At the first auditory synapse in mammals, one ribbon-type AZ of the IHC drives one postsynaptic spiral ganglion neuron (SGN) to spike at rates exceeding 100 Hz in silence and 1 kHz upon sound onset 1, 2 . Moreover, SGNs sustain firing rates of several hundred Hz during ongoing acoustic stimulation. In such a steady state, vesicle replenishment has to balance vesicle fusion at the IHC AZ. Accordingly, high rates of initial and sustained exocytosis have been found in hair cells [3][4][5][6][7][8] . Ribbon-type AZs of IHCs replenish readily releasable vesicles at hundreds of Hz over several seconds of stimulation, faster than ribbon synapses in the eye 9-15 and most non-ribbon-type AZs 16(but see ref. 17). This efficient vesicle re-supply maintains a large standing pool of fusion competent synaptic vesicles, which appears to be critical for reliable and temporally precise sound encoding [18][19][20] . and Otof +/+ mice ( Fig. 2a-b The observations of normal RRP size after resting the synapse for more than 30 seconds and of reduced vesicle re-supply during ongoing stimulation prompted us to explore RRP recovery from depletion in paired-pulse experiments (Fig. 3a). RRP recovery, assessed as the paired-pulse ratio for different inter-pulse intervals, was impaired in Otof Pga/Pga mice (Fig. 3b). This indicated a deficit in vesicle replenishment also in the rest period between stimuli. The ΔC m pattern elicited by trains of ten short (10 ms) depolarizations demonstrates the exocytosis phenotype found after a period of rest (30 s voltage-clamp at -84 mV): normal RRP exocytosis but subsequent failure (Fig. 3c). Studying the C m decline after exocytosis we observed normal endocytic membrane retrieval (Fig. 3d). Normal synaptic ultrastructure in Otof Pga/Pga IHCsIn order to explore whether a docking or a priming defect underlies the impairment of vesicle replenishment at Otof Pga/Pga IHC synapses, we studied their ultrastructure using electron microscopy (EM). Both, EM of single ultrathin sections (perpendicular to the 7 plasma membrane and the long axis of the ribbon; Supplementary Fig. 2) as well as EM tomography (Fig. 3e-f High-resolution EM tomography ( Fig. 3e-f) was used to measure the distance of membrane-proximal synaptic vesicles (labeled orange in Fig. 3f) from the plasma membrane under both conditions. The average membrane-membrane distance was approximately 6 nm regardless of condition and genotype (Supplementary Reduced rates but maintained size variability of EPSCsThe stark contrast between the absence of auditory neuron population responses in vivo (Fig. 1) (Fig. 4a-b). We pooled the data from recordings in 5. Fig. 4h-i; Kolmogorov-Smirnov test, p = 0.14). Moreover, we detected action potential generation by recording action currents in the loose-patch configuration (Fig. 4g). Together these results suggest that Otof Pga/Pga synapses should be capable of encoding sound into spiking activity in auditory nerve fibers, albeit at lower rates.In addition, we recorded from SGNs of Otof -/-mice of the same age a...

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the beta1 integrin family regulate myoblast fusion. beta1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of beta1-deficient myoblasts, suggesting that beta1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, beta1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of beta1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.

Mammals have an astonishing ability to sense and discriminate sounds of different frequencies and intensities. Fundamental for this process are mechanosensory hair cells in the inner ear that convert sound-induced vibrations into electrical signals. The study of genes that are linked to deafness has provided insights into the cell biological mechanisms that control hair cell development and their function as mechanosensors.

In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement.

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.