Absence of myosin-like immunoreactivity in stereocilia of cochlear hair cells

Drenckhahn, Detlev; Kellner, JD; Mannherz, Hans Georg; Gröschel‐Stewart, Ute; Kendrick‐Jones, John; Scholey, Jonathan M.

doi:10.1038/300531a0

Cited by 97 publications

(33 citation statements)

References 15 publications

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“…None of these antibodies reacted with any of the proteins on the hair borders even though they reacted strongly to brush border and gizzard proteins, respectively, not surprisingly as there are no bands from that preparation at their respective molecular weights. Immunoblots using the intact sensory epithelium, however, showed a strong reaction with the antimyosin antibodies; this is reasonable as this protein has been shown by immunofluorescence to be present in hair cells (10,11,16).…”

Section: Lmmunoblotsmentioning

confidence: 93%

“…Second, some investigators have reported the presence of a protein at certain locations in hair cells, yet others using a different antibody to the same protein, show that it appears to be absent. Two examples are: antifimbrin, localized to the stereocilia and cuticular plate by Slepecky and Chamberlin (22) and by Sobin and Flock (23), but only in the stereocilia by Drenckhahn et al (11); and antimyosin, in one case localized in stereocilia and cuticular plates (16), yet in another report only in the cuticular plate, not the stereocilia (10). Third, the antibody methods do not really tell us if the brush border and hair cell proteins are identical or if they just share common domains.…”

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confidence: 99%

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Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

Tilney

Stephens

et al. 1989

The Journal of Cell Biology

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Abstract. The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57-and 65-kD bands present in the sensory epithelium and the cytoskeleton.It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57-and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125/~ transverse striping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

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Section: Lmmunoblotsmentioning

confidence: 93%

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confidence: 99%

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

Tilney

Stephens

et al. 1989

The Journal of Cell Biology

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“…Stress fibers contain ␣-Actinin and nonmuscle Myosin type II (Lo et al, 2004;Lele and Kumar, 2007). Support cells in the Organ of Corti also seem to contain these proteins (Drenckhahn et al, 1982;Slepecky and Chamberlain, 1985;Donaudy et al, 2004). Therefore, we investigated the presence of these factors in Drosophila tendon cells.…”

Section: Nonmuscle Myosin Type II Associates With Cytoskeletal Arraysmentioning

confidence: 99%

Prominent Actin Fiber Arrays inDrosophilaTendon Cells Represent Architectural Elements Different from Stress Fibers

Silva

Hahn

Huber

et al. 2008

MBoC

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“…Although actin and fimbrin are major structural proteins in both structures, purified stereocilia and stereociliary cores did not contain bands at the molecular masses of the two other major microvilli core proteins, villin (95 kDa) and the 110-kDa protein. Villin is immunocytochemically absent from stereocilia (8,31). The 110-kDa protein is the core-membrane crosslinker in microvilli, binds CaM, and has myosin-like properties (reviewed in ref.…”

Section: Methodsmentioning

confidence: 99%

"Bundle blot" purification and initial protein characterization of hair cell stereocilia.

Shepherd

Barres

Corey

1989

Proc. Natl. Acad. Sci. U.S.A.

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Stereocilia were isolated from bullfrog (Rana catesbeiana) saccular hair cells by nitrocellulose adhesion. The high purity and high yield of the preparation were demonstrated by microscopy. SDS/PAGE of stereociliary proteins resolved 12-15 major bands. Actin, previously identified as a component of the stereociliary core, was identified in purified stereocilia as a band comi rating with authentic actin and by phalloidin labeling of intact isolated stereocilia. Fimbrin was identified in immunoblots of purified stereocilia. The most abundant other proteins migrated at 11, 14, 16-19, 27, and 36 kDa. Demembranated stereociliary cores consisted primarily of protein bands corresponding to actin and fimbrin and several proteins ranging from 43 to 63 kDa. Because the adaptation mechanism in hair cells is calcium-sensitive and seems localized to stereocilia, we sought evidence for calciumbinding proteins in stereocilia. Calmodulin and calbindin antibodies labeled stereocilia in intact cells. A protein band in purified stereocilia exhibited a Ca2+-dependent shift in electrophoretic mobility identical to that of authentic calmodulin, and the 27-kDa band may represent calbindin. These biochemical data demonstrate that stereocilia consist of a relatively small set of proteins. Most of these, including those involved in transduction and adaptation, are as yet uncharacterized. The availability ofpurified stereocilia should prove useful in further studies of structure-function relationships in these mechanically sensitive organelles.Hair cells, the receptor cells of the auditory and vestibular systems, transduce displacements of their apical hair bundle into electrical signals. The stereocilia constituting the bundle, which share structural properties with intestinal microvilli and growth-cone filopodia, are the mechanically sensitive organelles: displacement is thought to alter tension on filamentous links between their tips, which directly opens ion channels (1-3). The transduction current adapts to maintained stimuli, through a tension-altering mechanism (4, 5) thought to be situated in the tips of stereocilia (6).Although the physiology of transduction and adaptation is understood in some detail, the proteins involved in these processes are largely unknown. Because of the difficulty in purifying sufficient amounts of stereocilia, protein identification has been pursued through immunocytochemistry. Thus the structural proteins actin (7,8) To study the biochemistry of stereocilia we developed a purification technique that exploits the adhesion of the apical ends of stereocilia to nitrocellulose paper and the mechanical fiagility of the narrow basal ends. A related strategy has been used to adsorb small numbers of piscine stereocilia onto coverslips for ultrastructural studies (13). The nitrocellulose adhesion method, which we term "bundle blot" purification, gave a sufficiently high yield of pure stereocilia for biochemical analysis. Some results have appeared in preliminary form (14). leupeptin, 0.15 LM pepstatin...

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Absence of myosin-like immunoreactivity in stereocilia of cochlear hair cells

Cited by 97 publications

References 15 publications

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

Prominent Actin Fiber Arrays inDrosophilaTendon Cells Represent Architectural Elements Different from Stress Fibers

"Bundle blot" purification and initial protein characterization of hair cell stereocilia.

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