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The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15 N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips. Precise control of stereocilia length by auditory hair cells is vital for normal hearing. Drummond et al . follow in real-time the incorporation of actin into these structures and show that while the actin core is remarkably stable, and actin polymerization is limited to their distal tips.

Assembly of actin-based stereocilia is critical for cochlear hair cells to detect sound. To tune their mechanosensivity, stereocilia form bundles composed of graded rows of ascending height, necessitating the precise control of actin polymerization. Myosin 15 (MYO15A) drives hair bundle development by delivering critical proteins to growing stereocilia that regulate actin polymerization via an unknown mechanism. Here, we show that MYO15A is itself an actin nucleation-promoting factor. Moreover, a deafness-causing mutation in the MYO15A actin-binding interface inhibits nucleation activity but still preserves some movement on filaments in vitro and partial trafficking on stereocilia in vivo. Stereocilia fail to elongate correctly in this mutant mouse, providing evidence that MYO15A-driven actin nucleation contributes to hair bundle biogenesis. Our work shows that in addition to generating force and motility, the ATPase domain of MYO15A can directly regulate actin polymerization and that disrupting this activity can promote cytoskeletal disease, such as hearing loss. Actin filament polymerization is crucial for building sound-sensitive stereocilia in the cochlea. Here, the authors show that a myosin motor can nucleate actin filaments, revealing a mechanism for stereocilia growth and hereditary hearing loss.

Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. These auditory brainstem caspase-3 substrates were enriched for proteins previously shown to be cleaved by caspase-3, especially in non-apoptotic contexts. Functional annotation analysis revealed that our caspase-3 substrates were also enriched for proteins associated with several protein categories, including proteins found in extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem was highly enriched for our caspase-3 substrates. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of extracellular vesicle (EV) proteins.

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heatshocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

The members of the actin regulatory family of Ena/VASP proteins form stable tetramers. The vertebrate members of the Ena/VASP family, VASP, Mena and EVL, have many overlapping properties and expression patterns, but functional and regulatory differences between paralogues have been observed. The formation of mixed oligomers may serve a regulatory role to refine Ena/VASP activity. While it has been assumed that family members can form mixed oligomers, this possibility has not been investigated systematically. Using cells expressing controlled combinations of VASP, Mena and EVL, we evaluated the composition of Ena/VASP oligomers and found that VASP forms oligomers without apparent bias with itself, Mena or EVL. However, Mena and EVL showed only weak hetero-oligomerization, suggesting specificity in the association of Ena/VASP family members. Co-expression of VASP increased the ability of Mena and EVL to form mixed oligomers. Additionally, we found that the tetramerization domain (TD) at the C-termini of Ena/VASP proteins conferred the observed selectivity. Finally, we demonstrate that replacement of the TD with a synthetic tetramerizing coiled coil sequence supports homo-oligomerization and normal VASP subcellular localization.

Dendritic spines are signaling microcompartments that serve as the primary site of synapse formation in neurons. Actin assembly and myosin 2 contractility play major roles in the maturation of spines from filopodial precursors, as well as in the subsequent, activity-dependent changes in spine morphology that underly learning and memory. Myosin 18A is a myosin 2-like protein conserved from flies to man that lacks motor activity, is sub-stochiometric to myosin 2, and co-assembles with myosin 2 to make mixed filaments. Myosin 18A is alternatively spliced to create multiple isoforms that contain unique N- and C-terminal extensions harboring both recognizable and uncharacterized protein: protein interaction domains. These observations suggest that myosin 18A serves to recruit proteins to mixed filaments of myosin 2 and myosin 18A. One protein known to bind to myosin 18A is β-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42. Notably, β-Pix has been shown to promote spine maturation by activating both Arp2/3 complex-dependent branched actin filament assembly and myosin 2 contractility within spines. Here we show that myosin 18Aα is expressed in cerebellar Purkinje neurons and concentrates in spines along with myosin 2 and F-actin. Myosin 18Aα is targeted to spines by co-assembling with myosin 2 and by an actin binding site present in its N-terminal extension. miRNA-mediated knockdown of myosin 18Aα results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18Aα. Importantly, β-Pix co-localizes with myosin 18Aα in spines, and its spine localization is lost upon myosin 18A&α knockdown or when its myosin 18Aα binding site is deleted. Finally, we show that the spines of myosin 18Aα knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data demonstrate that mixed filaments of myosin 2 and myosin 18Aα form a complex with β-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of β-Pixs GEF activity.