Closure of supporting cell scar formations requires dynamic actin mechanisms

doi:10.1016/j.heares.2007.06.011

Hearing Research

Volume 232, Issues 1–2, October 2007, Pages 1-19

https://doi.org/10.1016/j.heares.2007.06.011 Get rights and content

Abstract

In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6 h), and allowed to recover (18 h). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery.

Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules.

Introduction

Aminoglycoside antibiotics, like gentamicin, are ototoxic drugs that induce hearing loss and vestibular disorders (Miller, 1985), and nephrotoxicity (Humes, 1999). Aminoglycosides selectively kill sensory hair cells that are then extruded from amphibian inner ear sensory epithelia (Baird et al., 1996), chick basilar papillae (Cotanche and Dopyera, 1990, Duncan et al., 2001) and mammalian vestibular organs (Duvall and Wersall, 1963, Lang and Liu, 1997, Li et al., 1995a). In the bullfrog saccule, each hair cell has a round apex and is surrounded by five to seven supporting cells that are characterized by their polygonal apical surfaces (Baird et al., 1996). When dying hair cells are extruded, the surrounding supporting cells expand their apical processes to seal the potential breach beneath the hair cell, and produce a supporting cell scar formation (Fig. 1), which is characterized by a multi-cellular actin ring (Steyger et al., 1997). This process is necessary to maintain the electro-chemical separation between endolymph and perilymph bathing the apical and baso-lateral membranes of cells at the surface of the scala media (Forge, 1985, Meiteles and Raphael, 1994).

The actin cytoskeleton participates in a variety of cellular activities, including cell division, cell motility, induction of cell polarity, and membrane trafficking (Brown and Song, 2001). Disruption of filamentous actin (F-actin) assembly interferes with the extrusion of dying epithelial cells in confluent epithelial cell cultures and subsequent epithelial closure (Rosenblatt et al., 2001). Inhibition of myosin function in these same cells also disrupts apoptotic cell extrusion. Therefore, we hypothesized that disruption of actin or myosin activity could interfere with the extrusion of dying hair cells and the closure of the supporting cell scar formations.

Li et al. (1995a) hypothesized that hair cell microtubules play an essential role during the extrusion process. Microtubule assembly is inhibited by colchicine binding at the polymerization end of the microtubule, resulting in the eventual depolymerization of the microtubular filament (Bergen and Borisy, 1983, Platts et al., 1999). Dynamic microtubular processes can also be disrupted by taxol which stabilizes microtubules by preventing microtubule depolymerization and normal turnover of tubulin subunits (Arnal and Wade, 1995). Therefore, we also tested the hypothesis that disruption of dynamic microtubular activity could interfere with the extrusion of dying hair cells and the closure of the supporting cell scar formations. Here we report the effects of six cytoskeletal disruption agents (summarized in Fig. 2) on the extrusion of bullfrog saccular hair cells during cytotoxic insult in vitro. The data demonstrate that only actin disruption agents significantly prevent hair cell extrusion, and concomitantly arrest the expansion of the supporting cell apical processes, preventing supporting cell scar formation.

Section snippets

Materials and methods

All reagents were from Sigma (Saint Louis, MO) unless otherwise stated.

Hair bundle density

The left ear provided the control saccular explant (incubated with or without cytoskeletal inhibitors) and the right ear the experimental saccular explant (gentamicin-treated with or without cytoskeletal disruption agents). At time-point 0, there was a statistically negligible difference (1.8%) in the density of hair bundles between the right ear (87.8 ± 2.2 bundles per 10,000 μm², n = 3) and left ear (89.4 ± 4.3 per 10,000 μm², n = 3, p > 0.05). After 24 h of incubation in ACM alone (left saccule), there

Discussion

Cytoskeletal disruption agents had three levels of effect on bullfrog saccular explants treated with aminoglycosides. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion, while actin disruption agents blocked scar formation, and largely prevented the extrusion of dying hair cells. These effects and other data are discussed below.

Acknowledgements

Funded by NIDCD R01 04555 (P.S.S.), 04555s (A.J.H.); P30 05983 and by the Oregon Lions Sight and Hearing Foundation. The authors thank Dennis Trune, Huy Bui and Juany Rehling for assistance with transmission light and electron microscopy, and Takatoshi Karasawa for comments on the manuscript.

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Research paperClosure of supporting cell scar formations requires dynamic actin mechanisms

Abstract

Introduction

Section snippets

Materials and methods

Hair bundle density

Discussion

Acknowledgements

Hear. Res.

Curr. Biol.

Curr. Biol.

Curr. Biol.

J. Biol. Chem.

J. Biol. Chem.

Hear. Res.

FEBS Lett.

Hear. Res.

J. Invest. Dermatol.

Mol. Cells

Exp. Cell. Res.

Hear. Res.

Neuropharmacology

J. Biol. Chem.

J. Biol. Chem.

Hear. Res.

Hear. Res.

J. Biol. Chem.

Curr. Biol.

Hear. Res.

Cell

Mol. Genet. Metab.

Differentiation

Curr. Biol.

Eur. J. Cell. Biol.

Int. J. Dev. Neurosci.

Hear. Res.

Mitotic and nonmitotic hair cell regeneration in the bullfrog vestibular otolith organs

Ann. NY Acad. Sci.

The actin cytoskeleton is required for the trafficking of the B cell antigen receptor to the late endosomes

Traffic

Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change

Nature

Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis

J. Cell. Biol.

Myosin light chain kinase plays a role in the regulation of epithelial cell survival

J. Cell. Sci.

Research paper
Closure of supporting cell scar formations requires dynamic actin mechanisms