Regeneration of broken tip links and restoration of mechanical transduction in hair cells

Abstract

A hair cell's tip links are thought to gate mechanoelectrical transduction channels. The susceptibility of tip links to acoustic trauma raises questions as to whether these fragile structures can be regenerated. We broke tip links with the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N,N-tetraacetic acid and found that they can regenerate, albeit imperfectly, over several hours. The time course of tip-link regeneration suggests that this process may underlie recovery from temporary threshold shifts induced by noise exposure. Cycloheximide does not block tip-link regeneration, indicating that new protein synthesis is not required. The calcium ionophore ionomycin prevents regeneration, suggesting regeneration normally may be stimulated by the reduction in stereociliary Ca 2؉ when gating springs rupture and transduction channels close. Supporting the equivalence of tip links with gating springs, mechanoelectrical transduction returns over the same time period as tip links; strikingly, adaptation is substantially reduced, even 24 hr after breaking tip links.Hair cells transduce displacements of their mechanically sensitive hair bundles into electrical signals (1, 2). A hair bundle consists of about 100 stereocilia, each of which contains hundreds of crosslinked actin filaments enshrouded by plasma membrane. Stereocilia are arrayed in rows of increasing height, producing a bundle that exhibits mirror symmetry along a central axis; this axis also corresponds to the bundle's axis of highest displacement sensitivity. When a bundle is displaced along this axis, the tip of each shorter stereocilium slides along the side of its tallest neighbor. Such displacements stretch elastic mechanical elements called gating springs, which open transduction channels (3). Optimally poised to be gating springs, tip links are 5 ϫ 150 nm extracellular filaments that stretch from the tip of a stereocilium to the side of its neighbor, parallel to the plane of mirror symmetry (4). Bundle displacements that stretch tip links also open transduction channels, and displacements that slacken tip links permit channels to close. Because 1,2-bis(O-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) eradicates tip links, simultaneously disrupting transduction by disengaging gating springs, tip links are thought to either be gating springs themselves or connect in series with the gating spring (5).A hair cell's transduction apparatus survives constant stimulation and occasionally experiences stimuli that can break tip links (6). If tip links indeed play an essential role in transduction, broken tip links should be replaced to maintain sensitivity to mechanical displacements. We intentionally broke tip links with BAPTA and found that they regenerate over a period of several hours. Regeneration is imperfect, as many links arise that connect stereociliary pairs not oriented along the plane of mirror symmetry. Although restoration of tip links does not require new protein synthesis, elevation of intracellular Ca 2ϩ levels effectively prev...

Three-dimensional Architecture of Hair-bundle Linkages Revealed by Electron-microscopic Tomography

“…The striking dichotomy that we observed in the lengths of tip links both in two-dimensional projection images and in three-dimensional tomographic reconstructions is also apparent in published images (Zhao et al 1996;Sollner et al 2004). Variations in tip-link dimensions cannot be attributed to tissue shrinkage, for in some instances long and short links coexisted in the same hair bundle.…”

“…The divergence of an auxiliary link from the main tip link may underlie the forking of tip links (Furness and Hackney 1985;Kachar et al 2000), for both filaments in the main link connect to the membrane surface. In our projection images, as well as in published micrographs (Zhao et al 1996;Siemens et al 2004), the sides of stereocilia often display globular extracellular densities that may reflect detached auxiliary links.…”

“…These filaments are probably connected to mechanically sensitive channels and are therefore essential to mechanoelectrical transduction. If exposed to lanthanoid ions or Ca 2+ chelators, tip links disappear concomitantly with the loss of responsiveness; as they regenerate over several hours, transduction recovers (Zhao et al 1996). When analyzed by high-resolution scanning electron microscopy, each tip link appears to comprise two filaments arranged in a right-handed, coiled-coil configuration (Kachar et al 2000).…”

Dynamics of Noise-Induced Cellular Injury and Repair in the Mouse Cochlea

“…These include (1) changes in the stereocilia bundle, including disarray (Mulroy and Whaley 1984), loss of tip links (Zhao et al 1996;Husbands et al 1999), or depolymerization of rootlets (Liberman and Dodds 1987); (2) excitotoxic effects on neural elements in the IHC area manifested as extreme swelling of peripheral terminals (Spoendlin 1971;Liberman and Mulroy 1982;Robertson 1983); (3) collapse of the supporting cells in the organ of Corti including both pillar cells (Nordmann et al 2000) and Hensen cells (Flock et al 1999); and (4) strial edema (Santi and Duvall 1978). It is not unlikely that all these changes make some contribution to threshold shift, depending on the severity of the exposure and the species involved.…”

The Involvement of Arl-5b in the Repair of Hair Cells in Sea Anemones

“…In the laboratory, hair cells from chickens lose mechanosensitivity after brief exposure to calcium-depleted buffers. Such exposure may mimic noise trauma, at least in part, because calcium-depleted buffers damage or destroy linkages interconnecting stereocilia (Assad et al 1991;Zhao et al 1996;Goodyear and Richardson 1999). Within 12 h, the traumatized hair cells replace missing tip links and recover mechanosensitivity (Zhao et al 1996).…”