Differential distribution of β- and γ-actin in guinea-pig cochlear sensory and supporting cells
Introduction
Actin is a major component of hair cells and supporting cells in the inner ear. In the mammalian cochlea, various properties of these cells change systematically along the length of the cochlear partition (see e.g. Lim, 1986). These changes contribute to the processes by which complex sounds are decomposed into their component frequencies (Robles and Ruggero, 2001). Morphological changes in actin-containing structures along the cochlear length have been reported (Carlisle et al., 1988) but whether specific variations occur in the types and amount of actin present has yet to be investigated.
Three subtypes of actin, α, β and γ, are distinguishable by their differing isoelectric points on 2D gels (Vandekerckhove and Weber, 1978, Vandekerckhove and Weber, 1981). Six functional actin genes have been described in humans, four of which encode the α- and γ-isoforms found in different muscle types (see e.g. Herman, 1993). The remaining two, ACTB and ACTG1, code for cytoplasmic β- and γ-actin which are found in a wide range of cell types including hair cells and supporting cells (Slepecky and Savage, 1994, Nakazawa et al., 1995, Hofer et al., 1997). Mutations of the human γ-actin gene cause progressive nonsyndromic sensorineural hearing loss, DFNA20/26 (Zhu et al., 2003, Wijk et al., 2003). Given the widespread distribution of γ-actin in the body, this is surprising and implies that it has a special role in hearing.
The cytoplasmic isoforms, β- and γ-actin, differ in proportion and distribution in various tissues (e.g. Otey et al., 1987) and interact with specific associated proteins (Sheterline et al., 1998). Changes in their expression result in cell phenotype changes, emphasizing their role in maintaining morphology (see review by Khaitlina, 2001). In the brain, β-actin is restricted to dynamic structures and is associated with cell processes e.g., dendritic spines and growth cone filopodia, whereas γ-actin is more ubiquitously distributed and occurs in relatively quiescent regions (Micheva et al., 1998).
In auditory hair cells, actin is found in the stereocilia, the cuticular plate and a circum-apical ring of filaments (Flock and Cheung, 1977, Flock et al., 1981, Tilney et al., 1980, Hirokawa and Tilney, 1982, Slepecky and Chamberlain, 1982, Slepecky and Chamberlain, 1983, Slepecky and Chamberlain, 1985, Slepecky and Chamberlain, 1986). These networks contain different cross-linking proteins (Drenckhahn et al., 1991). Mammalian outer hair cells (OHCs) contain, in addition, an F-actin cortical lattice that lies between the plasma lemma and the layers of sub-surface cisternae (Flock et al., 1986, Bannister et al., 1988, Holley and Ashmore, 1988, Holley and Ashmore, 1990). In guinea-pig OHCs at least, there is also an actin-rich infracuticular network (Carlisle et al., 1988).
The β-actin isoform is known to be involved in dynamic maintenance of the hair bundle in rodents (Schneider et al., 2002, Rzadzinska et al., 2004). In chick, Hofer et al. (1997) reported that β-actin occurs in the stereocilia but not the cuticular plate, whereas γ-actin occurs in both, and suggested that the ratio of β:γ determines the final size and length of stereocilia. In mammals, decreasing stereociliary length (see e.g. Lim, 1986) and increasing stiffness of the hair bundles (Flock and Strelioff, 1984) towards the cochlear base might reflect changes in the ratio. The possibility that this ratio influences the structure and function of subcellular regions in other cell types makes it worth examining isoform distribution in mammalian hair cells and supporting cells in detail. We have therefore investigated the distribution of each isoform in different cell types and in two different frequency regions in the organ of Corti of guinea pig using post-embedding immunogold labelling.
Section snippets
Animals and antibodies
Adult pigmented guinea pigs (500–850 g) exhibiting Preyer reflexes were killed with an overdose of sodium pentobarbitone (Vetalar®; 200 mg/kg IP), the bullae removed and opened. Cochleae were immediately perfused with, and immersed in, fixative for 2 h at room temperature and washed in phosphate buffered saline (PBS, pH 7.4). Subsequent processing varied according to the immunolabelling method. Animals were maintained and used in accordance with the “Principles of laboratory animal care” (NIH
Immunofluorescence
Immunofluorescence labelling of whole mounts for confocal microscopy revealed differential distributions of the two isoforms of actin in hair cells and supporting cells (Fig. 2(a)–(e)) which could be compared with distributions observed by post-embedding immunogold (Fig. 2(f)–(h)). Labelling for β-actin was most prominent in the pillar-cell foot and head and at the ends of the phalangeal process of the outer pillar cell (Fig. 2(a)–(c)) but the centre of the head region was unlabelled (Fig. 2
Post-embedding immunogold assessment of actin isoform distributions
We describe here relative distributions of β- and γ-actin isoforms in hair cells and supporting cells in the guinea-pig organ of Corti, thus extending previous immunocytochemical studies by Slepecky and Savage (1994) and Hofer et al. (1997). The semi-quantitative data were derived from immunogold labelling densities in equivalent cochlear regions from 5 animals. Consistent relative differences between cell types were found in all samples, even though comparative labelling densities for the two
Acknowledgements
Supported by the Wellcome Trust, Defeating Deafness and Tohoku University Hospital. We thank Mr. S. Murray and Mrs. K. Walker for technical assistance, and Dr. J. Bulinski for the γ-actin antibody.
References (62)
- et al.
The cortical lattice: a highly ordered system of subsurface filaments in guinea pig cochlear outer hair cells
Prog. Brain Res.
(1988) - et al.
Cross-links between stereocilia in the guinea pig cochlea
Hearing Res.
(1985) Comparing octaves, frequency ranges, and cochlear-map curvature across species
Hearing Res.
(1996)Actin isoforms
Curr. Opin. Cell Biol.
(1993)Functional specificity of actin isoforms
Int. Rev. Cytol.
(2001)Functional structure of the organ of Corti: a review
Hear Res.
(1986)- et al.
Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti
Hearing Res.
(1998) - et al.
The rosette complex in gerbil Deiters’ cells contains gamma-actin
Hearing Res.
(1995) - et al.
Immunocytochemical localization of 205 kDa microtubule-associated protein (205 kDa MAP) in the guinea pig organ of Corti
Brain Res.
(1992) - et al.
Actin-isoform pattern as a marker of normal or pathological smooth-muscle and fibroblastic tissues
Differentiation
(1987)
Distribution and polarity of actin in inner ear supporting cells
Hearing Res.
Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells
Hearing Res.
Expression of actin isoforms in the guinea pig organ of Corti: Muscle isoforms are not detected
Hearing Res.
Actin-binding and microtubule-associated proteins in the organ of Corti
Hearing Res.
Mechanics of microtubule bundles in pillar cells from the inner ear
Biophys. J.
At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide
J. Mol. Biol.
Mutations in the gamma-actin gene (ACTG1) are associated with dominant progressive deafness (DFNA20/26)
Am. J. Hum. Genet.
Phalloidin binding and rheological differences among actin isoforms
Biochemistry
Species differences in the distribution of infracuticular F-actin in outer hair cells of the cochlea
Hearing Res.
Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells
Cell Motil. Cytoskel.
Actin and microtubules in neurite initiation: are MAPs the missing link?
J. Neurobiol.
Three different actin filament assemblies occur in every hair cell: Each contains a specific actin crosslinking protein
J. Cell Biol.
Myosin-I isozymes in neonatal rodent auditory and vestibular epithelia
J. Assoc. Res. Otolaryngol.
Stiffness of the gerbil basilar membrane: radial and longitudinal variations
J. Neurophysiol.
Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes
Mol. Cell Biol.
Actin filaments in sensory hairs of inner ear receptor cells
J. Cell Biol.
Graded and nonlinear mechanical properties of sensory hairs in the mammalian hearing organ
Nature
Mechanisms of movement in outer hair cells and a possible structural basis
Arch. Otorhinolaryngol.
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.
Genetic insights into the morphogenesis of inner ear hair cells
Nature Rev. Gen.
Localization of myosin-Ibeta near both ends of tip links in frog saccular hair cells
J. Neurosci.
Cited by (52)
The actin cytoskeleton in hair bundle development and hearing loss
2023, Hearing ResearchThe Role of non-muscle actin paralogs in cell cycle progression and proliferation
2023, European Journal of Cell BiologyFrameshift mutation S368fs in the gene encoding cytoskeletal β-actin leads to ACTB-associated syndromic thrombocytopenia by impairing actin dynamics
2022, European Journal of Cell BiologyBuilding and repairing the stereocilia cytoskeleton in mammalian auditory hair cells
2019, Hearing ResearchCitation Excerpt :Hair cells express both β- and γ-actin isoforms. In chick and guinea pig cochlear hair cells, β-actin expression seems to be restricted to the hair bundle, while γ-actin is expressed in the hair bundle, the cuticular plate, and the lateral wall (Furness et al., 2005; Hofer et al., 1997). In adult cochlear hair cells from mouse and guinea pig, β- and γ-actin seem to be uniformly distributed along the entire stereocilia cytoskeleton (Perrin et al., 2010b), except at the stereocilia tips where γ-actin expression is significantly higher than β-actin, particularly in the outer hair cells (OHCs) (Furness et al., 2005; Patrinostro et al., 2018).
Cochlear connexin 30 homomeric and heteromeric channels exhibit distinct assembly mechanisms
2019, Mechanisms of DevelopmentFunctional Development of Hair Cells in the Mammalian Inner Ear
2014, Development of Auditory and Vestibular Systems: Fourth Edition