Article16 December 2002free access Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle Batiste Boëda Batiste Boëda Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Aziz El-Amraoui Aziz El-Amraoui Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Amel Bahloul Amel Bahloul UMR 144 CNRS/IC, Institut Curie, 26 rue d'Ulm, 75248 Paris, cedex 05, France Search for more papers by this author Richard Goodyear Richard Goodyear School of Biological Sciences, The University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Laurent Daviet Laurent Daviet Hybrigenics, 3–5 impasse Reille, 75014 Paris, France Search for more papers by this author Stéphane Blanchard Stéphane Blanchard Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Isabelle Perfettini Isabelle Perfettini Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Karl R. Fath Karl R. Fath Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany Present address: Queens College of CUNY, 65–30 Kissena Blvd, Flushing, NY, 11367 USA Search for more papers by this author Spencer Shorte Spencer Shorte Centre d'Imagerie Dynamique, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Jan Reiners Jan Reiners Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany Search for more papers by this author Anne Houdusse Anne Houdusse UMR 144 CNRS/IC, Institut Curie, 26 rue d'Ulm, 75248 Paris, cedex 05, France Search for more papers by this author Pierre Legrain Pierre Legrain Hybrigenics, 3–5 impasse Reille, 75014 Paris, France Search for more papers by this author Uwe Wolfrum Uwe Wolfrum Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany Search for more papers by this author Guy Richardson Guy Richardson School of Biological Sciences, The University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Christine Petit Corresponding Author Christine Petit Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Batiste Boëda Batiste Boëda Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Aziz El-Amraoui Aziz El-Amraoui Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Amel Bahloul Amel Bahloul UMR 144 CNRS/IC, Institut Curie, 26 rue d'Ulm, 75248 Paris, cedex 05, France Search for more papers by this author Richard Goodyear Richard Goodyear School of Biological Sciences, The University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Laurent Daviet Laurent Daviet Hybrigenics, 3–5 impasse Reille, 75014 Paris, France Search for more papers by this author Stéphane Blanchard Stéphane Blanchard Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Isabelle Perfettini Isabelle Perfettini Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Karl R. Fath Karl R. Fath Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany Present address: Queens College of CUNY, 65–30 Kissena Blvd, Flushing, NY, 11367 USA Search for more papers by this author Spencer Shorte Spencer Shorte Centre d'Imagerie Dynamique, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Jan Reiners Jan Reiners Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany Search for more papers by this author Anne Houdusse Anne Houdusse UMR 144 CNRS/IC, Institut Curie, 26 rue d'Ulm, 75248 Paris, cedex 05, France Search for more papers by this author Pierre Legrain Pierre Legrain Hybrigenics, 3–5 impasse Reille, 75014 Paris, France Search for more papers by this author Uwe Wolfrum Uwe Wolfrum Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany Search for more papers by this author Guy Richardson Guy Richardson School of Biological Sciences, The University of Sussex, Falmer, Brighton, BN1 9QG UK Search for more papers by this author Christine Petit Corresponding Author Christine Petit Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France Search for more papers by this author Author Information Batiste Boëda1, Aziz El-Amraoui1, Amel Bahloul2, Richard Goodyear3, Laurent Daviet4, Stéphane Blanchard1, Isabelle Perfettini1, Karl R. Fath5,6, Spencer Shorte7, Jan Reiners5, Anne Houdusse2, Pierre Legrain4, Uwe Wolfrum5, Guy Richardson3 and Christine Petit 1 1Unité de Génétique des Déficits Sensoriels, CNRS URA 1968, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France 2UMR 144 CNRS/IC, Institut Curie, 26 rue d'Ulm, 75248 Paris, cedex 05, France 3School of Biological Sciences, The University of Sussex, Falmer, Brighton, BN1 9QG UK 4Hybrigenics, 3–5 impasse Reille, 75014 Paris, France 5Institut für Zoologie, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany 6Present address: Queens College of CUNY, 65–30 Kissena Blvd, Flushing, NY, 11367 USA 7Centre d'Imagerie Dynamique, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, France ‡B.Boëda and A.El-Amraoui contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:6689-6699https://doi.org/10.1093/emboj/cdf689 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Usher syndrome (USH) is the most frequent cause of deaf-blindness in humans. Three clinical subtypes, USH1–3, have been described (reviewed in Petit, 2001). USH1 is the most severe form. It is characterized by severe to profound congenital sensorineural deafness, constant vestibular dysfunction (balance deficiency) and pre-pubertal onset retinitis pigmentosa. USH1 is genetically heterogeneous. Evidence for seven distinct genetic loci (USH1A–G) has been obtained, and four of the corresponding genes have been identified, namely USH1B, C, D and F (Petit, 2001; Mustapha et al., 2002). USH1B encodes the actin-based motor protein myosin VIIa (Weil et al., 1995). USH1C encodes harmonin (Bitner-Glindzicz et al., 2000; Verpy et al., 2000), a protein that contains PDZ domains (postsynaptic density, disc large, zonula occludens), modules known as organizers of submembranous protein complexes (Sheng and Sala, 2001). Alternatively spliced USH1C transcripts (Verpy et al., 2000) predict at least 10 protein isoforms which can be grouped into three subclasses, hereafter referred to as harmonin a, b, and c, and collectively as harmonin (Figure 1A). Finally, mutations in the genes encoding two cadherin-related proteins, cadherin 23 and protocadherin 15, underlie USH1D (Bolz et al., 2001; Bork et al., 2001) and USH1F (Ahmed et al., 2001; Alagramam et al., 2001a), respectively. Figure 1.(A) Predicted structures of harmonin a, b and c isoforms. Class a isoforms contain three PDZ domains and one coiled-coil (CC1) domain. The longest isoforms are grouped into class b; they contain an additional coiled-coil domain (CC2), and a proline, serine, threonine (PST)-rich region. Class c isoforms are the shortest; they contain only the first two PDZ domains and the first coiled-coil domain. (B) Schematic representation of an inner ear sensory hair cell. At the apical surface of the hair cell, a number of microvilli-like structures, called stereocilia, form the hair bundle. Each stereocilium contains a core of actin filaments (shown in red in the right panel). The central actin filaments of all the stereocilia insert into the cuticular plate, i.e. a dense meshwork of actin filaments lying beneath the apical cell surface. (C–F) Harmonin b in the vestibular hair cells (mouse). (C and D) At E14, harmonin b isoforms are only detected at the apical surface of the hair cells, whereas myosin VIIa staining delineates the whole cells. In a few cells, harmonin b immunoreactivity appears as a circle of bead-like foci located around the periphery of the cuticular plate (D and insets). At P8, harmonin b is located at the tips of the stereocilia (E, and detail in F), whereas myosin VIIa is present in both the hair cell bodies and stereocilia (E and F). (G–I) Harmonin in the cochlear hair cells (rat). (G) Schematic representation of the cochlear auditory organ (organ of Corti). It is made up of sensory cells (in red), namely the single row of inner hair cells (ihc) and the three rows of outer hair cells (ohc), and various types of supporting cells (in blue). (H and I) Distribution of harmonin isoforms in the rat organ of Corti at P4. Harmonin b is only detected in the hair bundle (H) of the sensory cells whereas, using the NW2 pan-harmonin antibody, harmonin isoforms a, b and c are detected in the cuticular plate as well as in the apical hair bundle (I). Asterisks indicate hair cell bodies. Bars: 10 μm in (C–E) and (H–I); and 5 μm in (F). Download figure Download PowerPoint Mouse mutants defective for myosin VIIa (shaker-1) (Mburu et al., 1997), cadherin 23 (waltzer) (Di Palma et al., 2001) and protocadherin 15 (Ames waltzer) (Alagramam et al., 2001b) have been reported. They are all deaf and exhibit vestibular dysfunction. In these mutants, the sensory cells of the cochlea (the auditory organ) and vestibular end organs (balance organs) display anomalies in the development of their hair bundle. This mechanosensory structure is composed of an ensemble of 30–300 microvilli called stereocilia, that project from the apical surface of the hair cell (DeRosier and Tilney, 2000) (Figure 1B). As in other microvilli, stereocilia are tightly packed with actin filaments that are uniformly polarized and have their barbed end, i.e. their faster growing end, located at the tip (DeRosier and Tilney, 2000). However, stereocilia have several specific morphological features: (i) they are wider and longer than the microvilli of 'conventional' epithelial cells and can contain up to 2000 actin filaments in some species; (ii) they taper off at their base, where they insert into the apical surface of the cell, with only a few centrally located actin filaments extending through this region to anchor them into the cuticular plate (Figure 1B, right panel), a dense meshwork of cross-linked actin filaments (DeRosier and Tilney, 1989); (iii) they are packed into rows of increasing height to form a highly organized and uniformly oriented hair bundle (Figure 1B); and (iv) they are connected by several different types of extracellular links (Pickles et al., 1984; Goodyear and Richardson, 1999). Upon deflection in response to the sound or acceleration stimulus, the stereocilia all pivot around their basal insertion points, causing opening or closing of the transduction channels, and thus fluctuations in the cell's membrane potential (Hudspeth, 1997). With the exception of some studies addressing the role of myosin VIIa (Gale et al., 2001; Tuxworth et al., 2001; El-Amraoui et al., 2002; Kros et al., 2002), the functions of the proteins underlying USH1 remain elusive. Here we address the role of harmonin in the inner ear. Because the three aforementioned mouse models of USH1 have disorganized hair cell stereocilia, and because harmonin, within the inner ear, is restricted to the sensory cells (Verpy et al., 2000), we hypothesized that this protein also is involved in the development of a coherent hair bundle. This prompted us to seek molecular partners of this protein. Our results implicate harmonin, cadherin 23 and myosin VIIa in a single functional network that underlies the formation of a coherent hair cell bundle. Results Harmonin in differentiating hair cell stereocilia We studied the distribution of harmonin during the period of hair bundle differentiation in the mouse and rat. In the mouse, stereocilia sprout from the apical surface of vestibular and cochlear hair cells at E13 and E15, respectively (Nishida et al., 1998; Denman-Johnson and Forge, 1999). In the cochlea, hair cell differentiation proceeds from the base to the cochlear apex and, by P4–P6, the hair bundles reach their final adult shape (Nishida et al., 1998). A previous analysis of harmonin transcripts (see Figure 1A) has indicated that class b isoforms are restricted largely to the inner ear, whereas a and c forms have a much broader expression throughout the entire organism (Verpy et al., 2000). We thus focused our analysis on the b isoforms. Two polyclonal antibodies directed against the harmonin PST domain, that is only present in b isoforms, were generated. They both gave the same cellular distribution pattern. In the mouse vestibule, harmonin b was detected from E13 onwards, and it was restricted to the emerging stereocilia of the hair cells (Figure 1C and D). Detailed analysis by confocal microscopy showed that between E13 and E16, the harmonin b labelling delineated the entire length of the stereocilia (not shown). Likewise in the cochlea, harmonin b was first detected at E15 in the differentiating hair bundles of the basal turn hair cells. From E15 to E17, the immunoreactivity extended towards the cochlear apex. From E16 in the vestibule (Supplementary figure 1A and B, available at The EMBO Journal Online) and P0 in the cochlea, the protein became concentrated at the apex of the maturing stereocilia (Figure 1E–I and data not shown). From P30 onwards, harmonin b was no longer detected in the cochlea. In the vestibule, the labelling only persisted in a few sensory hair cells, in both type I and type II hair cells (not shown). In contrast to the harmonin b expression profile, an antibody that recognizes an epitope common to the three harmonin isoform classes (a, b and c) detected the proteins both in the cuticular plate and along the stereocilia (Figure 1G–I), during the embryonic and postnatal life of the animal. Harmonin b is an F-actin-bundling protein To address the function of harmonin, a representative of each of the three isoform classes was first transfected into HeLa cells. In cells producing harmonin a (Figure 2A) or harmonin c (not shown), the protein was distributed uniformly throughout the cell body. In contrast, in cells expressing harmonin b, the protein was associated with filamentous structures (Figure 2B). Double immunolabels with antibodies to α-tubulin, β-tubulin, cytokeratin 18, vimentin or a pan cytokeratin antibody showed that harmonin b was not associated with microtubules or intermediate filaments (not shown). Rather, harmonin b was co-localized with actin filaments labelled with rhodamine–phalloidin (Figure 2B–D). To determine which domain(s) of harmonin b interact with the actin cytoskeleton, several truncated versions of the protein were expressed in HeLa cells. The shortest harmonin b fragment that co-localized with the actin filaments encompasses the second coiled-coil domain through the C-terminal end (CC2-Cter, amino acids 405–859) of the protein (not shown). In transfected cells, harmonin b-labelled structures were observed as either punctiform or long and curvy filaments (Figure 2B–D). Using three-dimensional spatial analysis, we found that the former are present almost exclusively at the cell plasma membrane–substrate interface, whereas the latter extended up to 10 μm throughout the cell, both being distinct from the actin stress fibres. We then studied the dynamics of green fluorescent protein (GFP)–harmonin b distribution in HeLa cells by digital fluorescence microscopy. The harmonin b label was first observed 6 h after transfection. At this stage, the protein was restricted to and highly concentrated at the distal end (barbed ends) of actin fibres, where they anchored to substrate adhesion sites (Figure 2E–J). Vinculin, a major component of the focal adhesion plaques (Zamir and Geiger, 2001) (Figure 2E, F and H–J), overlapped with the distal part of the harmonin b staining (Figure 2H and I). Observations performed at different intervals from 8 to 48 h after transfection indicated that harmonin b label extended further from the focal adhesion sites forming the long and curvy bundles. However, this changing pattern was slow as we observed only moderate modification of harmonin b-labelled structures in cells analysed by time lapse during a 4 h interval (not shown). To characterize further the behaviour of actin filaments in the presence of harmonin b, cells were treated with either latrunculin A (Figure 3B), which binds to and sequesters actin monomers, or cytochalasin D (Figure 3C), that binds to the barbed end of actin filaments and alters actin polymerization. With either of these drugs, both the cortical actin filaments and the stress fibres were disrupted, whereas the curvy, harmonin b-bound, actin filaments were unaffected (Figure 3A–C). Together, these results suggest that harmonin b stabilizes the actin filaments. Figure 2.Harmonin b induces F-actin bundling in HeLa cells. (A–D) HeLa cells were transiently transfected with either harmonin a (A) or harmonin b (B–D) cDNAs for 20 h. (A) Harmonin a is distributed throughout the cell body. (B–D) In contrast, overexpression of harmonin b (green) leads to the formation of harmonin b-immunoreactive long curvy bundles that co-localize with actin filaments (red). (E–J) HeLa cells transfected with harmonin b and collected 6 h after transfection. At this stage, most harmonin b labelling is present almost exclusively at the cell plasma membrane–substrate interface. A triple staining with rhodamine–phalloidin (red), anti-vinculin (green) and anti-harmonin b (blue) antibodies reveals that harmonin b decorates the extremities of actin stress fibres (violet in E–G). The enrichment of harmonin b labelling (blue) occurs close to the focal adhesion plaques visualized by anti-vinculin (green) staining (H–J). (F), (G), (I) and (J) are higher magnification views of the areas boxed in (E) and (H), respectively. Harmonin b and F-actin are both enriched at the distal ends of actin fibres (violet in F and G), and they slightly overlapped with the vinculin staining (I and J). Bars: 10 μm. Download figure Download PowerPoint Figure 3.(A–C) Harmonin b induces a resistance of actin filament to latrunculin A and cytochalasin D. (A) Transfected HeLa cells labelled for F-actin (red) and harmonin b (green). Actin stress fibres are observed in transfected cells that produce harmonin b as well as in the surrounding untransfected cells. (B and C) The disruptive effect of latrunculin A (B) and cytochalasin D (C) on actin filaments is not observed in transfected cells that produce harmonin b. With either of these drugs, both the cortical actin filaments and the harmonin b-unlabelled stress fibres were disrupted, whereas the har monin b-associated actin fibres were unaffected. (D–F) Harmonin b is an actin-bundling protein. Light microscopy (E) and electron microscopy (F and G) demonstration of F-actin bundle formation. (D) In the presence of GST-tagged CC2-Cter harmonin fragment, large and long F-actin bundles are observed. No bundled F-actin is observed in the absence of this protein (inset in D). In vitro electron microscopy analysis showed that actin filaments assemble into bundles in the presence (E), but not in the absence (F) of harmonin b. Bars: 10 μm in (A–C), 5 μm in (D) and 60 nm in (E) and (F). Download figure Download PowerPoint To determine how harmonin b interacts with F-actin, we then carried out in vitro binding assays (see Materials and methods). Harmonin b and rhodamine–phalloidin-labelled actin filaments were incubated together, and samples were visualized by light microscopy or were negatively stained for electron microscopy. We found that in the presence of the GST-tagged CC2-Cter harmonin b fragment (Figure 3D) or harmonin b (Figure 3E and F), actin filaments collected into large bundles. Upon GST cleavage by thrombin, the CC2-Cter harmonin b fragment also promoted F-actin bundling (Supplementary figure 3A). This actin-bundling activity of harmonin b was not affected by a high calcium concentration (10 mM) or calcium chelating agents (10 mM EGTA) (not shown). To determine whether harmonin b associates with these actin bundles, we mixed the GST-tagged CC2-Cter harmonin b fragment with actin filaments to allow bundling, then the bundles were separated from soluble proteins and single actin filaments by low-speed centrifugation. In the presence of the GST–CC2-Cter harmonin, the vast majority of F-actin was recovered in the pellet (Supplementary figure 3B). Finally, we demonstrated the direct binding of the CC2-Cter harmonin b fragment to F-actin by using CC2-Cter- or GST-coated beads incubated with actin filaments (Supplementary figure 3C and D). These results demonstrate that harmonin b has an actin-bundling activity that is independent of calcium. Cadherin 23 is present in the stereocilia and binds to harmonin b Mutations of the gene encoding cadherin 23 cause USH1D in man. To study the distribution of cadherin 23 in the mouse developing inner ear, we raised three polyclonal antibodies against extracellular or intracellular regions of the protein (see Materials and methods). Just like harmonin b, cadherin 23 was detected along the entire stereocilia as soon as they emerge, and subsequently became restricted to their apical regions (Figure 4A–F), as confirmed by immunoelectron microscopy on P3 cochlear hair cells (Figure 4G and H). Both in the vestibule and in the cochlea, the temporal pattern was also identical to that of harmonin b. From P30, cadherin 23 was no longer detected in the cochlear stereocilia and only persisted in a few vestibular type I and type II hair cells. The disappearance of cadherin 23 from the stereocilia of adult mice indicates that the protein is not a component of the apical links of mature hair bundles. Figure 4.Cadherin 23 in the differentiating hair cells (mouse). (A and B) In the vestibule, at E14, cadherin 23 is only detected at the apical surface of the hair cells (A), whereas myosin VIIa is detected in the entire hair cells (B). Up to E16, cadherin 23 delineates the entire length of the hair bundle, as shown by double staining for cadherin 23 and F-actin (C). (D–F) In a tangential section of a P8 vestibular macula, cadherin 23 (green) is also detected in the hair bundles, mainly at the tips of the stereocilia, visualized by F-actin staining (red). (G and H) Electron microscopy of a hair bundle from a P3 mouse cochlear hair cell. Cadherin 23 is detected between adjacent stereocilia, especially at their apical ends, as shown at higher magnification (arrowheads in H). Bars: 20 μm in (A) and (B); 3 μm in (C); 10 μm in (D); and (E), 5 μm in (F) and 100 nm in (G) and (H). Download figure Download PowerPoint The similar spatio-temporal distributions of harmonin b and cadherin 23 in the stereocilia suggested that these molecules may interact. We therefore examined whether such an interaction could be detected in co-transfected HeLa cells (Figure 5). In cells producing the cadherin 23 cytodomain (amino acids 3086–3354) and harmonin b, the two proteins entirely co-localized throughout the cell cytoplasm (not shown). To determine whether cadherin 23 could recruit harmonin b to the cell membrane, we analysed its distribution in the presence of the hEcad–cad23 chimeric protein. The hEcad–cad23 protein is composed of the extracellular and transmembrane domains of Ecadherin directly linked to the cytodomain of cadherin 23 (Figure 5A). In HeLa cells producing the two proteins, the hEcad–cad23 chimera was detected at the cell–cell contacts, where it recruited harmonin b. In contrast, harmonin b was absent at cell–cell contacts lacking the hEcad–cad23 chimera (Figure 5B). Moreover, the presence of hEcad–cad23 modified the harmonin b–actin pattern, i.e. long harmonin b-bound curvy filaments changed into punctate labelled structures (Figure 5B; Supplementary figure 5), thus suggesting an association between harmonin b and cadherin 23. Pull-down assays were then performed. Extracts of transfected HEK293 cells producing either harmonin a, harmonin b or the myosin VIIa tail (amino acids 848–2215) were incubated with immobilized GST-tagged cadherin 23 cytodomain (amino acids 3086–3354) or GST alone. Harmonins a and b were recovered with the GST-tagged cadherin 23 cytodomain, but not with GST alone. In contrast, the myosin VIIa tail was not recovered (Figure 6A). The interaction between cadherin 23 and harmonin was analysed further by an in vitro binding assay (Figure 6B). Different harmonin fragments were produced (see Materials and methods) and incubated with immobilized biotin-tagged cadherin 23 cytodomain. Both the PDZ1–PDZ2 peptide (amino acids 138–403) of harmonin and the PDZ2 domain alone (amino acids 189–307) bound to the cadherin 23 cytodomain, whereas binding was not observed with either PDZ1 or PDZ3 (Figure 6B and E). Figure 5.Harmonin b co-localizes with hEcad–cad23 in co-transfected HeLa cells. (A) Schematic diagram of the hEcad–cad23 chimera: it is composed of the five extracellular cadherin repeats (EC) and the transmembrane domain (TM) of human Ecadherin (hEcad) fused to the human cadherin 23 cytodomain (cad23). (B) HeLa cells were transiently transfected with either hEcad–cad23 or harmonin b cDNAs for 20 h. The hEcad–cad23 chimeric protein is targeted to cell–cell contacts (green; revealed by Pcad-C) essentially at the junctions between two transfected cells (arrowheads). Harmonin b (V5-tagged) is also recruited to these contacts, where it co-localized with the hEcad–cad23 chimera and with actin filaments (red). Neither the hEcad–cad23 chimera nor harmonin b is enriched at cell–cell contacts established with untransfected cells (arrows). In the cell cytoplasm, the presence of the hEcad–cad23 protein shifts the filamentous pattern usually observed with harmonin b, to punctiform actin-rich structures. Asterisks indicate HeLa cell bodies. Bar: 20 μm. Download figure Download PowerPoint Figure 6.Harmonin binds to cadherin 23 and myosin VIIa. (A) Pull-down assay. Extracts of transfected HEK293 cells producing either harmonin a, harmonin b or the myosin VIIa tail were incubated with immobilized GST-tagged cadherin 23 cytodomain or GST alone. The harmonins a and b bind to the GST-tagged cadherin 23 cytodomain but not to GST alone. The myosin VIIa tail does not bind to cadherin 23. (B–D) In vitro binding assays. (B) Characterization of the harmonin–cadherin 23 interaction domain. Different harmonin fragments were incubated with immobilized biotin-tagged cadherin 23 cytodomain. Only the PDZ1–PDZ2 peptide (amino acids 138–403) of harmonin and the PDZ2 domain alone (amino acids 189–307) bound to the cadherin 23 cytodomain. Biotin-tagged CAT (chloramphenicol acetyltransferase) was used as a negative control. (C) The C-terminal MyTH4 + FERM repeat of myosin VIIa (myosin VIIa-Cter) is incubated with different GST-tagged proteins. The myosin VIIa-Cter interacts with GST-tagged harmonin a, and with GST–MyRIP (used as a positive control), whereas it fails to bind to GST. The ezrin FERM domain (used as a negative control) does not bind to GST–harmonin a. (D) Characterization of the harmonin–myosin VIIa interaction domain. The myosin VIIa-Cter was incubated with different immobilized GST-tagged harmonin fragments. The PDZ1 domain of harmonin, but neither PDZ2 nor PDZ3, binds to myosin VIIa. (E) Schematic diagram illustrating how harmonin b could interact with myosin VIIa, cadherin 23 and F-actin. Acronyms: FERM (4.1, ezrin, radixin, moesin); MyTH4 (myosin tail homology 4); SH3 (src homology-3), IQ (isoleucine–glutamine motifs); EC (extracellular cadherin repeats).
