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An ultrastructural study of the guinea pig tectorial membrane ‘type A’ protofibril
289
Hearing Research, 46 (1990) 289-292 Elsevier
HEARES 01386
Short Communication
An ultrastructural
study of the guinea pig tectorial membrane ‘ type A’ protofibril
Toshio Arima lv3,David J. Lim 3, Hiroshi Kawaguchi I, Yosaburo Shibata * and Takuya Uemura 1 Departments of ’ Otolaryngology and 2 Anatomy, Faculty of Medicine. Kyushu University, Fukuoka, Japan and 3 Otological Research Laboratories, Ohio State University school of Medicine, Columbus, Ohio, U.S.A. (Received 13 November 1989; accepted 14 February 1990)
Fine structural features of the ‘type A’ protofibrils in the guinea pig tectorial membrane were examined using negative staining and deep-etching techniques. Negative-stained samples of fragmented tectorial membrane were composed of several fine filamentous subunits showing the clear banding pattern of the type A protofibrils. Deep-etched replicas of the EGTA (ethylene glycol bis-N,N,N’,N’-tetraacetic acid)-treated samples showed fine surface structure consisting of several linear arrays of filamentous elements on the extracellular fibrils, which is interpreted to be type A protofibrils. Tectorial membrane; Collagen, type II; Ear, inner
Although the biochemical properties of the tectorial membrane have been well-investigated, only recently, Thalmann et al. (1987) reported that the major protein component of the tectorial membrane is type II collagen. Richardson et al. (1987) further showed that the tectorial membrane was composed of collagens and non-collagenous polypeptides, and also demonstrated the presence of three types of collagen, type II, IX and V, using both immunoblotting and immunohistochemical techniques. Khalkhali-Ellis (1987) showed the presence of glycoprotein in the tectorial membrane. A previous ultrastructural study of the tectorial membrane described the two types of protofibrils; type A and B (Kronester-Frei, 1978). Type A protofibrils are lo-11 nm diameter filaments with straight, unbranched appearance and type B protofibrils are described as branched and coiled
Correspondence to: Toshio Arima, Department of Otolaryngology, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Fukuoka 812, Japan. 0378-5955/90/$03.50
with a diameter of 15-20 nm. Using deep-etching methods, Thalmann et al. (1987) compared the fine structural features of the tectorial membrane to the bovine elastic cartilage and found structural similarities between the type A protofibril and the type II collagen and between the Type B protofibril and the proteoglycan matrix, respectively. Hasko and Richardson (1988) described non-collageous components, called striated sheet, corresponding to the type B protofibril and also showed that the type A protofibrils were digested with bacterial collagenase. However, the fine structure and the definite nature of the type A protofibril are still unclear. In the present study, we examined the ultrastructural features of the extracellular fibrils in the tectorial membrane, using nagative staining and rapid-freeze, deep-etching techniques. Our observations demonstrated that the fine fibrils, corresponding to the type A protofibrils, in the tectorial membrane had a clear banding pattern and showed a substructure which consisted of several fine filamentous elements. These structural fea-
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
290
Fig. 1. A negatively
Fig. 2. A deep-etched
stained
sample of fragmented tectorial fine filamentous
membranes. Each fibril has clear banding elements (arrows). x 227000.
pattern
and consists
of several
replica of the tectorial membrane which was treated with 3 mM EGTA. Note that some fibrils have several fine linear arrays on their surfaces (arrowheads). x 192000.
291
tures are identical to those of the type II collagen fibril found in the matrix of cartilage (Vaughan et al., 1988; Mendler et al., 1989). Adult guinea pigs with normal Preyer’s reflex were used in this study. For negative staining, fresh tectorial membranes were removed, collected in a 5-ml syringe containing phosphate-buffered saline (PBS) and dissociated by passing the solution several times through a 26-gauge needle to make small fragments of the tectorial membranes (Arima et al., 1985). The small fragments were mounted on collodion-coated 300-mesh copper grids and negatively stained with 4% uranyl acetate. For freeze-etching studies, temporal bones were dissected out and co&leas were treated with 1% triton X-100 in Hepes buffer (100 mM KCl, 5 mM MgCl,, 3mM EGTA 30 mM Hepes, pH 7.2) for 10 min and fixed with 2.5% glutaraldehyde in the same buffer for 60 min. The treated organ of Corti was removed, mounted on slices of fixed lung placed on copper specimen holder disks, frozen-rapidly using liquid helium (Arima et al., 1987). The frozen tissues were cut with a razor blade, deeply-etched for 5 min at -95°C at 0.120.18 X 10e3 Pa, rotary-shadowed with platinum by double-axis rotary shadowing (Shibata et al., 1983) and backed with carbon at an angle of 90 O. After shadowing, tissues were dissolved in household bleach containing sodium hypochlorite. The replicas were mounted on 300-mesh grids and examined in a JeoI 100s electron microscope at 100 kV. A photo of the deep-etch replica was printed in reverse contrast. Negatively stained samples of fragmented tectorial membranes showed the type A fibrils (12-14 run in diameter) with clear banding patterns. The banding periodicity was lo-12 nm. Each fibril consisted of several fine filaments (Fig. 1). Deepetch replicas of the tectorial membrane sometimes showed the several linear filamentous arrays on the surface of the fine fibrils in the tectorial membrane. The diameter of the fibrils in the replicas was 17-18 run (Fig. 2). Since a rotary replication technique for deep-etch replicas usually increases the diameter of filaments, 12-14 MI was the true diameter of the type A protofibril. The presence of type II collagen in the tectorial membrane has been reported, using immunohistochemical (Tomoda et al., 1984) and biochemical
techniques (Thalmann et al., 1987; Richardson et al., 1987), and it has been suggested that fibrils corresponding to the type A protofibril may be type II collagen. In the present study, we demonstrated the clear banding pattern and the substructure consisted of several filamentous elements of the type A protofhrils. Considering imrntmohistochemical and biochemical data, it is most likely that the fine fibrils having clear banding pattern in the tectorial membrane are type II collagen fibrils. Thalmann et al. (1987) examined fresh, nontreated tectorial membrane using rapid-freeze, deep-etch techniques, and described the fine structure of the fibrous elements. However, they failed to detect the substructure of the major fibrils. In the present study, we used the samples treated with 3 mM EGTA for deep-etching study and demonstrated the substructure consisted of several linear arrays on the surface of the major fibrils in the tectorial membrane. Hasko and Richardson (1988) reported that the EGTA-treatment caused the disorganization of the non-collagenous components in the tectorial membrane. In the fresh, non-treated samples, we were unable to detect the substructure of the major fibrils. In this study, a reduction of non-collageous matrix by EGTAtreatment was useful to detect the substructure of the fibrils. Morphologically similar fibrils have also been described in the connective tissues of the vestibule (Hamilton, 1967), spiral ligament (Kimura and Schuknecht, 1970), and the basilar membrane (Cabezudo, 1978). Tomoda et al. (1984) demonstrated the presence of type II collagen in various portions in the inner ear including the tectorial membrane, using immunohistochemical techniques. Considering above two observations, such fine extracellular fibrils in the various portions in the inner ear could possibly be type II collagen. Acknowledgments This study was supportd by a grant from Deafness Research Foundation and by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 01771346).
292
References Arima, T., Shibata, Y. and Yamamoto, T. (1985) Three-dimensional visualization of basal body structures and some cytoskeletal components in the apical zone of tracheal ciliated cells. J. Ultrastruct. Res. 93 61-70. Arima, T., Uemura, T. and Yamamoto, T. (1987) Three-dimensional visualization of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membraneous organelles. Hear. Res. 25, 61-68. Cabezudo L.M. (1978) The ultrastructure of the basilar membrane in the cat. Acta Otolaringol. 86, 160-175. Hamilton, D.W. (1967) Perilymphatic fibrocytes in the vestibule of the inner ear. Anat. Res. 157, 627-639. Hasko, J.A., Richardson, G.P. (1988) The ultrastructural organization and properties of the mouse tectorial membrane matrix. Hear. Res. 35, 21-38. Khalkhali-Ellis, Z., Hemming, F.W. and Steel, K.P. (1987) Glycoconjugates in the tectorial membrane. Hear. Res. 25. 185-191. Kimura, R.S. and Schuknecht, H.F. (1970) The ultrastructure of the human stria vascularis. Part I. Acta Otolaryngol. 69, 415-427.
Kronester-Frei, A. (1978) Ultrastructure of the different zones of the tectorial membrane. Cell Tissue Res. 193, 11-23. Mendler, M. Eich-Bender, S.G., Vaughan, L., Winterhalter. K.H. and Bruckner, P. (1989) Cartilage contains mixed fibrils of collagen types II, IX, and XI. J. Cell Biol. 108. 191-197. Richardson G.P., Russel. I.J., Duance, V.C. and Bailey, A.J. (1987) Polypeptide composition of the mammalian tectorial membrane. Hear. Res. 25, 45-60. Shibata, Y., Arima, T. and Yamamoto, T. (1983) Double-axis rotary replication for deep-etching. J. Microsc. 136, 121123. Thahnann, I., Thallinger, G., Crouch, E.C.. Comegys, T.H.. Barrett, T. and Thahnann, R. (1987) Composition and supramolecular organization of the tcctorial membrane. Laryngoscope 97, 357-367. Tomoda, K., Yamashita. T., Kumazawa, T. and Yoo, T.J. (1984) Type II collagen distribution in the middle and inner ear: Immunohistochemical studies. Ear Res. Jpn. 15, 199202. Vaughan, L., Mendler, M., Huber, S., Bruckner, P., Winterhalter, K.H.. Irwin, MI. and Mayne, R. (1988) D-periodic distribution of collagen type IX along cartilage fib&. J. Cell Biol. 106, 991-997.
Hearing Research, 46 (1990) 289-292 Elsevier
HEARES 01386
Short Communication
An ultrastructural
study of the guinea pig tectorial membrane ‘ type A’ protofibril
Toshio Arima lv3,David J. Lim 3, Hiroshi Kawaguchi I, Yosaburo Shibata * and Takuya Uemura 1 Departments of ’ Otolaryngology and 2 Anatomy, Faculty of Medicine. Kyushu University, Fukuoka, Japan and 3 Otological Research Laboratories, Ohio State University school of Medicine, Columbus, Ohio, U.S.A. (Received 13 November 1989; accepted 14 February 1990)
Fine structural features of the ‘type A’ protofibrils in the guinea pig tectorial membrane were examined using negative staining and deep-etching techniques. Negative-stained samples of fragmented tectorial membrane were composed of several fine filamentous subunits showing the clear banding pattern of the type A protofibrils. Deep-etched replicas of the EGTA (ethylene glycol bis-N,N,N’,N’-tetraacetic acid)-treated samples showed fine surface structure consisting of several linear arrays of filamentous elements on the extracellular fibrils, which is interpreted to be type A protofibrils. Tectorial membrane; Collagen, type II; Ear, inner
Although the biochemical properties of the tectorial membrane have been well-investigated, only recently, Thalmann et al. (1987) reported that the major protein component of the tectorial membrane is type II collagen. Richardson et al. (1987) further showed that the tectorial membrane was composed of collagens and non-collagenous polypeptides, and also demonstrated the presence of three types of collagen, type II, IX and V, using both immunoblotting and immunohistochemical techniques. Khalkhali-Ellis (1987) showed the presence of glycoprotein in the tectorial membrane. A previous ultrastructural study of the tectorial membrane described the two types of protofibrils; type A and B (Kronester-Frei, 1978). Type A protofibrils are lo-11 nm diameter filaments with straight, unbranched appearance and type B protofibrils are described as branched and coiled
Correspondence to: Toshio Arima, Department of Otolaryngology, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Fukuoka 812, Japan. 0378-5955/90/$03.50
with a diameter of 15-20 nm. Using deep-etching methods, Thalmann et al. (1987) compared the fine structural features of the tectorial membrane to the bovine elastic cartilage and found structural similarities between the type A protofibril and the type II collagen and between the Type B protofibril and the proteoglycan matrix, respectively. Hasko and Richardson (1988) described non-collageous components, called striated sheet, corresponding to the type B protofibril and also showed that the type A protofibrils were digested with bacterial collagenase. However, the fine structure and the definite nature of the type A protofibril are still unclear. In the present study, we examined the ultrastructural features of the extracellular fibrils in the tectorial membrane, using nagative staining and rapid-freeze, deep-etching techniques. Our observations demonstrated that the fine fibrils, corresponding to the type A protofibrils, in the tectorial membrane had a clear banding pattern and showed a substructure which consisted of several fine filamentous elements. These structural fea-
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
290
Fig. 1. A negatively
Fig. 2. A deep-etched
stained
sample of fragmented tectorial fine filamentous
membranes. Each fibril has clear banding elements (arrows). x 227000.
pattern
and consists
of several
replica of the tectorial membrane which was treated with 3 mM EGTA. Note that some fibrils have several fine linear arrays on their surfaces (arrowheads). x 192000.
291
tures are identical to those of the type II collagen fibril found in the matrix of cartilage (Vaughan et al., 1988; Mendler et al., 1989). Adult guinea pigs with normal Preyer’s reflex were used in this study. For negative staining, fresh tectorial membranes were removed, collected in a 5-ml syringe containing phosphate-buffered saline (PBS) and dissociated by passing the solution several times through a 26-gauge needle to make small fragments of the tectorial membranes (Arima et al., 1985). The small fragments were mounted on collodion-coated 300-mesh copper grids and negatively stained with 4% uranyl acetate. For freeze-etching studies, temporal bones were dissected out and co&leas were treated with 1% triton X-100 in Hepes buffer (100 mM KCl, 5 mM MgCl,, 3mM EGTA 30 mM Hepes, pH 7.2) for 10 min and fixed with 2.5% glutaraldehyde in the same buffer for 60 min. The treated organ of Corti was removed, mounted on slices of fixed lung placed on copper specimen holder disks, frozen-rapidly using liquid helium (Arima et al., 1987). The frozen tissues were cut with a razor blade, deeply-etched for 5 min at -95°C at 0.120.18 X 10e3 Pa, rotary-shadowed with platinum by double-axis rotary shadowing (Shibata et al., 1983) and backed with carbon at an angle of 90 O. After shadowing, tissues were dissolved in household bleach containing sodium hypochlorite. The replicas were mounted on 300-mesh grids and examined in a JeoI 100s electron microscope at 100 kV. A photo of the deep-etch replica was printed in reverse contrast. Negatively stained samples of fragmented tectorial membranes showed the type A fibrils (12-14 run in diameter) with clear banding patterns. The banding periodicity was lo-12 nm. Each fibril consisted of several fine filaments (Fig. 1). Deepetch replicas of the tectorial membrane sometimes showed the several linear filamentous arrays on the surface of the fine fibrils in the tectorial membrane. The diameter of the fibrils in the replicas was 17-18 run (Fig. 2). Since a rotary replication technique for deep-etch replicas usually increases the diameter of filaments, 12-14 MI was the true diameter of the type A protofibril. The presence of type II collagen in the tectorial membrane has been reported, using immunohistochemical (Tomoda et al., 1984) and biochemical
techniques (Thalmann et al., 1987; Richardson et al., 1987), and it has been suggested that fibrils corresponding to the type A protofibril may be type II collagen. In the present study, we demonstrated the clear banding pattern and the substructure consisted of several filamentous elements of the type A protofhrils. Considering imrntmohistochemical and biochemical data, it is most likely that the fine fibrils having clear banding pattern in the tectorial membrane are type II collagen fibrils. Thalmann et al. (1987) examined fresh, nontreated tectorial membrane using rapid-freeze, deep-etch techniques, and described the fine structure of the fibrous elements. However, they failed to detect the substructure of the major fibrils. In the present study, we used the samples treated with 3 mM EGTA for deep-etching study and demonstrated the substructure consisted of several linear arrays on the surface of the major fibrils in the tectorial membrane. Hasko and Richardson (1988) reported that the EGTA-treatment caused the disorganization of the non-collagenous components in the tectorial membrane. In the fresh, non-treated samples, we were unable to detect the substructure of the major fibrils. In this study, a reduction of non-collageous matrix by EGTAtreatment was useful to detect the substructure of the fibrils. Morphologically similar fibrils have also been described in the connective tissues of the vestibule (Hamilton, 1967), spiral ligament (Kimura and Schuknecht, 1970), and the basilar membrane (Cabezudo, 1978). Tomoda et al. (1984) demonstrated the presence of type II collagen in various portions in the inner ear including the tectorial membrane, using immunohistochemical techniques. Considering above two observations, such fine extracellular fibrils in the various portions in the inner ear could possibly be type II collagen. Acknowledgments This study was supportd by a grant from Deafness Research Foundation and by a Grantin-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 01771346).
292
References Arima, T., Shibata, Y. and Yamamoto, T. (1985) Three-dimensional visualization of basal body structures and some cytoskeletal components in the apical zone of tracheal ciliated cells. J. Ultrastruct. Res. 93 61-70. Arima, T., Uemura, T. and Yamamoto, T. (1987) Three-dimensional visualization of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membraneous organelles. Hear. Res. 25, 61-68. Cabezudo L.M. (1978) The ultrastructure of the basilar membrane in the cat. Acta Otolaringol. 86, 160-175. Hamilton, D.W. (1967) Perilymphatic fibrocytes in the vestibule of the inner ear. Anat. Res. 157, 627-639. Hasko, J.A., Richardson, G.P. (1988) The ultrastructural organization and properties of the mouse tectorial membrane matrix. Hear. Res. 35, 21-38. Khalkhali-Ellis, Z., Hemming, F.W. and Steel, K.P. (1987) Glycoconjugates in the tectorial membrane. Hear. Res. 25. 185-191. Kimura, R.S. and Schuknecht, H.F. (1970) The ultrastructure of the human stria vascularis. Part I. Acta Otolaryngol. 69, 415-427.
Kronester-Frei, A. (1978) Ultrastructure of the different zones of the tectorial membrane. Cell Tissue Res. 193, 11-23. Mendler, M. Eich-Bender, S.G., Vaughan, L., Winterhalter. K.H. and Bruckner, P. (1989) Cartilage contains mixed fibrils of collagen types II, IX, and XI. J. Cell Biol. 108. 191-197. Richardson G.P., Russel. I.J., Duance, V.C. and Bailey, A.J. (1987) Polypeptide composition of the mammalian tectorial membrane. Hear. Res. 25, 45-60. Shibata, Y., Arima, T. and Yamamoto, T. (1983) Double-axis rotary replication for deep-etching. J. Microsc. 136, 121123. Thahnann, I., Thallinger, G., Crouch, E.C.. Comegys, T.H.. Barrett, T. and Thahnann, R. (1987) Composition and supramolecular organization of the tcctorial membrane. Laryngoscope 97, 357-367. Tomoda, K., Yamashita. T., Kumazawa, T. and Yoo, T.J. (1984) Type II collagen distribution in the middle and inner ear: Immunohistochemical studies. Ear Res. Jpn. 15, 199202. Vaughan, L., Mendler, M., Huber, S., Bruckner, P., Winterhalter, K.H.. Irwin, MI. and Mayne, R. (1988) D-periodic distribution of collagen type IX along cartilage fib&. J. Cell Biol. 106, 991-997.