ARTICLE |
Correspondence to: Vladimir Tsuprun, University of Minnesota, Rm. 109, Lions Research Bldg., 2001 Sixth St. SE, Minneapolis, MN 55455. E-mail: tsupr001@tc.umn.edu
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Summary |
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The structure and symmetry of chinchilla outer hair cell (OHC) stereocilia side and attachment links were investigated by transmission electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. The side links run laterally between and across the rows of the stereocilia and connect the stereocilia together within the bundle. Attachment links form a crown-like array around the tips of only the tallest OHC stereocilia and attach these stereocilia to the Type B fibrils of the tectorial membrane. Computer averaging of the side links from tannic acid-treated tissues showed a central dense region of the link between adjacent stereocilia and a smaller dense portion at the plasma membrane end of the link. Computer averaging of Cuprolinic blue-treated tissues showed low electron density of the central region of the link, and the plasma membrane ends of the link were electron dense. After tannic acid treatment, the attachment links showed a diffused radial distribution around the tips of the tallest OHC stereocilia. After Cuprolinic blue treatment, the attachment links appeared as electron-dense, membrane-bound granular structures arranged with radial symmetry. The central regions of the side links are reactive to tannic acid. These regions appear to contain neutral and basic residues of proteins and participate in side-by-side association of the side links in regular aggregates. Cuprolinic blue-reactive regions of the side and attachment links appear to contain acidic sulfated residues of glycoproteins or proteoglycans, which may be involved in the attachment of these links to the stereocilium membrane. (J Histochem Cytochem 50:493502, 2002)
Key Words: stereocilia links, transmission electron, microscopy, tannic acid, cuprolinic blue, image analysis
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Introduction |
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Deflection of hair cell stereocilia appears to result in the opening of mechano-electrical transduction channels (for review see
OHC stereocilia in mammals are arranged in bundles with pseudo-hexagonal symmetry in each bundle and are connected with several types of crosslinks. There are three types of extracellular links in OHC stereocilia (
The second set of stereocilia links is the side links, which run laterally between and across the rows of the stereocilia and connect the stereocilia to one another to maintain the morphological integrity of the bundle (
A third type of the link is called the attachment link (
The molecular composition of the extracellular components associated with the mammalian stereocilia is poorly understood. The plasma membrane of the stereocilium is covered with a coat of evenly distributed glycoconjugates (
The purpose of the present work was to investigate the structure, distribution, and symmetry of the stereocilia side and attachment links in chinchilla OHC and the relationship between the structural organization of these links and OHC stereocilia properties. TEM using tannic acid and Cuprolinic blue histochemical procedures and computer image analysis were used in this study.
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Materials and Methods |
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Samples of cochlear tissue were obtained from our tissue bank from normal chinchillas approximately 1 year old. The animals were deeply anesthetized with 0.2 ml ketamine/xylazine and then sacrificed by decapitation. Cochleas were removed from temporal bones and fixed by perfusion via round and oval windows. The care and use of the animals for this study were reviewed and approved by the University of Minnesota's Institutional Animal Care Committee.
Tannic acid and Cuprolinic blue treatments were used for a TEM study to obtain histological and structural information on the OHC stereocilia links. Tannic acid reacts with proteins irrespective of their electrical charge (
Tannic acid treatment was used as previously described (
Cochlear sections were examined using a JEOL 1010 electron microscope at 60 kV and at magnifications of x500015,000. Prints of micrographs were digitized using a flatbed scanner with a pixel size of 0.6 nm at specimen level. Image analysis was carried out on an SGI Onyx computer using SPIDER (System for Processing Image Data in Electron Microscopy) software. This software system was developed by Frank and co-workers and has been used for 2D and 3D image analysis of a large number of biological macromolecules in many laboratories (
Some micrographs show adjacent stereocilia that contain images of the side links in the same projection in the section. Because of image noise, the projected structure of the link cannot be determined visually from these micrographs with high reliability. The averaging of a number of repeated noisy images of the side links improves the signalnoise ratio to visualize more objectively its structural details. Individual links between stereocilia can deviate from ideal periodic lattice positions or have some changes in the rotational orientation, and their images must be aligned by relative translation and rotation before averaging. The procedure that determines shifts and angles and, accordingly, brings two particles into a common position of alignment using SPIDER software, uses the cross-correlation function for translational alignment and auto-correlation function for orientational alignment (
Image fragments of the same size (more than one period), containing individual links quasi-regularly distributed between adjacent stereocilia, were selected interactively for averaging from the images displayed on the monitor screen. Before averaging a circular mask was imposed on selected fragments to remove unnecessary background, and each selected image was floated within this mask using the average density and Gaussian filter (
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Results |
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Side Links
Longitudinal sections along a row of OHC stereocilia treated with tannic acid, show discrete bands of side links between adjacent stereocilia, and some diffuse material is visible over the stereocilia tips (Fig 1a). Fig 1b shows a horizontal section through a bundle of streocilia. An electron-dense core of compact actin filaments, called the rootlet (as they insert into cuticular plate), was observed only in the stereocilia corresponding to the longest row of the bundle in which the stereocilia have the longest rootlets. However, spot-like projections of actin filaments in each stereocilium indicate that this section crosses the stereocilia perpendicular to their axes. In horizontal sections, the side links formed a pseudo-hexagonal lattice. The length of the links varied and corresponded most frequently to the minimal distance between surfaces of adjacent stereocilia, as shown by arrows in Fig 1b. Some of the side-link strands projecting from the cell membrane have free ends and did not appear to connect to adjacent stereocilia. Most of the side links are composed of one to three strands, each containing central globular domains about 20 nm in diameter. High-power images show that these domains contact each other between adjacent stereocilia (Fig 1c and Fig 1d) or sometimes form linear aggregates (Fig 1d).
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Tip, side, and attachment links can also be observed in the longitudinal sections crossing the rows of stereocilia after Cuprolinic blue treatment (Fig 2a). Cuprolinic blue treatment provided an inverse staining pattern of the side links compared to the tannic acid treatment (Fig 2a). Darkly stained granular, membrane-associated structures were located, with spacing of about 20 nm between them. No central dense region, like that revealed after tannic acid treatment, was observed. In one of the rare cases, longitudinal section of the bundle showed two parallel rows of electron-dense cross-sections of the side links arranged with about 20-nm periodicity along the stereocilium axis (Fig 2b).
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Fig 2c shows a typical horizontal section of an OHC stereocilia bundle after Cuprolinic blue treatment and demonstrates that the distribution of the side links appears very close to mirror symmetry. The projections of the side links consist of one, two, or three strands. Membrane-bound ends of the links are seen as electron-dense, globular regions and the central portions of the strands were only slightly stained or are not stained at all.
High-power images of longitudinal sections of the OHC stereocilia after tannic acid (Fig 3a) and Cuprolinic blue (Fig 3b) treatments show linear arrays of the side links between adjacent stereocilia in their rows, with a quasi-regular spacing of about 20 nm along the shaft of stereocilium. Some variations of staining and spacing between individual side-link images were observed after both procedures. For image enhancement of the side-link projections, repeated noisy fragments between stereocilia were aligned and averaged using SPIDER software.
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For tannic acid treatment, we selected 10 image fragments of an original image from the area between adjacent stereocilia (Fig 3a), with each fragment of about 40-nm height and containing about two individual side-link images. The average of these fragments (Fig 3c) was similar to that of the original image and clearly showed the large central dense region (large arrowhead) and smaller low-density regions (small arrowheads) of the side link located near the surface of the stereocilium plasma membrane.
Images of the side links after Cuprolinic blue treatment were also computer-aligned and averaged. The average of six individual fragments (Fig 3d) showed that the distribution of electron density of the link was inverse to that obtained after tannic acid treatment (Fig 3c). Membrane-bound ends of the link were seen as regions of high electron density, whereas, the central portion of the link demonstrated much lower electron density.
Attachment Links
At high magnification, the presence of attachment links around the tips of the tallest stereocilia was observed in longitudinal sections after tannic acid treatment, with a bald area at the extreme tip of the stereocilium (Fig 4a). In horizontal sections (Fig 4b), the attachment links showed a radial distribution at some distance from the stereocilium membrane (Fig 4b). A different staining pattern was produced after Cuprolinic blue treatment. Membrane-associated granular, electron-dense structures 1525 nm in diameter were observed around the tips of the tallest stereocilia in longitudinal (Fig 4c) and horizontal sections (Fig 4d). Sometimes the attachment links could be seen together with the side links in horizontal sections, and both link types had a similar morphological appearance (Fig 4d). However, the attachment links demonstrated a radial distribution around the tips of only the tallest OHC stereocilia, whereas, the side links were arranged with a pseudo-hexagonal symmetry between stereocilia of the whole bundle (Fig 1b and Fig 2c).
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Horizontal sections of some stereocilia showed a strong radial symmetry of granular structures (Fig 4e and Fig 4f). Two of these images, with the best correlation coefficients between them, were aligned (within internal and external radiuses around location of granular structures) and averaged using one image as reference. The average (Fig 4g) was very similar to the original images (Fig 4e and Fig 4f) and clearly showed a radial symmetry in distribution of electron-dense, granular projections.
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Discussion |
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A diagram summarizing symmetric organization of OHC stereocilia links and their histochemical reactivity in the chinchilla is shown in Fig 5. The helical structure of the tip links has been described previously (
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Computer image analysis of tannic acid-treated tissues showed the central dense regions of the side links between adjacent stereocilia and much smaller dense portions of the links attached to the plasma membrane. Some of the side links appeared to consist of linear aggregates of globular domains (Fig 1d), which may correspond to the central regions of the side links. Computer image analysis of OHC stereocilia after Cuprolinic blue treatment showed electron-dense regions on both ends of the side links, whereas their central areas were only lightly stained. The different staining patterns after tannic acid and Cuprolinic blue treatments may indicate compositional differences between central and membrane-associated regions of the side links. Tannic acid contains phenolic and carboxyl groups and, when used in combination with glutaraldehyde and osmium tetroxide, it interacts with many different types of proteins, glycoproteins, and other components, regardless of their electrical charge (
The results of this study may be compared with the data on composition, structure, and properties of stereocilia links obtained for other species. Different morphological and compositional types of crosslink have been found between the stereocilia in avian hair cells. A 275-kD protein, hair cell antigen (HCA), was demonstrated in avian sensory hair cells (
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Acknowledgments |
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Supported by 1R03 DC 04464-01A1 and NS12125 grants from NIDCD.
The computations were performed on an SGI Origin 3800 computer at the Supercomputer Institute of the University of Minnesota.
Received for publication July 30, 2001; accepted November 27, 2001.
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