Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.

Quorum sensing contributes to activated IgM-secreting B cell homeostasis.

Maintenance of plasma IgM levels is critical for immune system function and homeostasis in humans and mice. However, the mechanisms that control homeostasis of the activated IgM-secreting B cells are unknown. After adoptive transfer into immune-deficient hosts, B lymphocytes expand poorly, but fully reconstitute the pool of natural IgM-secreting cells and circulating IgM levels. By using sequential cell transfers and B cell populations from several mutant mice, we were able to identify novel mechanisms regulating the size of the IgM-secreting B cell pool. Contrary to previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells having overlapping survival requirements, homeostasis of the innate IgM-secreting B cell pool is also achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess the density of activated B cells by sensing their secreted IgG. This process involves the FcγRIIB, a low-affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation, and its intracellular effector the inositol phosphatase SHIP. As a result of the engagement of this inhibitory pathway, the number of activated IgM-secreting B cells is kept under control. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled B cell activation and autoimmunity.

Otoferlin interacts with myosin VI: implications for maintenance of the basolateral synaptic structure of the inner hair cell.

Otoferlin has been proposed to be the Ca(2+) sensor in hair cell exocytosis, compensating for the classical synaptic fusion proteins synaptotagmin-1 and synaptotagmin-2. In the present study, yeast two-hybrid assays reveal myosin VI as a novel otoferlin binding partner. Co-immunoprecipitation assay and co-expression suggest an interaction of both proteins within the basolateral part of inner hair cells (IHCs). Comparison of otoferlin mutants and myosin VI mutant mice indicates non-complementary and complementary roles of myosin VI and otoferlin for synaptic maturation: (i) IHCs from otoferlin mutant mice exhibited a decoupling of CtBP2/RIBEYE and Ca(V)1.3 and severe reduction of exocytosis. (ii) Myosin VI mutant IHCs failed to transport BK channels to the membrane of the apical cell regions, and the exocytotic Ca(2+) efficiency did not mature. (iii) Otoferlin and myosin VI mutant IHCs showed a reduced basolateral synaptic surface area and altered active zone topography. Membrane infoldings in otoferlin mutant IHCs indicated disturbed transport of endocytotic membranes and link the above morphological changes to a complementary role of otoferlin and myosin VI in transport of intracellular compartments to the basolateral IHC membrane.