Elsevier

Hearing Research

Volume 126, Issues 1–2, December 1998, Pages 151-160
Hearing Research

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti

https://doi.org/10.1016/S0378-5955(98)00164-6Get rights and content

Abstract

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to α-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.

Introduction

Determining the organisation of cytoskeletal proteins in the different cell types in the organ of Corti is important if its mechanical properties are to be fully understood. For this reason, the distributions of many of them have been, and continue to be, explored. One such protein is spectrin, a heterodimeric molecule first identified in the submembranous cytoskeleton of erythrocytes. It has now been found in a wide range of nonerythroid cells and tissues, most notably in brain in a form generally referred to as `fodrin' (see, e.g. Burridge et al., 1982; Carlin et al., 1983; Glenney et al., 1983; Fach et al., 1985; Shimo-Oka and Atsumi, 1986; Zagon et al., 1986; Black et al., 1988; Fugimoto and Ogawa, 1989; Fugimoto et al., 1991; Isayama et al., 1993). Spectrin is thought to play an important role in the establishment and maintenance of cell shape and is frequently found in association with actin, microtubules and other major structural proteins (see review by Bennett, 1990). It consists of α- (molecular mass 240 kDa) and β- (220 kDa) polypeptide chains that coil around each other in a double helix with tetramers being formed by the end-to-end association of two heterodimers (Shotton et al., 1979).

In the guinea-pig organ of Corti, spectrin has been identified at the light microscopic level in intercellular junctions, the cuticular plates and lateral walls of inner hair cells (IHCs) and outer hair cells (OHCs), in the infracuticular network of OHCs, and in the supporting cells especially in their phalangeal processes (Ylikoski et al., 1990, Ylikoski et al., 1992; Drenckhahn et al., 1991; Slepecky and Ulfendahl, 1992; Raphael et al., 1994). However, the precise ultrastructural distribution of spectrin has only been reported so far in the OHCs where it has been found in the cuticular plate and beneath the plasma membrane of the lateral walls in a structure known as the cortical lattice (Nishida et al., 1993).

The cortical lattice originally attracted interest because it was proposed to play a role in the motile behaviour of OHCs (Bannister et al., 1988; Holley and Ashmore, 1988a). Whilst the IHCs appear to act as the main sensory cells of the cochlea, the OHCs may actively exert forces that enhance its response to sound (for review, see Dallos, 1993). The plasma membrane is currently thought to be the site of the force generation (Holley and Ashmore, 1988b; Holley et al., 1991; Kalinec et al., 1992) and to act in conjunction with the lattice to produce length changes in the OHCs, e.g. when they are subjected to depolarisation. It has been suggested that the lattice consists of circumferentially arranged actin filaments cross-linked by thin filaments of spectrin, forming an anisotropic sheet that is stiffer circumferentially than longitudinally (Holley and Ashmore, 1990; Holley et al., 1992). In this arrangement, it is likely that spectrin confers elasticity on the lattice and provides a means by which it can be anchored to the membrane (Holley and Ashmore, 1990; Holley, 1991). An additional suggestion is that spectrin may be linked to stretch-activated ion channels in OHCs which may sense membrane tension and regulate intracellular pressure, thus playing a role in both sensor and motor mechanisms (Sachs, 1988; Ding et al., 1991).

The cortical lattice of the OHCs has been the subject of a number of previous investigations. However, a meshwork of filaments that resembles it has been found beneath the lateral plasma membrane in the neck region of guinea-pig IHCs (Furness and Hackney, 1990). It is not yet known whether this meshwork contains similar cytoskeletal proteins to the cortical lattice of OHCs but since spectrin has been detected in IHC lateral walls at the light microscopic level (Ylikoski et al., 1990, Ylikoski et al., 1992; Raphael et al., 1994), it may be a component of this structure too. Knowing this, and how it is distributed in the supporting cells is important for understanding the mechanical properties of the tissue as a whole. In this study therefore, immunogold labelling with a monoclonal antibody to α-spectrin has been used to compare the distribution of spectrin in the IHCs with that in supporting cells and OHCs.

Section snippets

Animals and antibodies

Nineteen adult pigmented guinea pigs weighing 600–900 g and possessing normal Preyer reflexes were used in this study. Animals were maintained and used in accordance with the `Principles of Laboratory Animal Care' (NIH publication No. 85-23, revised 1983) and the Animals (Scientific Procedures) Act, 1986. The primary antibody was a monoclonal anti-α-spectrin, IgG1 (Chemicon International, UK).

Immunoblotting

To check the specificity of the antibody, Western blots of the guinea-pig organ of Corti were prepared

Immunoblotting

In Western blots of freshly dissected organ of Corti, the anti-α-spectrin antibody labelled a prominent band in the 240 kDa region corresponding to that expected for α-spectrin. Four weaker bands were also detected at approximately 220, 174, 150 and 89 kDa (Fig. 1). In blots of guinea-pig blood labelled with the anti-α-spectrin antibody, a prominent band also corresponding to α-spectrin was seen at 240 kDa and two weak bands similar to those seen in both organ of Corti blots were noted at 150

Discussion

This investigation provides further evidence for the presence of spectrin in the organ of Corti thus confirming previous light and electron microscopic observations (e.g. Ylikoski et al., 1990, Ylikoski et al., 1992; Slepecky and Ulfendahl, 1992; Nishida et al., 1993; Raphael et al., 1994). It also provides additional ultrastructural information about the location of this cytoskeletal protein in the IHCs and supporting cells.

The immunoblotting confirms that the antibody predominantly labels

Acknowledgements

Supported by The Wellcome Trust and Defeating Deafness: The Hearing Research Trust. We thank Mrs K. Walker and Mrs J. Kott for technical assistance. Thanks are also due to Dr M.G. Evans for his comments on the manuscript.

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