Understanding Actin Dynamics and Its Regulatory Mechanisms in Developing Stereocilia
Stereocilia, the actin-based protrusions of auditory hair cells, are essential for converting mechanical stimuli into electrical signals. Their formation and maintenance require tightly regulated actin dynamics, including elongation and widening through filamentous actin (F-actin) incorporation. Here, we show that newly expressed actin first incorporates at stereocilia tips and then extends along the shaft to promote widening, with additional filaments assembling at the periphery of a stable actin core. Notably, we detected signals of both barbed and pointed ends present at stereocilia tips, revealing a previously unrecognized population of short actin filaments that contribute to stereocilia growth. Overexpression of actin further enriched short filaments along the shaft, linking their abundance to widening. Short filament levels correlated with the presence of the unconventional myosins MYO3A/B and MYO15A at stereocilia tips, implicating these motors in generating or stabilizing filaments required for elongation and widening. Focusing on class III myosins, we demonstrate that MYO3A is the predominant paralog in cochlear inner hair cells, indispensable for determining bundle dimensions and preventing overgrowth. Although MYO3B accumulates at tips in the absence of MYO3A, it provides only partial compensation, failing to maintain short filament populations or transport cargo such as ESPN-L1 to stereocilia tips. Strikingly, simultaneous deletion of MYO3A and ESPN1 eliminated MYO3B from stereocilia tips and sharply reduced tip width, suggesting that MYO3 complexes regulate actin incorporation specifically at the upper shaft. Moreover, kinase-deficient MYO3 mutants produced excessively long stereocilia and disrupted the distribution of MYO7A and EPS8L2, uncovering an unexpected role for the kinase domain in controlling tip-localized actin dynamics. Together, these findings establish a mechanism by which stereocilia widen and uncover MYO3A as a central regulator of tip widening in cochlear hair cells, providing mechanistic insights into how its dysfunction leads to DFNB30-associated hearing loss.
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