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

Abstract

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.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

“…Other mutations involving proteins associated with cochlear function and/or maturation either cause the loss of hair cells or specific structural or functional deficits during defined periods of development, ranging from embryonic to postnatal stages (5). In mutants in which hair cells remain viable, there is generally a failure in the down-regulation of immature hair cell biophysical characteristics and in the acquisition of adult properties (6)(7)(8)(9). The data presented here for miR-96 exemplify a coherent developmental brake that prevents the general biophysical and morphological differentiation between IHCs and OHCs that normally occurs from around birth.…”

Myosin VI and its cargo adaptors – linking endocytosis and autophagy

“…For example, the region containing the RRL motif interacts with nuclear dot protein 52 (NDP52), Traf6-binding protein (T6BP), optineurin and GAIP-interacting protein C-terminus (GIPC), and the region encompassing the WWY motif is required for binding to Tom1, Dab2 and lemur tyrosine kinase-2 (LMTK2) (Bunn et al, 1999;Chibalina et al, 2007;Morris et al, 2002;Morriswood et al, 2007;Sahlender et al, 2005;Spudich et al, 2007;Tumbarello et al, 2012). For other myosin VI adaptor molecules, such as SAP97 (Wu et al, 2002), otoferlin (Heidrych et al, 2009), phospholipase Cd3 (Sakurai et al, 2011) and Dock7 (Majewski et al, 2012), the exact binding domain has not yet been determined. The two subdomains containing the RRL and WWY protein-protein interaction sites are linked by a phospholipid binding motif that interacts specifically and with high affinity to PtdIns(4,5)P2 (Spudich et al, 2007).…”

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

“…However, otoferlin is distinct from synaptotagmin I in that it contains multiple fusion-active C2 domains linked in series and possesses longer linkers between each C2 domain that may endow otoferlin with flexibility and reach. When combined with the findings that otoferlin binds myosin VI and calcium channels (Heidrych et al, 2009;Ramakrishnan et al, 2009), it is tempting to speculate that the protein links vesicles to SNAREs and calcium channels in a docked and primed state. Given our finding that most of the C2 domains of otoferlin can directly regulate membrane fusion and the recent proposal that synaptic vesicles bound to ribbons are capable of compound fusion (Matthews and Sterling, 2008), the scenarios by which otoferlin might operate become numerous.…”