Keratin filament deployment and cytoskeletal networking in a sensory epithelium that vibrates during hearing

Mogensen, Mette; Henderson, Craig G.; Mackie, John B.; Lane, E. Birgitte; Garrod, David R.; Tucker, John B.

doi:10.1002/(sici)1097-0169(1998)41:2<138::aid-cm5>3.0.co;2-a

Cited by 27 publications

(13 citation statements)

References 40 publications

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“…Although they provide mechanical stability to cells, cytokeratins probably serve other roles as well, including the organization of organelles and the nucleus (15)(16)(17). Cytokeratins, including cytokeratins 8, 9, and 19 and otokeratin, are known to occur in cells of the inner ear (13,(18)(19)(20)(21)(22)(23). A definitive subclassification of our inner-ear cytokeratin within the type II cytokeratin family is difficult because of the sequence conservation within this family and the lack of anuran cytokeratin sequences to use for comparison.…”

Section: Discussionmentioning

confidence: 99%

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Cyr

Bell

Hudspeth

2000

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

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Extensive biochemical characterization of cells in the inner ear has been hampered by a lack of tools with which to identify inner-ear proteins. By using a single-chain antibody fragment isolated from a bacteriophage-displayed library, we have identified a cytokeratin that is abundant in nonsensory cells of the frog inner ear. Although the progenitors of hair cells exhibit strong immunoreactivity to this cytokeratin, the signal declines in immature hair cells and vanishes as the cells mature. The correlation between diminished immunoreactivity and hair-cell differentiation indicates that the cytokeratin is down-regulated during the transition from a nonsensory to a sensory cell and suggests that the marker is an early index of hair-cell differentiation. O ur awareness of faint sounds and weak accelerations rests on the sensitivity of hair cells, which respond to subnanometer deflections of their hair bundles (1). The exquisite sensitivity of hair cells unfortunately renders them acutely susceptible to trauma from acoustical overstimulation. In addition, aminoglycoside antibiotics and some chemotherapeutic agents can damage or kill these sensory cells. Because they are postmitotic, hair cells can be replaced only by the differentiation of precursors. Although hair cells do not regenerate in the adult mammalian cochlea, replacement does occur in mammalian vestibular organs and more extensively in nonmammalian receptor organs (2, 3).Little is known about the proteins involved in hair-cell differentiation and function. Although the identification and characterization of inner-ear proteins are impeded by the paucity of cells, the isolation of specific reagents directed against inner-ear proteins makes biochemical studies feasible. To generate such reagents, we produced a bacteriophage-displayed antibodyfragment library directed against proteins of the bullfrog inner ear (4). From this library, we isolated a single-chain Fv fragment (scFv) that specifically labels a protein expressed almost exclusively in the inner ear. We describe here the further characterization and identification of this protein as a cytokeratin whose expression declines during hair-cell differentiation. Materials and MethodsThe production of a library of bacteriophage-displayed scFvs and the selection of a clone producing an scFv with a high specificity for an inner-ear antigen have been described (4). For the present study, soluble scFvs from clone scFv-278 were isolated by the procedures in that reference.Immunocytochemistry on Saccular Whole Mounts. Bullfrog sacculi were isolated and the otolithic membranes were mechanically removed. Sacculi were fixed for 1 h at 4°C in 4% (wt͞vol) formaldehyde in PBS (68 mM NaCl͞58 mM Na 2 HPO 4 ͞17 mM NaH 2 PO 4 , pH 7.4), rinsed in PBS, permeabilized for 1 h in PBS containing 1% (vol͞vol) Triton X-100, and washed in PBS. Samples were blocked for 2 h in PBS containing 2.5% (vol͞vol) Liquid Block (Amersham Pharmacia) and 3% (vol͞vol) goat serum, then incubated overnight at 4°C in a mixture of 1.8 mg͞liter aff...

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Section: Discussionmentioning

confidence: 99%

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Cyr

Bell

Hudspeth

2000

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

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“…Therefore, the transient role for microtubules in OHC stiffness might act as a foundation for organization of the actin-based cuticular plate and, in part, for the organization of stereocilia. Surrounding these sensory hair cells are many types of nonsensory supporting cells, which are thought to have either metabolic (Wangemann and Liu, 1996;Kikuchi et al, 2000) or structural (Tolomeo and Holley, 1997;Mogensen et al, 1998) roles in hearing. To explore the structural role of pillar cells, we investigated the impact of both actin filaments and microtubules.…”

Section: Research Articlementioning

confidence: 99%

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea

et al. 2012

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SUMMARYCorrect patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses. We also explored the cytoskeletal organization of developing sensory and non-sensory cells, and used pharmacological modulation of cytoskeletal elements to show that the developmental increase of hair cell stiffness is a direct result of actin filaments, whereas the development of supporting cell surface mechanical properties depends on the extent of microtubule acetylation. Finally, this study found that the fibroblast growth factor signaling pathway is necessary for the developmental time course of cell surface mechanical properties, in part owing to the effects on microtubule structure.

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“…5b), the OHCs contract with a lead of 90°, thereby drawing the BM and the reticular lamina together. In this configuration, the outer pillar cell behaves as a rigid strut (26,27) extending from a broad foot via an articulated joint. The outer pillar cell resists the contractions of the OHCs so that when the BM is pulled up toward the scala media, the foot of the outer pillar cell rotates slightly and pushes the joint between itself and the inner pillar cell downwards toward the scala tympani.…”

Section: Ohcs Boost the Vibrations Of The Bm At The Time Of Maximum Bmentioning

confidence: 99%

The spatial and temporal representation of a tone on the guinea pig basilar membrane

Nilsen

Russell

2000

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

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In the mammalian cochlea, the basilar membrane's (BM) mechanical responses are amplified, and frequency tuning is sharpened through active feedback from the electromotile outer hair cells (OHCs). To be effective, OHC feedback must be delivered to the correct region of the BM and introduced at the appropriate time in each cycle of BM displacement. To investigate when OHCs contribute to cochlear amplification, a laser-diode interferometer was used to measure tone-evoked BM displacements in the basal turn of the guinea pig cochlea. Measurements were made at multiple sites across the width of the BM, which are tuned to the same characteristic frequency (CF). In response to CF tones, the largest displacements occur in the OHC region and phase lead those measured beneath the outer pillar cells and adjacent to the spiral ligament by about 90°. Postmortem, responses beneath the OHCs are reduced by up to 65 dB, and all regions across the width of the BM move in unison. We suggest that OHCs amplify BM responses to CF tones when the BM is moving at maximum velocity. In regions of the BM where OHCs contribute to its motion, the responses are compressive and nonlinear. We measured the distribution of nonlinear compressive vibrations along the length of the BM in response to a single frequency tone and estimated that OHC amplification is restricted to a 1.25-to 1.40-mm length of BM centered on the CF place.cochlear amplifier ͉ outer hair cell ͉ frequency selectivity ͉ laser-diode interferometry v on Békésy (1) discovered that sound-induced stapes movement causes a wave of basilar membrane (BM) displacement to travel from the base of the cochlea to the apex because of a pressure difference set up across the cochlear partition. The length of the BM is graded in stiffness, and the cochlear partition can be modeled as a series of weakly coupled sections, each section comprised of a rigid mass connected to the sides of the cochlea by springs that decrease in stiffness toward the apex (2). The BM will resonate when the mechanical impedance caused by the stiffness of the springs cancels that caused by the mass of the section (1). At a point just basal to the resonant place, a peak of displacement will occur on the BM. At a point just apical to the resonant place, the BM impedance is insufficient to maintain a pressure difference across the cochlear partition, and the traveling wave dies out. The location of the displacement peak depends on stimulus frequency, so that each segment of the BM is tuned to a characteristic frequency (CF). von Békésy measured BM movement under postmortem conditions and found that the mechanical responses were broadly tuned and insensitive. Subsequent measurements made from sensitive cochleae in vivo have revealed that, in fact, BM displacement responses are sharply tuned and very sensitive at low sound-pressure levels (3-6).Contributions from the electromotile outer hair cells (OHCs) (7-10) appear to be essential for cochlear sensitivity and frequency tuning (11,12). Many current models of cochlear functi...

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Keratin filament deployment and cytoskeletal networking in a sensory epithelium that vibrates during hearing

Cited by 27 publications

References 40 publications

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Identification with a recombinant antibody of an inner-ear cytokeratin, a marker for hair-cell differentiation

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea

The spatial and temporal representation of a tone on the guinea pig basilar membrane

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