Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids

Cadherin-23 is a component of early transient lateral links of the auditory sensory cells' hair bundle, the mechanoreceptive structure to sound. This protein also makes up the upper part of the tip links that control gating of the mechanoelectrical transduction channels. We addressed the issue of the molecular complex that anchors these links to the hair bundle F-actin core. By using surface plasmon resonance assays, we show that the cytoplasmic regions of the two cadherin-23 isoforms that do or do not contain the exon68-encoded peptide directly interact with harmonin, a submembrane PDZ (post-synaptic density, disc large, zonula occludens) domain-containing protein, with unusually high affinity. This interaction involves the harmonin Nter-PDZ1 supramodule, but not the C-terminal PDZ-binding motif of cadherin-23. We establish that cadherin-23 directly binds to the tail of myosin VIIa. Moreover, cadherin-23, harmonin and myosin VIIa can form a ternary complex, which suggests that myosin VIIa applies tension forces on hair bundle links. We also show that the cadherin-23 cytoplasmic region, harmonin and myosin VIIa interact with phospholipids on synthetic liposomes. Harmonin and the cytoplasmic region of cadherin-23, both independently and as a binary complex, can bind specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which may account for the role of this phospholipid in the adaptation of mechanoelectrical transduction in the hair bundle. The distributions of cadherin-23, harmonin, myosin VIIa and PI(4,5)P2 in the growing and mature auditory hair bundles as well as the abnormal locations of harmonin and myosin VIIa in cadherin-23 null mutant mice strongly support the functional relevance of these interactions

Caractérisation fonctionnelle et structurale de trois myosines non conventionnelles (la myosine VI, la myosine VIIa et la myosine IB d'Acanthamoeba)

Résumé français (1000 caractères maxi)Résumé anglais (idem)ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Molecular characterization of canine parvovirus-2 variants circulating in Tunisia

International audienceCanine parvovirus type 2 (CPV2) emerged in 1978 as a highly contagious and very serious disease in dogs. The characterization of CPV2 antigenic types is exclusively based on the identification of the amino acid residue at position 426 of the capsid protein VP2. Currently, three antigenic types CPV-2a (asparagine N-426), CPV-2b (aspartic acid D-426) and CPV-2c (glutamic acid E-426) are circulating worldwide. In Tunisia, despite the fact that many clinical and few serological investigations clearly indicate that CPV is widespread and of major concerns in the local dog population, no molecular and antigenic type characterization of circulating variants has been carried out. This investigation showed that most of clinically presumed CPV infections were confirmed by classical or real-time PCR. When no real-time PCR facilities were affordable, classical PCR as reported here in association with restriction fragment length polymorphism (RFLP) with MboI and MboII can be very useful for screening and diagnosing CPV infections. A total of 50 variants were characterized by sequencing and an almost even representation of the different antigenic types, including CPV-2c and slightly more type 2b, were evidenced. Characterization of the Tunisian variants by MGB probe assays as reported was inefficient for most of CPV-2a variants because of their typical nucleotide mutation C-1269. Phylogenetic analysis showed that the Tunisian variants underwent evolution for a relatively long period of time inside the country. The analysis also showed some crossings of the different antigenic types, leaving both genotypic and phenotypic characteristic mutations

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa.

International audienceBy using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa.

International audienceBy using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process

Conformational switch of harmonin, a submembrane scaffold protein of the hair cell mechanoelectrical transduction machinery

International audienceMutations in the gene encoding harmonin, a multi-PDZ domain-containing submembrane protein, cause Usher syndrome type 1 (congenital deafness and balance disorder, and early-onset sight loss). The structure of the protein and biological activities of its three different classes of splice isoforms (a, b, and c) remain poorly understood. Combining biochemical and biophysical analyses , we show that harmonin-a1 can switch between open and closed conforma-tions through intramolecular binding of its C-terminal PDZ-binding motif to its N-terminal supramodule NTD-PDZ1 and through a flexible PDZ2-PDZ3 linker. This conformational switch presumably extends to most harmonin iso-forms, and it is expected to have an impact on the interaction with some binding partners, as shown here for cadherin-related 23, another component of the hair cell mechanoelectrical transduction machinery

Myosin VIIa, harmonin and cadherin23, three Usher I gene products cooperate to shape the sensory hair bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia

Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice.: Usher 1 retinal pathogenesis

International audienceThe mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients