase-conjugated anti-rabbit antibodies (Biosys), diluted
156
Fig. 1. Fodrin-immunoreactive euticular plates (CP) were identified in l /.~msections under the light microscope; (A) Section through the long axis of the cells show the strong immunostainingof the CP; (B) Section parallel to the surface of the epithelium showing fodrin immunostaining in the CP and non-im.,n.unoreactive~toplasmic areas (arrows). Bars = 10/zm.
0
•
¢i
Fig. 2. (A) Semithin section of crista ampuilares epithelium showing the immunoreactive extent of the CP laterally along the plasma membranes of the cell, either on one side, or on both the sides (arrows). (B) Electron micrograph of type II hair cell (ceil'indicated by arrow in Fig. 2A showing two immunolabeled striated organelles (arrows). They are separated from the immunopositive CP and in close association with the lateral cell membranes. (C) A labeled striated organelle (arrow) with dark and light bands attached to the CP. (D) UItrathin section through two hair cells, showing a labeled CP and a striated organelle (arrow). (A) Scale bar = 10/~m. (B-D) Ultrathin sections counterstained with lead citrate. Scale bars -- 1 p.m.
157
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,~r
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Fig. 3. (A) and (B) Ultrathin sections of non immunostained normal tissue. (A) The neck of a t~lae ! hair cell contains a striated organelle (arrow) associated with the plasma membrane arid numerous microtubules (thin arrow) nc: nerve calyx; (B) A type !I hair cell showing same arrangement of a striated organelle associated with the plasma membrane; (C), (D) and (E) Immunostained ultrathin sections counterstained with lead citrate; (C) Same view as in Fig.(B) of a strongly immunostained striated organelle; (D) A type I cell with separate labeled CP and labeled striated organelle (arrow);, (E) Probably type I hair cell with labeled CP. Scale' bars = 0.5/tm.
Fig. 4. Ultrathin sections without lead staining showing labelling of cuticular plates and SO with fodrin antibodies in type II (A) and I (B) hair cells.
158
1 : 200, for 2 h at room temperature. Bound peroxidase complexes were visualised by reaction with 0.05% diaminobenzidine (Sigma) and 0.01% hydrogen peroxide in 0.1 M Tris buffer, pH 7.4. The tissues were then postfixed in 1% OsO4, dehydrated and embedded into Epon 812. In control sections, the fodrin antibodies were replaced by non-immune rabbit serum (0.3-1.25 /~g:ml). One-/~m thick sections were used to locate the immunoreactive structures under the light microscope. Consecutive ultrathin sections were collected and stained with lead citrate. Some sections also were examined without lead staining as a control to identify the dark reaction product against a light, unstained, background. Conventional ultrastructural studies were carried out on receptors from two 1 month-old rats that had been perfused intracardially with a mixture of 4% paraformaldehyde/2.5% glutaraldehyde. Ultrathin sections of the receptors were prepared by standard methods.
Results
Fodrin immunoreactivity is clearly confined to the apical pole of the sensory hair cells on 1 /~m semithin sections under the light microscope (Fig. 1A, B). This strong labelling corresponds to the cuticular plates of both type I and type II hair cells identified on the semithin sections counterstained with to!uidine blue. No staining was detected in the stereocilia and in the supporting cells (Fig. 1A). Sections parallel to the surface of the epithelium through the apical portion of sensory cells showed fodrin immunoreactivity in the cuticular plates, with negative areas (Fig. 1B, arrows) corresponding to the cytoplasmic apical areas of the cell such as the area where the kinocilium is implantated (Fig. 1C). Some of hair cells contained an infracuticular immunoreactive structure that seemed to extend from the cuticular plate to the periphery of the cell along one or both sides of the plasma membrane (Fig. 2A). No immunoreactivity was observed on the control sections. Immunoelectron microscopy of fodrin revealed that the intense labelling with antibodies to fodrin is not only confined to the cuticular plates of the two types of hair cells but that SO also showed fodrin immunoreactivity (Figs. 2B, C, D; 3D). Immunoreactive SO were found either separated from the cuticular plate and apposed to the plasma membrane on both sides of the cells (Fig. 2B), or associated with the lower face of the cuticular plate and projecting down toward the base of the cell (Fig. 2C). Their striations, with electron-dense and -light bands, are clearly visible in the Figs. 2C, D. Slightly oblique cross sections of the sensory cells showed immunoreactive SO in close association with
the plasma membrane in type II cells (Figs. 2D, 3C). Immunoreactive SO were also seen in the same areas of type I cells (Fig. 3E). SO were present in most of the type II ceils (Fig. 3B) and many type I cells in electron micrographs of serial sections of the vestibular epithelium not treated by immunocytochemistry. They were continuous with the infracuticular plate region (not shown) or isolated and in close association with the cell membrane, as shown in Fig. 3A. In this type I cell, SO were present in the neck region where it seems to be associated with the dense population of microtubules. The immunoreactive pattern of cuticular plates and SO can be easily detected on sections without staining (Fig. 4).
Discussion
The present results confirm our previous report (Scarfone et al., 1988) of fodrin immunoreactivity in the cuticular plate of the vestibular hair cells. Furthermore, they also show that fodrin immunoreactivity is associated with the infracuticular striated structures commonly described as 'striated organelles'. Fodrin is a member of the spectrin family of rodshaped actin-binding proteins. These proteins are major constituents of the erythrocyte submembraneous skeleton (for reviews see Bennett, 1985; Mangeat, 1988). They are also present in association with actin filaments in several non-erythroid cell types (Rodman et al., 1986; Kobayashi and Hirokawa, 1988; Fujimoto and Ogawa, 1989a,b). They have been shown to crosslink the actin rootlets to each other and to the plasma membrane in the terminal web of the intestinal brush border cells and in gastric parietal cells (Glenney et al., 1983; Hirokawa et al., 1983; Mooseker, 1983; Mizuno et al., 1989). Fodrin, or brain-spectrin, can be associated to the plasma membrane or to intracellular structures as the cytoskeleton; it can also exist free throughout the cytoplasmic matrix of neurons (Fach et al., 1985; Shimo-Oka and Atsumi, 1986; Zagon et al., 1986). Several authors have shown that fodrin can change from a submembraneous cytoskeletal position to a diffuse distribution in the cytoplasmic matrix (Glenney et al., 1982; Carlin e t a ! . , 1983; Perrin et Aunis, 1985; Yoneda et al., 1990). The changes in its iocalisation according to physiological state have been linked to stimuli that would increase the cytosolic calcium concentration (Perrin and Aunis, 1985; Mercier et al., 1989). The cuticular plate of vestibular hair cells, like those of the cochlear hair cells, contains cytoskeletal and contractile proteins such as actin, tropomyosin and myosin (Sobin and Flock, 1983; Drenckhahn et al., 1985; Sans et al., 1989). Their presence suggests the existence of active processes. Shape changes, probably
159
involving a reorganisation of the apical cytockeleton of vestibular hair cells, have been observed (Scarfone et al., 1989) and there is evidence suggesting that these changes can be induced by K + depolarisation (Didier et al., 1990; Zenner et al., 1990; Valat et al., 1991). K + depolarisation also induces a rise in cytosolic calcium concentration in vestibular cells (Ohmori, 1988; Chabert et al., 1991). Thus, changes in the shape of vestibular cells parallel, at least during K + depolarisation, a rise in intracellular Ca ~+ concentration. We have previously suggested that the presence of fodrin in the cuticular plate could signify that the rigidity of this structure is controlled by intracellular C a 2+ concentration via a change in the fodrin affinity for actin (Scarfone et al., 1988). This study demonstrates the presence of fodrin in the infracuticular region of vestibular cells in association with SO. This suggests that SO also could be modified by a rise in intracellular Ca 2+ and thus be involved in such dynamic changes as those that occur during mechano-electric transduction. It has been suggested that the SO in the subapical part of vestibular hair cells are implicated in active mechanisms, because of their structure and arrangement (Ross and Bourne, 1983; Sans~- 1989). As in the cuticular plate, fodrin could be responsible for the Ca2+-dependent cross-linking of the actin filaments of the SO and could also play a role in their interaction with submembraneous skeleton, as SO are frequently observed in close association with the cell membrane. Similarly, SO are known to associale with microtubules (Hoshino, 1975; Jorgensen, 1982) and fodrin can bind to microtubules (Ishikawa et al., 1983; Fach et al., 1985; Shimo-Oka and Atsumi,1986). Hence, fodrin could promote dynamic interactions between SO and microtubules. These later form an important bundle in the neck of vestibular hair cells (Favre and Sans, 1983; Favre et al., 1986) and are implicated in shape changes of the hair cells (Scaffone et al., 1989).
Acknowledgments We wish to thank B. Moniot for her excellent technical assistance and B. Axnaud for photographic work.
References Bennett, V. (1985) The membrane sl~eleton of human erythrocytes and its implications for more complex cells. Ann. Rev. Biochem. 54, 273-304. Carlin R.K., Bartelt, D.C. and Siekevitz, P. (1983) Identification of fodrin as a major calmodnlin-binding protein in postsynaptic density preparationsJ. Cell. Biol. 96, 443-448. Chabbert, C., Devau, G., Sladeczek, F., Lehouelleur, J. and Sans, A. (1991) lntracellular free calcium in isolated vestibular hair cells and potassium iontophoresis. NeuroReport 2, 243-246.
Didier, A, Decory, L. and Cazals, Y. (1990) Evidence for potassiuminduced motility in type I vestibular hair cells. Hear. Res. 46, 171-176. Drenckhahn, D., Sh~ifer, T. and Prinz, M. (1985) Actin, myosin and associated proteins in the vertebrate auditory and vestibular organs. Immunocytochemical and biochemical studies. In: D. Drescher (Ed.), Auditory Biochemistry, Charles C Thomas, Springfield, IL. pp. 312-335. Engstriim, H.,Bergstrom, B. and Ades, H.W. (1972) Macula utriculi and macule saccul'i in the squirrel monkey. Aeta Otolaryngol Suppl. 301, 75. Favre, D. and Sans, A. (1983) Organization and density of microtubules in the vestibular sensory cells in the cat. Acta Otolaryngol. 96, 15-20. Favre, D., Dem~mes, D. and Sans, A. (1986) Mierotubule organization and synaptogenesis in the vestibular sensory cells. Dev. Brain Res. 25, 137-142. Fach, B.L., Graham, S.K., and Keates, R.A.B. (1985) Association of fodrin with brain microtubules. Can. J. Bioehem. Cell Biol. 63, 372-381. Flock, A., Cheung, H.C., Flock, B. and Utter, G. (1981) Three sets of actin filaments in sensory cells of the inner ear. Identification and functional orientation determined by gel electrophoresis, immunofluorescence and electron microscopy. J. Neurocytol. 10, 133-147. Friedman, I., Cawthorne, T., McLay, K. and Bird, E.S. (1963) Electron microscopic observations on "the human membranous labyrinth with particular reference to M6ni6re's disease. J. Ultrastruet. Res. 9, 123-138. Fujimot.o, T. and Ogawa, K. (1989a) Retrieving vesicles in secretioninduced rat chromaffin cells contain fodrin. J. Histochem. Cytochem.37, 1589-1599. Fujimoto, i. and Ogawa, K. (1989b) lmmunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium. J. Histochem. Cytochem. 37, 1345-1352. Glenney, J.R., Glenney, P. and Weber, K. (1983) The spectrin related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of the intestinal epithelial cells. J. Cell Biol. 96, 1491-1496. Hirokawa, N., Cheney, R.E. and Willard, M. (1983) Location of a protein of the spectriu-fodrin-TW 260/240 f~,alily in the mouse intestinal brush border. Cell 32, 953-965. Hoshino, T. (1975) An electron microscopic study of the otolithic maculae of the lamprey (Enthosphenus japonicus). Acta Otolaryngol. 80, 43-53. Ishikawa, M., Murofushi, H. and Sakai, H. (1983) Bundling of microtubules in vitro by fodrin. J. Biochem. 94, 1209-1217. Jorgensen, J.M. (1982) Microtubules and laminated structures in inner ear hair cells. Acta Otolaryngol. 94, 241-248. Kimura, R.S. (1966) Hairs of the cochlear sensory cells and their attachment to the rectorial membrane. Acta Otolaryngol. 61, 55. Kobayashi, N. and Hirokawa, N. (1988) Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell. Cell Motil. Cytoskel. 11, 167-177. Mangeat, P.H. (1988) Interaction of biological membranes with the cytoskeletal framework of living cells. Biol. Cell. 64, 261-281. Mercier, F., Reggio, H., Deviiliers, G. and Bataille, D. (1989) Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of aetin and speetrin upon stimulation of gastric acid secretion. 108, 441-453. Mizuno, M, Fujimoto, T. and Ogawa, K. (1989) Distribution of F-actin, fodrin, and ankyrin in gastric parietal cells of the rat. Acta Histoehem. Cytochem. 22, 593-603. Mooseker, M.S. (1983) Actin binding proteins of the brush border Cell 35, 11-13. Ohmori, H. (1988) Mechanical stimulation and Fura-2 fluorescence in th~. hair bundle of dissociated hair cells of the chick. J. Physiol. 399, 115-137.
160 Perrin, D. and Aunis, D. (1985) Reorganization of fodrin induced by stimulation in secretory cells. Nature (Lond.), 315, 589. Rodman, J.S., Moosker, M. and Farq,thar, M.G. (1986) Cytosqueletal proteins of the rat kidney proximal tubule brush border. Eur. J. Cell Biol. 42, 319-327. Ross, M. (1982) Striated organelles in hair cells of rat inner ear maculas: description and indicaton for transduction. Physiologist 25, Suppl S 113-S 114. Ross, M. and Bourne, C. (1983) Interrelated striated elements in vestibular hair cells of the rat. Science 220, 622-623. Sans, A. (1989) Uitrastructural study of striated organelles in vestibular sensory cells of human fetuses. Anat. Embryol. 179, 457-463. Sans, A., Atger, P., Cavadore, C. and Cavadore, J.C. (1989) Immunocytochemical localization of myosin, tropomyosin and actin in vestibular hair cells of human fetuses and cats. Hear. Res. 40, 117-126. Scarfone, E., Dem~mes, D., Perrin, D., Aunis, D. and Sans, A. (1988) Fodrin (brain spectrin) immunocytochemical localization in vestibular hair cells. Neurosci. Lett. 93, 13-18. Scarfone, E., UIfendahl, M. and Flock, A. (1989) Type I vestibular hair cells undergo microtubules related shape changes. I.E.B. Paris. Abstract. 1989. Shimo-oka, T. and Atsumi, S. (1986) Localization of spectrin in chicken and monkey ventral horns by immunoelectron microscopy. J. Neurocytol. 15, 715-723.
Slepecky, N. and Chamberlain, S.C. (1982) Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea. Cell Tissue Res. 224, 15-24. Slepecloy, N., Hamernik, R.P. and Henderson, D. (1981) The consistent occurrence of a stria!ed organeile (Friedman body) in the inner cells of the normal chinchilla. Acta Otolaryngol. 91, 189198. Sobin, A. and Flock, A. (1983) lmmunohistochemical identification and localization of actin and fimbrin in vestibular hair cells in the normal guinea pig and in a strain of the walzing guinea pig. Acta Otolaryngol. 96,407-412. Spoendlin, H. (1966) The organization of the cochlear receptor. Adv. Oto. Rhino. Laryngol, 13, 19. Valat, J., Griguer, C., Lehouelleur, J. and Sans, A. (1991) Motile responses of isolated guinea pig vestibular hair cells. Neurose. Lett. 127, 231-236. Yoneda, K., Fujimoto, T., Imamura, S. and Ogawa, K. (1990) Distribution of fodrin in the keratinocyte in vivo and in vitro. J.Invest. Dermatol. 94, 724-729. Zenner, H.P., Zimmermann, U. and Gitter, A.H. (1990) Cell poten~'ial and motility of isolated mammalian vestibular sensory cells. Hear. Res. 50, 289-294. Zagon, I.S., Higbee, R. Riederer, B.M. and Goodman, S.R. (1986) Spectrin subtypes in mammalian brain: an immunoelectron microscopic study.J. Neurosc. 6, 2977-2986.
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00 H E A R E S 01769
Fodrin immunocytochemical localization in the striated organelles of the rat vestibular hair cells D. Dem~mes and E. Scarfone INSERM U.254. Laboratoire de Neurophysiologie Sensorielle, Montpellier. France (Received 9 October 1991; Revision received 26 February 1992; Accepted 2 March 1992)
The immunocytochemical distribution of a spectrin-related protein, fodrin was studied at the electron microscopic level in the rat vestib."-lar hair cells. As previously demonstrated [Scarfone et al., Neurosci. Lett. 93, 13-18, 1988], an intense immunoreactivity was found in the cuticular plates. We demonstrate furthermore, here, for the first time the association of foddn immunoreactivity with the striated infracuticular structures called striated organelles (SO). Fodrin was found in striated structures clearly identified as SO in both Type I and Type 1I hair cells. SO were labelled regardless of their location, subcuticular or associated with the plasma membrane of the cells. We suggest that fodrin, as in the cuticular plate, could participate to the Ca -'+ dependent cross-linking of the actin filaments of the striated organelles and could play a role in their interaction with the submembraneous cytoskeleton. Fodrin; Vestibular hair cell; lmmunocytochemical; Distribution; Striated organelles
Introduction
Inner ear sensory cells of human and several animal species contain at the level of the cuticular plate and in the infracuticular region striated structures called striated organelles (SO) (Slepecky et al., 1981; Slepecky and Chamberlain, 1982; Sans, 1989). These SO were found to be structural components of normal hair cells from healthy vestibular and cochlear receptors despite having previously been associated with pathological or ageing phenomena (Friedman et al., 1963; Kimura, 1966; Spoendlin, 1966; Engtr0m et al., 1972). Slepecky and Chamberlain (1982) have demonstrated S-1 myosin decoration of actin filaments in the dense bands of SO situated at the base of the cuticular plate of hair cells in the cochlea of the chinchilla and suggested that SO coud be involved in the active processes of the cochlea. Motile processes associated with sensory transduction have also been proposed to occur in vestibular hair cells and SO, because of their structure and their location at the apex of the cells, have been implicated in them (Ross, 1982; Ross and Bourne, 1983; Sans, 1989) A spectrin-related protein, a fodrin, has recently been found in vestibular end organs by immunoblotting and localised in the cuticular plate of vestibular hair
Correspondance to: DanieHe DemC:mes, INSERM U.254, U.S.T.L.~ Laboratoire de Neurophysiologie Sensorielle, C.P. 089, Place E. Bataillon, 34095 Montpellier Cedex 5, France.
cells by immunofluorescence and immunoelectron microscopy (Scarfone et al., 1988). Cytoskeletal and contractile proteins such as actin (Flock et al., 1981), myosin and troporayosin (Sans et al., 1989) were found in the same area of the sensory cells. We have suggested that fodrin participates in the cuticular plate organisation by cross-linking actin filaments and by anchoring the cuticular plate to the apical membrane (Scarfone et al., 1988). The fact that SO are also partly constituted of actin filaments and are frequently closely associated with the plasma membrane may indicate that these SO may also contain fodfin. We have therefore carried out a thorough ultrastructural study of the immunolocalization of fodrin in the SO of vestibular hair cells,
Material and Methods
Young adult rats were transcardially perfused with freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The vestibular receptors were dissected out, postfixed for 1 h in the same fixative then rinsed in the phosphate buffer. They were immersed twice in acetone at -30°C and then thawed in phosphate buffer. Pre-embedding immunocytochemistry was carried out by incubating the receptors with affinity-purified anti-fodrin antibodies (Perrin and Aunis, 1985), diluted 3 - 2 0 / z g / m l , for 15 h at 4°C. The receptors were then transferred to horseradish peroxidase-conjugated anti-rabbit antibodies (Biosys), diluted
156
Fig. 1. Fodrin-immunoreactive euticular plates (CP) were identified in l /.~msections under the light microscope; (A) Section through the long axis of the cells show the strong immunostainingof the CP; (B) Section parallel to the surface of the epithelium showing fodrin immunostaining in the CP and non-im.,n.unoreactive~toplasmic areas (arrows). Bars = 10/zm.
0
•
¢i
Fig. 2. (A) Semithin section of crista ampuilares epithelium showing the immunoreactive extent of the CP laterally along the plasma membranes of the cell, either on one side, or on both the sides (arrows). (B) Electron micrograph of type II hair cell (ceil'indicated by arrow in Fig. 2A showing two immunolabeled striated organelles (arrows). They are separated from the immunopositive CP and in close association with the lateral cell membranes. (C) A labeled striated organelle (arrow) with dark and light bands attached to the CP. (D) UItrathin section through two hair cells, showing a labeled CP and a striated organelle (arrow). (A) Scale bar = 10/~m. (B-D) Ultrathin sections counterstained with lead citrate. Scale bars -- 1 p.m.
157
~.
,~r
~
Fig. 3. (A) and (B) Ultrathin sections of non immunostained normal tissue. (A) The neck of a t~lae ! hair cell contains a striated organelle (arrow) associated with the plasma membrane arid numerous microtubules (thin arrow) nc: nerve calyx; (B) A type !I hair cell showing same arrangement of a striated organelle associated with the plasma membrane; (C), (D) and (E) Immunostained ultrathin sections counterstained with lead citrate; (C) Same view as in Fig.(B) of a strongly immunostained striated organelle; (D) A type I cell with separate labeled CP and labeled striated organelle (arrow);, (E) Probably type I hair cell with labeled CP. Scale' bars = 0.5/tm.
Fig. 4. Ultrathin sections without lead staining showing labelling of cuticular plates and SO with fodrin antibodies in type II (A) and I (B) hair cells.
158
1 : 200, for 2 h at room temperature. Bound peroxidase complexes were visualised by reaction with 0.05% diaminobenzidine (Sigma) and 0.01% hydrogen peroxide in 0.1 M Tris buffer, pH 7.4. The tissues were then postfixed in 1% OsO4, dehydrated and embedded into Epon 812. In control sections, the fodrin antibodies were replaced by non-immune rabbit serum (0.3-1.25 /~g:ml). One-/~m thick sections were used to locate the immunoreactive structures under the light microscope. Consecutive ultrathin sections were collected and stained with lead citrate. Some sections also were examined without lead staining as a control to identify the dark reaction product against a light, unstained, background. Conventional ultrastructural studies were carried out on receptors from two 1 month-old rats that had been perfused intracardially with a mixture of 4% paraformaldehyde/2.5% glutaraldehyde. Ultrathin sections of the receptors were prepared by standard methods.
Results
Fodrin immunoreactivity is clearly confined to the apical pole of the sensory hair cells on 1 /~m semithin sections under the light microscope (Fig. 1A, B). This strong labelling corresponds to the cuticular plates of both type I and type II hair cells identified on the semithin sections counterstained with to!uidine blue. No staining was detected in the stereocilia and in the supporting cells (Fig. 1A). Sections parallel to the surface of the epithelium through the apical portion of sensory cells showed fodrin immunoreactivity in the cuticular plates, with negative areas (Fig. 1B, arrows) corresponding to the cytoplasmic apical areas of the cell such as the area where the kinocilium is implantated (Fig. 1C). Some of hair cells contained an infracuticular immunoreactive structure that seemed to extend from the cuticular plate to the periphery of the cell along one or both sides of the plasma membrane (Fig. 2A). No immunoreactivity was observed on the control sections. Immunoelectron microscopy of fodrin revealed that the intense labelling with antibodies to fodrin is not only confined to the cuticular plates of the two types of hair cells but that SO also showed fodrin immunoreactivity (Figs. 2B, C, D; 3D). Immunoreactive SO were found either separated from the cuticular plate and apposed to the plasma membrane on both sides of the cells (Fig. 2B), or associated with the lower face of the cuticular plate and projecting down toward the base of the cell (Fig. 2C). Their striations, with electron-dense and -light bands, are clearly visible in the Figs. 2C, D. Slightly oblique cross sections of the sensory cells showed immunoreactive SO in close association with
the plasma membrane in type II cells (Figs. 2D, 3C). Immunoreactive SO were also seen in the same areas of type I cells (Fig. 3E). SO were present in most of the type II ceils (Fig. 3B) and many type I cells in electron micrographs of serial sections of the vestibular epithelium not treated by immunocytochemistry. They were continuous with the infracuticular plate region (not shown) or isolated and in close association with the cell membrane, as shown in Fig. 3A. In this type I cell, SO were present in the neck region where it seems to be associated with the dense population of microtubules. The immunoreactive pattern of cuticular plates and SO can be easily detected on sections without staining (Fig. 4).
Discussion
The present results confirm our previous report (Scarfone et al., 1988) of fodrin immunoreactivity in the cuticular plate of the vestibular hair cells. Furthermore, they also show that fodrin immunoreactivity is associated with the infracuticular striated structures commonly described as 'striated organelles'. Fodrin is a member of the spectrin family of rodshaped actin-binding proteins. These proteins are major constituents of the erythrocyte submembraneous skeleton (for reviews see Bennett, 1985; Mangeat, 1988). They are also present in association with actin filaments in several non-erythroid cell types (Rodman et al., 1986; Kobayashi and Hirokawa, 1988; Fujimoto and Ogawa, 1989a,b). They have been shown to crosslink the actin rootlets to each other and to the plasma membrane in the terminal web of the intestinal brush border cells and in gastric parietal cells (Glenney et al., 1983; Hirokawa et al., 1983; Mooseker, 1983; Mizuno et al., 1989). Fodrin, or brain-spectrin, can be associated to the plasma membrane or to intracellular structures as the cytoskeleton; it can also exist free throughout the cytoplasmic matrix of neurons (Fach et al., 1985; Shimo-Oka and Atsumi, 1986; Zagon et al., 1986). Several authors have shown that fodrin can change from a submembraneous cytoskeletal position to a diffuse distribution in the cytoplasmic matrix (Glenney et al., 1982; Carlin e t a ! . , 1983; Perrin et Aunis, 1985; Yoneda et al., 1990). The changes in its iocalisation according to physiological state have been linked to stimuli that would increase the cytosolic calcium concentration (Perrin and Aunis, 1985; Mercier et al., 1989). The cuticular plate of vestibular hair cells, like those of the cochlear hair cells, contains cytoskeletal and contractile proteins such as actin, tropomyosin and myosin (Sobin and Flock, 1983; Drenckhahn et al., 1985; Sans et al., 1989). Their presence suggests the existence of active processes. Shape changes, probably
159
involving a reorganisation of the apical cytockeleton of vestibular hair cells, have been observed (Scarfone et al., 1989) and there is evidence suggesting that these changes can be induced by K + depolarisation (Didier et al., 1990; Zenner et al., 1990; Valat et al., 1991). K + depolarisation also induces a rise in cytosolic calcium concentration in vestibular cells (Ohmori, 1988; Chabert et al., 1991). Thus, changes in the shape of vestibular cells parallel, at least during K + depolarisation, a rise in intracellular Ca ~+ concentration. We have previously suggested that the presence of fodrin in the cuticular plate could signify that the rigidity of this structure is controlled by intracellular C a 2+ concentration via a change in the fodrin affinity for actin (Scarfone et al., 1988). This study demonstrates the presence of fodrin in the infracuticular region of vestibular cells in association with SO. This suggests that SO also could be modified by a rise in intracellular Ca 2+ and thus be involved in such dynamic changes as those that occur during mechano-electric transduction. It has been suggested that the SO in the subapical part of vestibular hair cells are implicated in active mechanisms, because of their structure and arrangement (Ross and Bourne, 1983; Sans~- 1989). As in the cuticular plate, fodrin could be responsible for the Ca2+-dependent cross-linking of the actin filaments of the SO and could also play a role in their interaction with submembraneous skeleton, as SO are frequently observed in close association with the cell membrane. Similarly, SO are known to associale with microtubules (Hoshino, 1975; Jorgensen, 1982) and fodrin can bind to microtubules (Ishikawa et al., 1983; Fach et al., 1985; Shimo-Oka and Atsumi,1986). Hence, fodrin could promote dynamic interactions between SO and microtubules. These later form an important bundle in the neck of vestibular hair cells (Favre and Sans, 1983; Favre et al., 1986) and are implicated in shape changes of the hair cells (Scaffone et al., 1989).
Acknowledgments We wish to thank B. Moniot for her excellent technical assistance and B. Axnaud for photographic work.
References Bennett, V. (1985) The membrane sl~eleton of human erythrocytes and its implications for more complex cells. Ann. Rev. Biochem. 54, 273-304. Carlin R.K., Bartelt, D.C. and Siekevitz, P. (1983) Identification of fodrin as a major calmodnlin-binding protein in postsynaptic density preparationsJ. Cell. Biol. 96, 443-448. Chabbert, C., Devau, G., Sladeczek, F., Lehouelleur, J. and Sans, A. (1991) lntracellular free calcium in isolated vestibular hair cells and potassium iontophoresis. NeuroReport 2, 243-246.
Didier, A, Decory, L. and Cazals, Y. (1990) Evidence for potassiuminduced motility in type I vestibular hair cells. Hear. Res. 46, 171-176. Drenckhahn, D., Sh~ifer, T. and Prinz, M. (1985) Actin, myosin and associated proteins in the vertebrate auditory and vestibular organs. Immunocytochemical and biochemical studies. In: D. Drescher (Ed.), Auditory Biochemistry, Charles C Thomas, Springfield, IL. pp. 312-335. Engstriim, H.,Bergstrom, B. and Ades, H.W. (1972) Macula utriculi and macule saccul'i in the squirrel monkey. Aeta Otolaryngol Suppl. 301, 75. Favre, D. and Sans, A. (1983) Organization and density of microtubules in the vestibular sensory cells in the cat. Acta Otolaryngol. 96, 15-20. Favre, D., Dem~mes, D. and Sans, A. (1986) Mierotubule organization and synaptogenesis in the vestibular sensory cells. Dev. Brain Res. 25, 137-142. Fach, B.L., Graham, S.K., and Keates, R.A.B. (1985) Association of fodrin with brain microtubules. Can. J. Bioehem. Cell Biol. 63, 372-381. Flock, A., Cheung, H.C., Flock, B. and Utter, G. (1981) Three sets of actin filaments in sensory cells of the inner ear. Identification and functional orientation determined by gel electrophoresis, immunofluorescence and electron microscopy. J. Neurocytol. 10, 133-147. Friedman, I., Cawthorne, T., McLay, K. and Bird, E.S. (1963) Electron microscopic observations on "the human membranous labyrinth with particular reference to M6ni6re's disease. J. Ultrastruet. Res. 9, 123-138. Fujimot.o, T. and Ogawa, K. (1989a) Retrieving vesicles in secretioninduced rat chromaffin cells contain fodrin. J. Histochem. Cytochem.37, 1589-1599. Fujimoto, i. and Ogawa, K. (1989b) lmmunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium. J. Histochem. Cytochem. 37, 1345-1352. Glenney, J.R., Glenney, P. and Weber, K. (1983) The spectrin related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of the intestinal epithelial cells. J. Cell Biol. 96, 1491-1496. Hirokawa, N., Cheney, R.E. and Willard, M. (1983) Location of a protein of the spectriu-fodrin-TW 260/240 f~,alily in the mouse intestinal brush border. Cell 32, 953-965. Hoshino, T. (1975) An electron microscopic study of the otolithic maculae of the lamprey (Enthosphenus japonicus). Acta Otolaryngol. 80, 43-53. Ishikawa, M., Murofushi, H. and Sakai, H. (1983) Bundling of microtubules in vitro by fodrin. J. Biochem. 94, 1209-1217. Jorgensen, J.M. (1982) Microtubules and laminated structures in inner ear hair cells. Acta Otolaryngol. 94, 241-248. Kimura, R.S. (1966) Hairs of the cochlear sensory cells and their attachment to the rectorial membrane. Acta Otolaryngol. 61, 55. Kobayashi, N. and Hirokawa, N. (1988) Cytoskeletal architecture and immunocytochemical localization of fodrin in the terminal web of the ciliated epithelial cell. Cell Motil. Cytoskel. 11, 167-177. Mangeat, P.H. (1988) Interaction of biological membranes with the cytoskeletal framework of living cells. Biol. Cell. 64, 261-281. Mercier, F., Reggio, H., Deviiliers, G. and Bataille, D. (1989) Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of aetin and speetrin upon stimulation of gastric acid secretion. 108, 441-453. Mizuno, M, Fujimoto, T. and Ogawa, K. (1989) Distribution of F-actin, fodrin, and ankyrin in gastric parietal cells of the rat. Acta Histoehem. Cytochem. 22, 593-603. Mooseker, M.S. (1983) Actin binding proteins of the brush border Cell 35, 11-13. Ohmori, H. (1988) Mechanical stimulation and Fura-2 fluorescence in th~. hair bundle of dissociated hair cells of the chick. J. Physiol. 399, 115-137.
160 Perrin, D. and Aunis, D. (1985) Reorganization of fodrin induced by stimulation in secretory cells. Nature (Lond.), 315, 589. Rodman, J.S., Moosker, M. and Farq,thar, M.G. (1986) Cytosqueletal proteins of the rat kidney proximal tubule brush border. Eur. J. Cell Biol. 42, 319-327. Ross, M. (1982) Striated organelles in hair cells of rat inner ear maculas: description and indicaton for transduction. Physiologist 25, Suppl S 113-S 114. Ross, M. and Bourne, C. (1983) Interrelated striated elements in vestibular hair cells of the rat. Science 220, 622-623. Sans, A. (1989) Uitrastructural study of striated organelles in vestibular sensory cells of human fetuses. Anat. Embryol. 179, 457-463. Sans, A., Atger, P., Cavadore, C. and Cavadore, J.C. (1989) Immunocytochemical localization of myosin, tropomyosin and actin in vestibular hair cells of human fetuses and cats. Hear. Res. 40, 117-126. Scarfone, E., Dem~mes, D., Perrin, D., Aunis, D. and Sans, A. (1988) Fodrin (brain spectrin) immunocytochemical localization in vestibular hair cells. Neurosci. Lett. 93, 13-18. Scarfone, E., UIfendahl, M. and Flock, A. (1989) Type I vestibular hair cells undergo microtubules related shape changes. I.E.B. Paris. Abstract. 1989. Shimo-oka, T. and Atsumi, S. (1986) Localization of spectrin in chicken and monkey ventral horns by immunoelectron microscopy. J. Neurocytol. 15, 715-723.
Slepecky, N. and Chamberlain, S.C. (1982) Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea. Cell Tissue Res. 224, 15-24. Slepecloy, N., Hamernik, R.P. and Henderson, D. (1981) The consistent occurrence of a stria!ed organeile (Friedman body) in the inner cells of the normal chinchilla. Acta Otolaryngol. 91, 189198. Sobin, A. and Flock, A. (1983) lmmunohistochemical identification and localization of actin and fimbrin in vestibular hair cells in the normal guinea pig and in a strain of the walzing guinea pig. Acta Otolaryngol. 96,407-412. Spoendlin, H. (1966) The organization of the cochlear receptor. Adv. Oto. Rhino. Laryngol, 13, 19. Valat, J., Griguer, C., Lehouelleur, J. and Sans, A. (1991) Motile responses of isolated guinea pig vestibular hair cells. Neurose. Lett. 127, 231-236. Yoneda, K., Fujimoto, T., Imamura, S. and Ogawa, K. (1990) Distribution of fodrin in the keratinocyte in vivo and in vitro. J.Invest. Dermatol. 94, 724-729. Zenner, H.P., Zimmermann, U. and Gitter, A.H. (1990) Cell poten~'ial and motility of isolated mammalian vestibular sensory cells. Hear. Res. 50, 289-294. Zagon, I.S., Higbee, R. Riederer, B.M. and Goodman, S.R. (1986) Spectrin subtypes in mammalian brain: an immunoelectron microscopic study.J. Neurosc. 6, 2977-2986.