Abstract
Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.
Highlights
Dynamic actin turnover and rearrangement alter overall cell geometry and polarity, as well as local membrane topology, to form distinct cellular structures (Campellone and Welch, 2010)
Cdc42 immunoreactivity was detected in trichloroacetic acid (TCA)-fixed inner hair cells (IHCs) and outer hair cells (OHCs) at P0, with intense reactivity in stereocilia of Cdc42flox/flox mice (Fig. 1B), which was consistent with a report stating that Cdc42 is a hair bundle protein (Shin et al, 2013)
Here, we demonstrated for the first time the localization and activation of Cdc42 at the stereociliary membranes and apical junctional complexes (AJCs) in cochlear hair cells by the following two methods: exogenous expression of adenovirus-encoded GFP–Cdc42 in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells in an explant organ of Corti from Cdc42-FRET biosensor transgenic mice
Summary
Dynamic actin turnover and rearrangement alter overall cell geometry and polarity, as well as local membrane topology, to form distinct cellular structures (Campellone and Welch, 2010). In some structures, such as lamellipodia and the Listeria comet tail, actin filaments are highly dynamic with a turnover rate measured in seconds (Ponti et al, 2004; Theriot et al, 1992), whereas they are relatively stable in stereocilia (Schwander et al, 2010) and apical junctional complexes (AJCs) (Ivanov et al, 2005). The mode and effect of actin turnover at AJCs remain unknown (Nunes et al, 2006)
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