Summary At the presynaptic active zone, Ca2+ influx triggers fusion of synaptic vesicles. It is not well understood how Ca2+-channel clustering and synaptic vesicle docking are organized. Here we studied structure and function of hair cell ribbon synapses following genetic disruption of the presynaptic scaffold protein Bassoon. Mutant synapses - mostly lacking the ribbon - showed a reduction in membrane-proximal vesicles, with ribbonless synapses affected more than ribbon-occupied synapses. Ca2+-channels were also fewer at mutant synapses and appeared in abnormally shaped clusters. Ribbon absence reduced Ca2+-channel numbers at mutant and wild-type synapses. Fast and sustained exocytosis were reduced notwithstanding normal coupling of the remaining Ca2+-channels to exocytosis. In-vitro recordings revealed a slight impairment of vesicle replenishment. Mechanistic modeling of the in-vivo data independently supported morphological and functional in-vitro findings. We conclude that Bassoon and the ribbon (1) create a large number of release sites by organizing Ca2+-channels and vesicles, and (2) promote vesicle replenishment.

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Harmonin is a scaffolding protein required for normal mechanosensory function in hair cells. Here, we describe a novel presynaptic association of harmonin and Cav1.3 Ca2+ channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

Six point mutations in non-muscle ␥-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each ␥-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.Hearing depends on sound-dependent distortion of specialized mechanosensory hair cells within the cochlea of the inner ear. In these cells, staircase arrangements of 20 -300 hair-like receptors called stereocilia protrude from the apical surface. Mechanical deflection of these structures results in the opening of gated ion channels located on the surface of these protrusions. The result is the conversion of sound-dependent distortion into the propagation of neural signals that are relayed to the brain (1).Hair cell function is contingent upon proper functioning of the actin cytoskeleton. Each mature stereocilium contains a core of actin filaments closely packed into rigid hexagonal bundles cross-linked by several actin-binding proteins (2). These filaments not only regulate the three-dimensional aspects of the stereocilia such as height and diameter, but also provide the rigidity necessary for proper functioning of the stereocilium. The stereocilia are embedded in a dense gel-like meshwork of actin filaments with random polarity called the cuticular plate (3). This structure provides a base in which the stereocilia are anchored and assists in maintaining their erect positions. Encircling each hair cell near its apical surface is the zonula adherens, a circumferential belt of actin filaments that runs parallel to the plasma membrane (4). These rings form focal contacts with neighbor...