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Christine Petit - One of the best experts on this subject based on the ideXlab platform.
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Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis.
Molecular vision, 2016Co-Authors: Imen Ben-rebeh, Christine Petit, M'hamed Grati, Crystel Bonnet, Walid Bouassida, Imen Hadjamor, Hammadi Ayadi, Abdelmonem Ghorbel, Saber MasmoudiAbstract:Purpose Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, Cadherin-23, protoCadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively.
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The tip-link molecular complex of the auditory mechano-electrical transduction machinery.
Hearing research, 2015Co-Authors: Elise Pepermans, Christine PetitAbstract:Sound waves are converted into electrical signals by a process of mechano-electrical transduction (MET), which takes place in the hair bundle of cochlear hair cells. In response to the mechanical stimulus of the hair bundle, the tip-links, key components of the MET machinery, are tensioned and the MET channels open, which results in the generation of the cell receptor potential. Tip-links are composed of Cadherin-23 (Cdh23) and protoCadherin-15 (Pcdh15), both non-conventional Cadherins, that form the upper and the lower part of these links, respectively. Here, we review the various Pcdh15 isoforms present in the organ of Corti, their localization in the auditory hair bundles, their involvement in the molecular complex forming the tip-link, and their interactions with transmembrane molecules that are components of the lower MET machinery.
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Differential Distribution of Harmonin Isoforms and Their Possible Role in Usher-1 Protein Complexes in Mammalian
2013Co-Authors: Photoreceptor Cells, Aziz El-amraoui, Jan Reiners, B Reidel, Christine Petit, Batiste Boëda, Irene Huber, Uwe WolfrumAbstract:PURPOSE. Human Usher syndrome is the most common form of combined deafness and blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction, and prepubertal onset retinitis pigmentosa. Previous studies have shown that the USH1-proteins myosin VIIa, harmonin, and Cadherin 23 interact and form a functional network during hair cell differentiation in the inner ear. The purpose of the present study was to analyze the molecular and cellular functions of these USH1 proteins in the mammalian retina. METHODS. Antibodies to USH1 proteins were generated and used in Western blot analysis of subcellular photoreceptor fractions and immunofluorescence and electron microscopy of the retina. RESULTS. Splice variants of harmonin were differentially expresse
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Coupling of the mechanotransduction machinery and stereocilia F-actin polymerization in the cochlear hair bundles
Bioarchitecture, 2011Co-Authors: Elisa Caberlotto, Vincent Michel, Jacques Boutet De Monvel, Christine PetitAbstract:Mechanoelectrical transduction (MET), the conversion of mechanical stimuli into electrical signals, operated by the sensory hair cells of the inner ear enables hearing and balance perception. Crucial to this process are the tip-links, oblique fibrous filaments that interconnect the stereocilia within the hair bundle and mechanically gate MET channels. In a recent study, we found a complete regression of the short and medium but not of the high stereocilia row upon the disappearance of the tip-link, caused by the loss of one of its components, Cadherin-23, or of one of its anchoring proteins, sans, in engineered mutant mice. This indicates the existence of a coupling between the MET and F-actin polymerization machineries at the tips of two smallest stereocilia rows of the hair bundle. Here, we report about these results and discuss the possible roles of the tip-link tension on stereocilia F-actin polymerization, acting either directly or via Ca2+-dependent mechanisms involving the gating of MET channels.
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Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids
Human molecular genetics, 2010Co-Authors: Amel Bahloul, Vincent Michel, Jean-pierre Hardelin, Sylvie Nouaille, Sylviane Hoos, Anne Houdusse, Patrick England, Christine PetitAbstract: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)P(2)), 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)P(2) 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.
Marcos Sotomayor - One of the best experts on this subject based on the ideXlab platform.
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Structural determinants of protoCadherin-15 mechanics and function in hearing and balance perception.
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Deepanshu Choudhary, Yoshie Narui, Brandon L. Neel, Lahiru N. Wimalasena, Carissa F. Klanseck, Pedro De-la-torre, Conghui Chen, Raul Araya-secchi, Elakkiya Tamilselvan, Marcos SotomayorAbstract:The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. ProtoCadherin-15 and Cadherin-23, two atypically large Cadherins with 11 and 27 extracellular Cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protoCadherin-15 + Cadherin-23 heterotetrameric complex at 2.9-A resolution, depicting a parallel homodimer of protoCadherin-15 EC1-3 molecules forming an antiparallel complex with two Cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protoCadherin-15 fragments used to build complete high-resolution models of the monomeric protoCadherin-15 ectodomain. Molecular dynamics simulations and validated crystal contacts are used to propose models for the complete extracellular protoCadherin-15 parallel homodimer and the tip-link bond. Steered molecular dynamics simulations of these models suggest conditions in which a structurally diverse and multimodal protoCadherin-15 ectodomain can act as a stiff or soft gating spring. These results reveal the structural determinants of tip-link-mediated inner-ear sensory perception and elucidate protoCadherin-15's structural and adhesive properties relevant in disease.
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Structural determinants of protoCadherin-15 elasticity and function in inner-ear mechanotransduction
2019Co-Authors: Deepanshu Choudhary, Yoshie Narui, Brandon L. Neel, Lahiru N. Wimalasena, Carissa F. Klanseck, Pedro De-la-torre, Conghui Chen, Raul Araya-secchi, Elakkiya Tamilselvan, Marcos SotomayorAbstract:ProtoCadherin-15 (PCDH15), an atypical member of the Cadherin superfamily, is essential for vertebrate hearing and its dysfunction has been associated with deafness and progressive blindness. The PCDH15 ectodomain, made of eleven extracellular Cadherin (EC1-11) repeats and a membrane adjacent domain (MAD12), assembles as a parallel homodimer that interacts with Cadherin-23 (CDH23) to form the tip link, a fine filament necessary for inner-ear mechanotransduction. Here we report X-ray crystal structures of a PCDH15 + CDH23 heterotetrameric complex and ten PCDH15 fragments that were used to build complete high-resolution models of the monomeric PCDH15 ectodomain. Using molecular dynamics (MD) simulations and validated crystal contacts we propose models for complete PCDH15 parallel homodimers and the tip-link bond. Steered MD simulations of these models predict their strength and suggest conditions in which a multimodal PCDH15 ectodomain can act as a stiff or soft gating spring. These results provide a detailed view of the first molecular steps in inner-ear sensory transduction.
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Beyond Cell–Cell Adhesion: Sensational Cadherins for Hearing and Balance
Cold Spring Harbor perspectives in biology, 2018Co-Authors: Avinash Jaiganesh, Raul Araya-secchi, Yoshie Narui, Marcos SotomayorAbstract:Cadherins form a large family of proteins often involved in calcium-dependent cellular adhesion. Although classical members of the family can provide a physical bond between cells, a subset of special Cadherins use their extracellular domains to interlink apical specializations of single epithelial sensory cells. Two of these Cadherins, Cadherin-23 (CDH23) and protoCadherin-15 (PCDH15), form extracellular "tip link" filaments that connect apical bundles of stereocilia on hair cells essential for inner-ear mechanotransduction. As these bundles deflect in response to mechanical stimuli from sound or head movements, tip links gate hair-cell mechanosensitive channels to initiate sensory perception. Here, we review the unusual and diverse structural properties of these tip-link Cadherins and the functional significance of their deafness-related missense mutations. Based on the structural features of CDH23 and PCDH15, we discuss the elasticity of tip links and models that bridge the gap between the nanomechanics of Cadherins and the micromechanics of hair-cell bundles during inner-ear mechanotransduction.
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Zooming in on Cadherin-23: Structural Diversity and Potential Mechanisms of Inherited Deafness.
Structure (London England : 1993), 2018Co-Authors: Avinash Jaiganesh, Pedro De-la-torre, Conghui Chen, Aniket Patel, Domenic J. Termine, Florencia Velez-cortes, Marcos SotomayorAbstract:Summary Cadherin-23 (CDH23) is an essential component of hair-cell tip links, fine filaments that mediate inner-ear mechanotransduction. The extracellular domain of CDH23 forms about three-fourths of the tip link with 27 extracellular Cadherin (EC) repeats that are structurally similar but not identical to each other. Calcium (Ca2+) coordination at the EC linker regions is key for tip-link elasticity and function. There are ∼116 sites in CDH23 affected by deafness-causing mutations, many of which alter conserved Ca2+-binding residues. Here we present crystal structures showing 18 CDH23 EC repeats, including the most and least conserved, a fragment carrying disease mutations, and EC repeats with non-canonical Ca2+-binding motif sequences and unusual secondary structure. Complementary experiments show deafness mutations' effects on stability and affinity for Ca2+. Additionally, a model of nine contiguous CDH23 EC repeats reveals helicity and potential parallel dimerization faces. Overall, our studies provide detailed structural insight into CDH23 function in mechanotransduction.
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Tuning Inner-Ear Tip-Link Affinity Through Alternatively Spliced Variants of ProtoCadherin-15
Biochemistry, 2018Co-Authors: Yoshie Narui, Marcos SotomayorAbstract:Human hearing relies upon the tip-to-tip interaction of two nonclassical Cadherins, protoCadherin-15 (PCDH15) and Cadherin-23 (CDH23). Together, these proteins form a filament called the tip link that connects neighboring stereocilia of mechanosensitive hair cells. As sound waves enter the cochlea, the stereocilia deflect and tension is applied to the tip link, opening nearby transduction channels. Disruption of the tip link by loud sound or calcium chelators eliminates transduction currents and illustrates that tip-link integrity is critical for mechanosensing. Tip-link remodeling after disruption is a dynamic process, which can lead to the formation of atypical complexes that incorporate alternatively spliced variants of PCDH15. These variants are categorized into six groups (N1–N6) based upon differences in the first two extracellular Cadherin (EC) repeats. Here, we characterized the two N-terminal EC repeats of all PCDH15 variants (pcdh15(N1) to pcdh15(N6)) and combined these variants to test complex ...
David P Corey - One of the best experts on this subject based on the ideXlab platform.
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Single Molecule Force Spectroscopy of Hair-Cell Tip-Link Proteins
Biophysical Journal, 2016Co-Authors: Mounir A. Koussa, Marcos Sotomayor, Wesley Philip Wong, Andrew Ward, David P CoreyAbstract:Mechanical stimuli from sound and head movements are converted into electrical signals by hair cells of the inner ear. Key to this process are tip links, fine protein filaments that convey mechanical force to hair-cell transduction channels. Tip links are made of two atypical Cadherins: protoCadherin-15 and Cadherin-23, positioned at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between the N-termini of these two proteins. The crystal structure of the interacting domain has been solved, and molecular dynamics simulations have provided an estimate of the force it can withstand. However the lack of adequate technology has prevented direct measurement of the unbinding force.We have developed molecular tools to facilitate single-molecule force spectroscopy of tip-link proteins, based on self-assembled DNA nanoswitches (Halvorsen et al., 2011). The tips of protoCadherin-15 and Cadherin-23 are enzymatically conjugated to DNA oligos, which bind in selected locations to a single-stranded M13 DNA scaffold. The remaining single-stranded M13 is hybridized to complementary DNA oligomers. Once assembled, the nanoswitches are used in an optical tweezer system to measure the rupture forces of the tip link.Measured in this way, the unbinding force is about 35 piconewtons, a value consistent with extrapolations of the molecular dynamics simulations to slower loading rates, but disconcertingly low compared to what tip links must withstand physiologically. Tip links, however, have two strands, and thus two interaction domains in close proximity. Calculations suggest that this arrangement greatly increases the average lifetime of a tip link.
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Towards force spectroscopy of single tip-link bonds
2015Co-Authors: Mounir A. Koussa, Marcos Sotomayor, Wesley Philip Wong, David P CoreyAbstract:Inner-ear mechanotransduction relies on tip links, fine protein filaments made of Cadherin-23 and protoCadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link Cadherins are thought to form a heterotetrameric complex, with two Cadherin-23 molecules forming the upper part of the filament and two protoCadherin-15 molecules forming the lower end. The interaction between Cadherin-23 and protoCadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of Cadherin-23 and protoCadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature should allow us to identify single-molecule rupture events in pulling experiments.
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Single-Molecule Force-Spectroscopy of Inner Ear Proteins
Biophysical Journal, 2015Co-Authors: Mounir A. Koussa, Wesley Philip Wong, David P CoreyAbstract:Inner-ear mechanotransduction relies on tip links, fine protein filaments made of Cadherin-23 and protoCadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link Cadherins are thought to form a heterotetrameric complex, with two Cadherin-23 molecules forming the upper part of the filament and two protoCadherin-15 molecules forming the lower end. The interaction between Cadherin-23 and protoCadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of Cadherin-23 and protoCadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature allows us to identify single-molecule rupture events in pulling experiments.
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Molecular Mechanisms of Deafness Mutations Disrupting Tip-Link Function in Hair-Cell Mechanotransduction
Biophysical Journal, 2014Co-Authors: Marcos Sotomayor, Rachelle Gaudet, David P CoreyAbstract:Hair-cell tip links are fine filaments that directly convey mechanical force to inner ear mechanotransduction channels. These filaments are made of protoCadherin-15 (PCDH15) and Cadherin-23 (CDH23), two deafness-related proteins that feature long extracellular domains interacting tip-to-tip in a calcium dependent manner. Here we combine X-ray crystallography, molecular dynamics simulations, and binding experiments to explore the molecular mechanisms by which deafness mutations disrupt tip-link function in hair-cell mechanotransduction. We find that these mutations disrupt tip links through impaired interaction between PCDH15 and CDH23 (PCDH15-R113G and PCDH15-I108N), impaired calcium binding (CDH23-D1010G), subtle weakening of structural stability (CDH23-S47P), or impaired folding (PCDH15-D157G). Interestingly, the biochemical effects of each of these deafness mutations correlate with the severity of the reported inner-ear phenotype. Our results shed light on the molecular mechanisms of hair-cell sensory transduction and may help develop tailored treatments for Cadherin-mediated deafness.
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Force Spectroscopy of Tip Link Proteins: A Study of Inner-Ear Biophysics
Biophysical Journal, 2014Co-Authors: Mounir A. Koussa, Marcos Sotomayor, Wesley P. Wong, David P CoreyAbstract:Hair cells in the inner ear convert mechanical stimuli, in the vestibular and auditory systems, into electrical signals which can be processed by the brain. Hair cells are highly polarized with a unique elaboration of modified microvilli at their apical surface known as stereo cilia. Sensory stimuli such as sound waves of a particular frequency will result in oscillations of the stereociliary bundle at that frequency. Movements of this bundle couple mechanically to the channel. This force acts to gate the channel in turn resulting in a mechano-electrical transduction. The force is relayed to the channel by filaments known as tip links. Tip links are made of two atypical Cadherins, protoCadherin-15 and Cadherin-23 at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between two anti-parallel pairs of EC domains. Although much has been discerned about the biophysics of this system the lack of adequate technology has prevented the direct measurement of many of these properties. We have developed molecular tools to facilitate the single molecule study of these properties. Self-assembled DNA nanoswitches are functionalized with protoCadherin 15 and Cadherin 23 fragments using the enzyme sortase. In order to preserve protein function, protein-DNA coupling is performed under physiological conditions. In this two-step process, a small synthetic peptide is first coupled to a DNA oligo. Next, utilizing the enzyme sortase, the protein is coupled to the DNA-peptide chimera under physiological conditions. This strategy frontloads all of the protein-incompatible chemistry so that it is performed on an oligo and a synthetic peptide, which are far more tolerant of non-physiological conditions. Once assembled, the nanoswitches are then used in an optical tweezer system to measure the rupture forces of the tip link under different conditions.
Uwe Wolfrum - One of the best experts on this subject based on the ideXlab platform.
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*to whom correspondence should be addressed
2014Co-Authors: Avital Adato, Vincent Michel, Kumar N. Alagramam, Jan Reiners, Uwe Wolfrum, Dominique Weil, Yoshiaki Kikkawa, Hiromichi Yonekawa, Aziz El-amraouiAbstract:g.oxfordjournals.org/ D ow nloaded from 2 Defects in myosin VIIa, harmonin, a PDZ-domain protein, Cadherin 23, protoCadherin 15 and sans, a putative scaffolding protein, underlie five forms of Usher syndrome type I (USH1). Mouse mutants for all these proteins exhibit disorganization of their hair bundle, which is the mechanotransduction receptive structure of the inner ear sensory cells, the cochlear and vestibular hair cells. We have previously demonstrated that harmonin interacts with Cadherin 23 and myosin VIIa. Here we address the extent of interactions between the five known USH1 proteins. We establish the previously suggested sans-harmonin interaction and find that sans also binds to myosin VIIa. We show that sans can form homomeric structures and that harmonin b can interact with all harmonin isoforms. We reveal tha
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Differential Distribution of Harmonin Isoforms and Their Possible Role in Usher-1 Protein Complexes in Mammalian
2013Co-Authors: Photoreceptor Cells, Aziz El-amraoui, Jan Reiners, B Reidel, Christine Petit, Batiste Boëda, Irene Huber, Uwe WolfrumAbstract:PURPOSE. Human Usher syndrome is the most common form of combined deafness and blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction, and prepubertal onset retinitis pigmentosa. Previous studies have shown that the USH1-proteins myosin VIIa, harmonin, and Cadherin 23 interact and form a functional network during hair cell differentiation in the inner ear. The purpose of the present study was to analyze the molecular and cellular functions of these USH1 proteins in the mammalian retina. METHODS. Antibodies to USH1 proteins were generated and used in Western blot analysis of subcellular photoreceptor fractions and immunofluorescence and electron microscopy of the retina. RESULTS. Splice variants of harmonin were differentially expresse
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Expression of Cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D.
Molecular vision, 2009Co-Authors: Ayala Lagziel, Robert J Morell, Uwe Wolfrum, Nora Overlack, Steven L. Bernstein, Thomas B FriedmanAbstract:Purpose We compared Cadherin 23 (Cdh23) mRNA and protein variants in the inner ear and retina of wild-type and mutant mice and primates to better understand the pleiotropic effects of Cdh23 mutations, and specifically to understand the absence of retinal degeneration in Cdh23 mutant mice. Methods Semiquantitative real-time PCR was used to compare the level of expression of Cdh23 alternative transcripts in the inner ear and retina of wild-type and homozygous Cdh23(v-6J) (waltzer) mice. Antibodies generated against CDH23 isoforms were used in immunohistochemistry, immunohistology, electron microscopy, and western blot analyses of mouse and primate inner ear and retina to study the distribution of these isoforms in various cellular compartments. Results Cdh23 mRNA alternative splice variants were temporally and spatially regulated in the inner ear and retina. In the mature mouse retina, CDH23 isoforms were broadly expressed in various cellular compartments of the photoreceptor layer. The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells. The longest CDH23 protein isoform, CDH23_V1, appeared by western blotting to be the only one affected by the Cdh23(v-6J) mutation; it was expressed in the wild-type mouse inner ear, but not in the mouse retina. However, CDH23_V1 was detected in western blot analyses of monkey and human retinas. Conclusions The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts. Many of these isoforms continue to be expressed in waltzer mice. The longest CDH23 isoform (CDH23_V1), however, is not expressed in mutant mice and is necessary for normal inner ear function. The longest isoform is expressed in the retinas of primates, but not detected in the mouse retina. This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.
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a core cochlear phenotype in ush1 mouse mutants implicates fibrous links of the hair bundle in its cohesion orientation and differential growth
Development, 2008Co-Authors: Gaelle Lefevre, Vincent Michel, Jean-pierre Hardelin, Uwe Wolfrum, Dominique Weil, Lea Lepelletier, Emilie Bizard, Christine PetitAbstract:The planar polarity and staircase-like pattern of the hair bundle are essential to the mechanoelectrical transduction function of inner ear sensory cells. Mutations in genes encoding myosin VIIa, harmonin, Cadherin 23, protoCadherin 15 or sans cause Usher syndrome type I (USH1, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa leading to blindness) in humans and hair bundle disorganization in mice. Whether the USH1 proteins are involved in common hair bundle morphogenetic processes is unknown. Here, we show that mouse models for the five USH1 genetic forms share hair bundle morphological defects. Hair bundle fragmentation and misorientation (25-52° mean kinociliary deviation, depending on the mutant) were detected as early as embryonic day 17. Abnormal differential elongation of stereocilia rows occurred in the first postnatal days. In the emerging hair bundles, myosin VIIa, the actin-binding submembrane protein harmonin-b, and the interstereocilia-kinocilium lateral link components Cadherin 23 and protoCadherin 15, all concentrated at stereocilia tips, in accordance with their known in vitro interactions. Soon after birth, harmonin-b switched from the tip of the stereocilia to the upper end of the tip link, which also comprises Cadherin 23 and protoCadherin 15. This positional change did not occur in mice deficient for Cadherin 23 or protoCadherin 15. We suggest that tension forces applied to the early lateral links and to the tip link, both of which can be anchored to actin filaments via harmonin-b, play a key role in hair bundle cohesion and proper orientation for the former, and in stereociliary elongation for the latter.
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photoreceptor expression of the usher syndrome type 1 protein protoCadherin 15 ush1f and its interaction with the scaffold protein harmonin ush1c
Molecular Vision, 2005Co-Authors: Jan Reiners, Tina Marker, Karin Jurgens, B Reidel, Uwe WolfrumAbstract:Purpose: The human Usher syndrome (USH) is the most common form of deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal onset of retinitis pigmentosa. Five corresponding genes of the seven USH1 genes have been cloned over the years. Recent studies indicated that three USH1 proteins, namely myosin VIIa (USH1B), SANS (USH1G), and Cadherin 23 (USH1D) interact with the USH1C gene product harmonin. In these protein-protein complexes harmonin acts as the scaffold protein binding these USH1 molecules via its PDZ domains. The aim of the present study was to analyze whether or not the fifth identified USH1 protein protoCadherin 15 (Pcdh15) also binds to harmonin and where these putative protein complexes might be localized in mammalian rod and cone photoreceptor cells. Methods: In vitro binding assays (GST pull-down, yeast two-hybrid assay) were applied. Antibodies against bacterial expressed USH1 proteins were generated. Affinity purified antibodies were used in immunoblot analyses of brain fractions and isolated retinas, in immunofluorescence studies, and in immunoelectron microscopic studies of rodent retinas. Results: We showed that Pcdh15 (USH1F) interacted with harmonin PDZ2. Immunocytochemistry revealed that Pcdh15 is expressed in photoreceptor cells of the mammalian retina, where it is colocalized with harmonin, myosin VIIa, and Cadherin 23 at the synaptic terminal. Colocalization of Pcdh15 with harmonin was found at the base of the photoreceptor outer segment, where newly synthesized disk membranes are present. Conclusions: Our data indicate that harmonin-Pcdh15 interactions probably play a role in disk morphogenesis. Furthermore, we provide evidence that a complex composed of all USH1 molecules may assemble at the photoreceptor synapse. This USH protein complex can contribute to the cortical cytoskeletal matrices of the pre- and postsynaptic regions, which are thought to play a fundamental role in the structural and functional organization of the synaptic junction. Defects in any of the USH1-complex partners may result in photoreceptor dysfunction causing retinitis pigmentosa, the clinical phenotype in the retina of USH1 patients.
Sabyasachi Rakshit - One of the best experts on this subject based on the ideXlab platform.
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Weakening of interaction networks with aging in tip-link protein induces hearing loss.
The Biochemical journal, 2021Co-Authors: Surbhi Garg, Gayathri S. Singaraju, Amin Sagar, Rahul Dani, Naimat K Bari, Athi N. Naganathan, Sabyasachi RakshitAbstract:Age-related hearing loss (ARHL) is a common condition in humans marking the gradual decrease in hearing with age. Perturbations in the tip-link protein Cadherin-23 that absorbs the mechanical tension from sound and maintains the integrity of hearing is associated with ARHL. Here, in search of molecular origins for ARHL, we dissect the conformational behavior of Cadherin-23 along with the mutant S47P that progresses the hearing loss drastically. Using an array of experimental and computational approaches, we highlight a lower thermodynamic stability, significant weakening in the hydrogen-bond network and inter-residue correlations among β-strands, due to the S47P mutation. The loss in correlated motions translates to not only a remarkable two orders of magnitude slower folding in the mutant but also to a proportionately complex unfolding mechanism. We thus propose that loss in correlated motions within Cadherin-23 with aging may trigger ARHL, a molecular feature that likely holds true for other disease-mutations in β-strand-rich proteins.
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structural basis of the strong cell cell junction formed by Cadherin 23
FEBS Journal, 2020Co-Authors: Gayathri S. Singaraju, Amin Sagar, Ashwani Kumar, J. S. Samuel, Jagadish P. Hazra, M K Sannigrahi, Ragothaman M. Yennamalli, Fnu Ashish, Sabyasachi RakshitAbstract:Cadherin-23, a giant atypical Cadherin, forms homophilic interactions at the cell-cell junction of epithelial cells and heterophilic interactions with protoCadherin-15 at the tip-links of neuroepithelial cells. While the molecular structure of the heterodimer is solved, the homodimer structure is yet to be resolved. The homodimers play an essential role in cell-cell adhesion as the downregulation of Cadherin-23 in cancers loosen the intercellular junction resulting in faster-migration of cancer cells and a significant drop in patient survival. In vitro studies have measured a stronger aggregation-propensity of Cadherin-23 compared to typical E-Cadherin. Here, we deciphered the unique trans-homodimer structure of Cadherin-23 in solution, and show that it consists of two electrostatic-based interfaces extended up to two terminal domains. The interface is robust, with a low off-rate of ~8x10-4 s-1 that supports its strong aggregation-propensity. We identified a point-mutation, E78K, that disrupts this binding. Interestingly, a mutation at the interface was reported in skin cancer. Overall, the structural basis of the strong Cadherin-23 adhesion may have far-reaching applications in the fields of mechanobiology and cancer.
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Structural basis of the strong cell‐cell junction formed by Cadherin‐23
The FEBS journal, 2019Co-Authors: Gayathri S. Singaraju, Amin Sagar, Ashwani Kumar, J. S. Samuel, Jagadish P. Hazra, M K Sannigrahi, Ragothaman M. Yennamalli, Fnu Ashish, Sabyasachi RakshitAbstract:Cadherin-23, a giant atypical Cadherin, forms homophilic interactions at the cell-cell junction of epithelial cells and heterophilic interactions with protoCadherin-15 at the tip-links of neuroepithelial cells. While the molecular structure of the heterodimer is solved, the homodimer structure is yet to be resolved. The homodimers play an essential role in cell-cell adhesion as the downregulation of Cadherin-23 in cancers loosen the intercellular junction resulting in faster-migration of cancer cells and a significant drop in patient survival. In vitro studies have measured a stronger aggregation-propensity of Cadherin-23 compared to typical E-Cadherin. Here, we deciphered the unique trans-homodimer structure of Cadherin-23 in solution, and show that it consists of two electrostatic-based interfaces extended up to two terminal domains. The interface is robust, with a low off-rate of ~8x10-4 s-1 that supports its strong aggregation-propensity. We identified a point-mutation, E78K, that disrupts this binding. Interestingly, a mutation at the interface was reported in skin cancer. Overall, the structural basis of the strong Cadherin-23 adhesion may have far-reaching applications in the fields of mechanobiology and cancer.
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The strong propensity of Cadherin-23 for aggregation inhibits cell migration.
Molecular oncology, 2019Co-Authors: M K Sannigrahi, Cheerneni S. Srinivas, Nilesh Deokate, Sabyasachi RakshitAbstract:Cadherin-23 (Cdh23), a long-chain non-classical Cadherin, exhibits strong homophilic and heterophilic binding. The physiological relevance of strong heterophilic binding with protoCadherin-15 at neuroepithelial tip links is well-studied. However, the role of Cdh23 homodimers in physiology is less understood, despite its widespread expression at the cell boundaries of various human and mouse tissues, including kidney, muscle, testes, and heart. Here, we performed immunofluorescence studies that revealed that Cdh23 is present as distinct puncta at the cell-cell boundaries of cancer cells. Analysis of patient data and quantitative estimation of Cdh23 in human tissues (normal and tumor) also indicated that Cdh23 is down-regulated via promoter methylation in lung adenocarcinoma (AD) and esophageal squamous cell carcinoma (SCC) cells; we also observed a clear inverse correlation between Cdh23 expression and cancer metastasis. Using HEK293T cells and four types of cancer cells differentially expressing Cdh23, we observed that cell migration was faster in cells with reduced levels of Cdh23 expression. The cell migration rate in cancer cells is further accelerated by the presence of excretory isoforms of Cdh23, which loosen its cell-adhesion ability by competitive binding. Overall, our data indicate the role of Cdh23 as a suppressor of cell migration.
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The strong propensity of Cadherin‐23 for aggregation inhibits cell migration
Wiley, 2019Co-Authors: Malay K. Sannigrahi, Cheerneni S. Srinivas, Nilesh Deokate, Sabyasachi RakshitAbstract:Cadherin‐23 (Cdh23), a long‐chain non‐classical Cadherin, exhibits strong homophilic and heterophilic binding. The physiological relevance of strong heterophilic binding with protoCadherin‐15 at neuroepithelial tip links is well‐studied. However, the role of Cdh23 homodimers in physiology is less understood, despite its widespread expression at the cell boundaries of various human and mouse tissues, including kidney, muscle, testes, and heart. Here, we performed immunofluorescence studies that revealed that Cdh23 is present as distinct puncta at the cell–cell boundaries of cancer cells. Analysis of patient data and quantitative estimation of Cdh23 in human tissues (normal and tumor) also indicated that Cdh23 is down‐regulated via promoter methylation in lung adenocarcinoma (AD) and esophageal squamous cell carcinoma (SCC) cells; we also observed a clear inverse correlation between Cdh23 expression and cancer metastasis. Using HEK293T cells and four types of cancer cells differentially expressing Cdh23, we observed that cell migration was faster in cells with reduced levels of Cdh23 expression. The cell migration rate in cancer cells is further accelerated by the presence of excretory isoforms of Cdh23, which loosen its cell‐adhesion ability by competitive binding. Overall, our data indicate the role of Cdh23 as a suppressor of cell migration
