To understand how cells differentially use the dozens of myosin isozymes present in each genome, we examined the distribution of four unconventional myosin isozymes in the inner ear, a tissue that is particularly reliant on actin-rich structures and unconventional myosin isozymes. Of the four isozymes, each from a different class, three are expressed in the hair cells of amphibia and mammals. In stereocilia, constructed of cross-linked F-actin filaments, myosin-Iβ is found mostly near stereociliary tips, myosin-VI is largely absent, and myosin-VIIa colocalizes with crosslinks that connect adjacent stereocilia. In the cuticular plate, a meshwork of actin filaments, myosin-Iβ is excluded, myosin-VI is concentrated, and modest amounts of myosin-VIIa are present. These three myosin isozymes are excluded from other actin-rich domains, including the circumferential actin belt and the cortical actin network. A member of a fourth class, myosin-V, is not expressed in hair cells but is present at high levels in afferent nerve cells that innervate hair cells. Substantial amounts of myosins-Iβ, -VI, and -VIIa are located in a pericuticular necklace that is largely free of F-actin, squeezed between (but not associated with) actin of the cuticular plate and the circumferential belt. Our localization results suggest specific functions for three hair-cell myosin isozymes. As suggested previously, myosin-Iβ probably plays a role in adaptation; concentration of myosin-VI in cuticular plates and association with stereociliary rootlets suggest that this isozyme participates in rigidly anchoring stereocilia; and finally, colocalization with cross-links between adjacent stereocilia indicates that myosin-VIIa is required for the structural integrity of hair bundles.

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Unconventional Myosins in Inner-Ear Sensory Epithelia

The mechanical behaviour of the ciliary bundles of hair cells in the turtle cochlea was examined by deflecting them with flexible glass fibres of known compliance during simultaneous intracellular recording of the cell's membrane potential. Bundle motion was monitored through the attached fibre partially occluding a light beam incident on a photodiode array. The change in photocurrent was assumed to be proportional to bundle displacement. For deflexions of 1-100 nm towards the kinocilium, the stiffness of the ciliary bundles was estimated as about 6 X 10(-4) N/m, with the fibre attached to the top of the bundle. When the fibre was placed at different positions up the bundle, the stiffness decreased approximately as the inverse square of the distance from the ciliary base. This suggests that the bundles rotate about an axis close to the apical pole of the cell and have a rotational stiffness of about 2 X 10(-14) N. m/rad. Step displacements of the fixed end of the flexible fibre caused the hair cell's membrane potential to execute damped oscillations; the frequency of the oscillations in different cells ranged from 20 to 320 Hz. Displacements towards the kinocilium always produced membrane depolarization. The amplitude of the initial oscillation increased with displacements up to 100 nm and then saturated. For small displacements of a few nanometres, the hair cell's mechanoelectrical sensitivity was estimated as about 0.2 mV/nm. Force steps delivered by the flexible fibre caused the bundle position to undergo damped oscillations in synchrony with the receptor potential. The mechanical oscillations could be abolished with large depolarizing currents that attenuated the receptor potential. When placed against a bundle, a fibre's spontaneous motion increased and became quasi-sinusoidal with an amplitude several times that expected from the compliance of the system. It is suggested that the hair bundle drives the fibre. We conclude that turtle cochlear hair cells contain an active force generating mechanism.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1985.sp015750

The mechanical properties of ciliary bundles of turtle cochlear hair cells.

Functional structure of the organ of Corti: a review.

Properties of a mechano-electrical transduction channel were studied in enzymatically dissociated chick vestibular hair cells by using a whole-cell recording variation of the patch voltage-clamp technique The apical hair bundle was stimulated by a glass rod which moved along a one-dimensional axis when stimulated by either a triangular or a trapezoidal command voltage The motion of the glass rod was monitored optically using a photodiode In response to triangular stimuli, the hair cell generated a current of triangular wave form with occasional step-like spiky or zigzag-appearing events Control experiments confirmed that the current was generated only when the hair bundle was displaced towards the tallest stereocilium The mechano-sensitive current was blocked by streptomycin and by neomycin The blockage by streptomycin was clearly voltage dependent: the reduction of the current became larger with hyperpolarization of the membrane This suggests that the positively charged antibiotic molecules plug the mechanically gated channels From the evidence presented in 3 and 4 above, the mechano-sensitive current recorded here was identified as the mechano-electrical transduction (m-et) current The permeability of the m-et channel to various monovalent cations was determined from reversal potential measurements Since a CsCl-EGTA intracellular medium was used, all the permeabilities were calculated relative to PCs The sequence of permeabilities was Li greater than Na greater than or equal to K greater than or equal to Rb greater than Cs greater than choline greater than TMA greater than TEA External Ca ions were indispensable for the recording of transduction current and Sr ions could replace Ca ions without loss of the transduction activity The minimum [Ca]o for stable generation of the m-et current was 20 microM in Cs saline The addition of 50-200 microM-Ca to the isotonic Ba saline could maintain the m-et current The m-et current was observed in isotonic Ca and in Sr salines Isotonic Ba, Mg and Mn salines were enriched with 1-2 mM-Ca in order to generate the m-et current The permeabilities of the divalent cations relative to Cs were calculated from the reversal potentials, and the sequence of permeabilities among divalent cations was Ca greater than Sr greater than Ba greater than Mn greater than Mg Step-like m-et currents were observed in Cs saline The smallest step amplitude with clear resolution had a conductance of 497 +/- 45 pS (mean +/- SD, n = 7 cells) This is likely to be an elementary m-et channel conductance(ABSTRACT TRUNCATED AT 400 WORDS)

Mechano-electrical transduction currents in isolated vestibular hair cells of the chick.

Reversible contraction of isolated mammalian cochlear hair cells.

Movements of the stiff sensory hairs, the stereocilia, which extend outwards from the apical surface of the hair cells in the auditory organ, are thought to have a key role in the process of transduction of sound energy into electrical impulses. Each stereocilium is supported by a paracrystalline axial bundle of actin filaments, which have identical polarities and are extensively cross-linked1–4. Recent immunohistochemical observations on whole mounts of the guinea pig organ of Corti indicated myosin-like immunoreactivity in association with hair cell stereocilia5. Considering the steric situation, it is, however, difficult to imagine any functional interaction between the bundled actin filaments and the assumed myosin molecules. Here we report that in semi-thin, transverse sections of quick-frozen, freeze-dried and plastic-embedded guinea pig organ of Corti, myosin-like immunostaining was restricted to the apical cytoplasm of hair cells and was not detected along the stereocilia. With respect to the immunohistochemical distribution of actin, myosin and the muscular Z-line protein, α-actinin, the apex of hair cells strongly resembles the intestinal epithelial brush border6–7.

U. Gröschel-Stewart

Papers

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