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Marcos Sotomayor - One of the best experts on this subject based on the ideXlab platform.
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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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A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15.
Biophysical journal, 2018Co-Authors: Pedro De-la-torre, Deepanshu Choudhary, Yoshie Narui, Raul Araya-secchi, Marcos SotomayorAbstract:Abstract The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) “repeats” that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of nonclassical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has 11 EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size-exclusion chromatography coupled to multiangle laser light scattering and small-angle x-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the “giant” FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.
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A Mechanically Weak Extracellular Membrane-Adjacent Domain Induces Dimerization of Protocadherin-15
2018Co-Authors: Pedro De-la-torre, Deepanshu Choudhary, Yoshie Narui, Raul Araya-secchi, Marcos SotomayorAbstract:The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) repeats that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of non-classical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has eleven EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size exclusion chromatography coupled to multi-angle laser light scattering and small-angle X-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the "giant" FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.
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Beyond Cell–Cell Adhesion: Sensational Cadherins for Hearing and Balance
Cold Spring Harbor perspectives in biology, 2018Co-Authors: Avinash Jaiganesh, Yoshie Narui, Raul Araya-secchi, 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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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 ...
Kumar N. Alagramam - One of the best experts on this subject based on the ideXlab platform.
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Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15.
PloS one, 2017Co-Authors: Shanthini Mahendrasingam, Robert Fettiplace, Kumar N. Alagramam, Ellen Cross, David N. FurnessAbstract:Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15.
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Distributions of PCDH15-CD1, -CD2, -CD3 and -ectodomain (ECD) antigen HL5614 in the organ of Corti of av6J mice at P3.
2013Co-Authors: Kumar N. Alagramam, David N. Furness, Richard J. Goodyear, Ruishuang Geng, Alexander F. J. Van Aken, Walter Marcotti, Corné J. Kros, Guy P. RichardsonAbstract:Hair bundles from +/av6J (A, C, E, G) and av6J/av6J (B, D, F, H) mice were stained with antibody PB303 to PCDH15-CD1 (A, B), antibody PB464-2B to PCDH15-CD2 (C, D), antibody PB375 to PCDH15-CD3 (E, F) and antibody HL5614 to PCDH15-ectodmain (G, H). Images are from the basal (A, B, E, F) and apical (C, D, G, H) coils. Staining seen with antibodies to the CD1 (A, B), CD2 (C, D) and CD3 (E, F) isoforms of PCDH15 is similar in +/av6J (A, C, E) and av6J/av6J (B, D, F) mice. Staining seen with HL5614 to the PCDH15 ectodomain in av6J/av6J (H) hair bundles is weak in comparison to that seen in +/av6J hair bundles (G) and is most prominent in the region of the kinocilium (arrows). I, Inner hair cell; O1, O2, O3, outer hair cells in rows 1, 2, and 3 respectively. Scale bar = 20 µm.
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Mutations in Protocadherin 15 and Cadherin 23 Affect Tip Links and Mechanotransduction in Mammalian Sensory Hair Cells
PloS one, 2011Co-Authors: Kumar N. Alagramam, David N. Furness, Richard J. Goodyear, Ruishuang Geng, Alexander F. J. Van Aken, Walter Marcotti, Corné J. Kros, Guy P. RichardsonAbstract:Immunocytochemical studies have shown that protocadherin-15 (PCDH15) and cadherin-23 (CDH23) are associated with tip links, structures thought to gate the mechanotransducer channels of hair cells in the sensory epithelia of the inner ear. The present report describes functional and structural analyses of hair cells from PCDH15av3J (av3J), PCDH15av6J (av6J) and Cdh23v2J (v2J) mice. The av3J and v2J mice carry point mutations that are predicted to introduce premature stop codons in the transcripts for PCDH15 and Cdh23, respectively, and av6J mice have an in-frame deletion predicted to remove most of the 9th cadherin ectodomain from PCDH15. Severe disruption of hair-bundle morphology is observed throughout the early-postnatal cochlea in av3J/av3J and v2J/v2J mice. In contrast, only mild-to-moderate bundle disruption is evident in the av6J/av6J mice. Hair cells from av3J/av3J mice are unaffected by aminoglycosides and fail to load with [3H]-gentamicin or FM1-43, compounds that permeate the hair cell's mechanotransducer channels. In contrast, hair cells from av6J/av6J mice load with both FM1-43 and [3H]-gentamicin, and are aminoglycoside sensitive. Transducer currents can be recorded from hair cells of all three mutants but are reduced in amplitude in all mutants and have abnormal directional sensitivity in the av3J/av3J and v2J/v2J mutants. Scanning electron microscopy of early postnatal cochlear hair cells reveals tip-link like links in av6J/av6J mice, substantially reduced numbers of links in the av3J/av3J mice and virtually none in the v2J/v2J mice. Analysis of mature vestibular hair bundles reveals an absence of tip links in the av3J/av3J and v2J/v2J mice and a reduction in av6J/av6J mice. These results therefore provide genetic evidence consistent with PCDH15 and CDH23 being part of the tip-link complex and necessary for normal mechanotransduction.
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A nonsynonymous SNP within PCDH15 is associated with lipid traits in familial combined hyperlipidemia
Human genetics, 2009Co-Authors: Adriana Huertas-vazquez, Ruishuang Geng, Christopher L. Plaisier, Blake E. Haas, Jenny C. Lee, Marleen M. J. Van Greevenbroek, Carla J. H. Van Der Kallen, Tjerk W.a. De Bruin, Marja-riitta Taskinen, Kumar N. AlagramamAbstract:Familial combined hyperlipidemia (FCHL) is a common lipid disorder characterized by the presence of multiple lipoprotein phenotypes that increase the risk of premature coronary heart disease. In a previous study, we identified an intragenic microsatellite marker within the protocadherin 15 (PCDH15) gene to be associated with high triglycerides (TGs) in Finnish dyslipidemic families. In this study we analyzed all four known nonsynonymous SNPs within PCDH15 in 1,268 individuals from Finnish and Dutch multigenerational families with FCHL. Association analyses of quantitative traits for SNPs were performed using the QTDT test. The nonsynonymous SNP rs10825269 resulted in a P = 0.0006 for the quantitative TG trait. Additional evidence for association was observed with the same SNP for apolipoprotein B levels (apo-B) (P = 0.0001) and total cholesterol (TC) levels (P = 0.001). None of the other three SNPs tested showed a significant association with any lipid-related trait. We investigated the expression of PCDH15 in different human tissues and observed that PCDH15 is expressed in several tissues including liver and pancreas. In addition, we measured the plasma lipid levels in mice with loss-of-function mutations in PCDH15 (PCDH15av-Tg and PCDH15av-3J) to investigate possible abnormalities in their lipid profile. We observed a significant difference in plasma TG and TC concentrations for the PCDH15av-3J carriers when compared with the wild type (P = 0.013 and P = 0.044, respectively). Our study suggests that PCDH15 is associated with lipid abnormalities.
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Development of Outer Hair Cells in Ames Waltzer Mice: Mutation in Protocadherin 15 Affects Development of Cuticular Plate and Associated Structures
Anatomical record (Hoboken N.J. : 2007), 2008Co-Authors: Yayoi S. Kikkawa, Karen S. Pawlowski, Charles G. Wright, Kumar N. AlagramamAbstract:The Ames waltzer (av) mouse mutant harbors a mutation in the protocadherin 15 gene (PCDH15) and is a model for deafness in Usher syndrome 1F and nonsyndromic deafness DFNB23. Mutation in PCDH15 affects stereocilia morphogenesis and polarity. Disruptions of apical cellular components in outer hair cells have also been described in av mutants. Organization of stereocilia and cell polarization may be dependent on proper orientation of structural components residing in the apical portion of the cell during development. We used electron and immunofluorescent microscopy to examine structural maturation of outer hair cells in av3J mice with emphasis on the fonticulus, basal body/centriole complex, actin mesh, and the microtubule network during initiation of bundle organization, between embryonic day (E) 16.5 and postnatal day 5 (P5). We found major ultrastructural rearrangements near the hair cell surface in av3J mice. Earliest changes were in kinocilia, basal body, and stereocilia positioning and microtubule arrangement once the kinocilia had lateralized to the side of the cell (between E16.5 and postnatal day [P] 0, before cuticular plate formation and stereocilia elongation). By P0, the developing fonticulus in av mice appeared enlarged, with a normal vesicle density. Stereocilia bundle disorganization increased after P0, with disruptions of the actin mesh within the cuticular plate. These observations support the hypothesis that mutations in PCDH15 in av3J mice adversely affect coordinated maturation of apical cell components, resulting in disturbed stereocilia bundle polarity in av mice.
Thomas B Friedman - One of the best experts on this subject based on the ideXlab platform.
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Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells
PLoS biology, 2013Co-Authors: Artur A. Indzhykulian, Thomas B Friedman, Zubair M Ahmed, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Inna A. Belyantseva, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca2+-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.
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Formation of a transient PCDH15–PCDH15 tip link and its subsequent replacement with PCDH15–CDH23 link.
2013Co-Authors: Artur A. Indzhykulian, Thomas B Friedman, Zubair M Ahmed, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Inna A. Belyantseva, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:The model assumes that BAPTA disrupts PCDH15–CDH23 bonds [39] and MET channels are bound to or located near the lower end of the tip link [3]. The transduction channel becomes nonfunctional after tip link disruption. Therefore, its location immediately after BAPTA treatment is unknown, although illustrated as present at the tip of stereocilium. Alternatively, MET channels may migrate away from the tip of a stereocilium as a complex with PCDH15 molecules. Harmonin-based complexes linking CDH23 to the cytoskeleton [41] are shown as blue circles near the upper end of the tip link.
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Regenerating stereocilia links appear at the tips but not at the bottom of stereocilia.
2013Co-Authors: Artur A. Indzhykulian, Thomas B Friedman, Zubair M Ahmed, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Inna A. Belyantseva, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:(A–B) Conventional (secondary electron) SEM images of IHC stereocilia before (A) and immediately after (B) link disruption. Dashed rectangles indicate the areas magnified in insets. Arrows point to the tip links. (C) Distribution of the links along the height of stereocilia (0%, bottom; 100%, top) in the third (shortest) row at different stages of link recovery. (D) Backscatter SEM image of IHC bundle immuno-labeled with anti-PDCH15 antibody, HL5614 (10 nm gold particles seen as white dots). (E) The same as in (D), but primary antibody was omitted. (F) Percentage of immuno-gold particles observed on links of second and third row stereocilia in IHCs at two different dilutions of HL5614. (G) Representative images of HL5614 labeling in third row stereocilia immediately and 20 min after BAPTA treatment. (H) Cumulative distribution of PCDH15 immuno-gold particles on third row stereocilium in control and during link recovery. For each time point, 50–70 stereocilia images were scaled to a common template (dashed line) and the location of every gold particle was shown by a semitransparent grey circle. (I) Distribution of PCDH15 immuno-gold particles along the height of third row stereocilia. Data in panels (C), (F), and (I) are shown as mean ± SE. Age of the cells: P3–4 plus 2–3 days in vitro (P3–4+2–3 div).
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Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome
Human Genetics, 2008Co-Authors: Zubair M Ahmed, Saima Anwar, Polina P. Belyantseva, Hanka Venselaar, Muhammad Qasim, Saima Riazuddin, Thomas B FriedmanAbstract:Mutations of PCDH15 , encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be composed of 35 exons and encodes a variety of isoforms with 3–11 ectodomains (ECs), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations, we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1, we identified homozygous mutant alleles (one missense, one splice site, three nonsense and two deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date, in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F , which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15 , mutations in the introns or regulatory elements of PCDH15 , or an additional locus for type I USH at chromosome 10q21.1.
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Ames Waltzer deaf mice have reduced electroretinogram amplitudes and complex alternative splicing of PCDH15 transcripts.
Investigative ophthalmology & visual science, 2006Co-Authors: Ricky J. L. Haywood-watson, Zubair M Ahmed, Lori L. Hampton, James F. Battey, Sten Kjellstrom, Ronald A. Bush, Yuichiro Takada, Paul A. Sieving, Thomas B FriedmanAbstract:Purpose Mutations of PCDH15, the gene encoding protocadherin 15, cause either nonsyndromic deafness DFNB23 or Usher syndrome type 1F (USH1F) in humans and deafness with balance problems in Ames waltzer (av) mice. Persons with USH1 usually begin to exhibit signs of retinitis pigmentosa (RP) in early adolescence, but av mice are reported to have functional retinas. In this study, the auditory, visual and molecular biological phenotype of PCDH15av-5J and PCDH15av-Jfb mice is characterized, and their usefulness as animal models of USH1 is evaluated. Methods Hearing thresholds of mice between 6 and 10 weeks of age were measured by auditory brain stem response (ABR). Immunohistochemistry and histology were used to examine the effect of homozygosity of PCDH15av-5J on stereocilia bundles of inner ear hair cells and on the photoreceptor cells of the retina. Scotopic and photopic Ganzfeld ERGs were recorded from homozygous PCDH15av-5J and PCDH15av-Jfb mice at different ages. Heterozygous littermates served as control subjects. Measurements of the width of the outer nuclear layer (ONL) and the length of rod photoreceptor outer segment (ROS) were made. Results Homozygous PCDH15av-5J mice have profound hearing loss and disorganized stereocilia bundles of inner ear hair cells. Compared with heterozygous littermates, homozygous PCDH15av-5J and PCDH15av-Jfb mutant mice had scotopic ERG amplitudes consistently reduced by approximately 40% at all light intensities. The b-to-a-wave ratio confirmed that the a- and b-waves were reduced proportionally in homozygous mutant mice. Histologic measurements of retinal sections revealed no significant differences in either the ONL width or the ROS length as a function of genotype. The protocadherin 15 labeling pattern with antisera PB303 in the retina of both heterozygous and homozygous PCDH15av-5J mice was indistinguishable from the wild type. Wild-type PCDH15 have many alternatively spliced isoforms. A novel isoform was found in the retina of homozygous PCDH15av-5J mice, which appears to circumvent the effect of the mutant allele (IVS14-2A-->G), which causes skipping of exon 14, a shift in the translation reading frame and a premature stop codon in exon 15. Conclusions PCDH15(av-5J) and PCDH15(av-Jfb) mice do not faithfully mimic the RP found in USH1 due to mutations of PCDH15, but have significantly attenuated ERG function in the absence of histologic change. The decline in ERG amplitude with a preserved b-to-a-wave ratio suggests a role for PCDH15 in retinal function and/or generation of the ERG potentials. Understanding the molecular mechanism by which av mice circumvent degeneration of the retina might offer insights into potential therapies for USH1.
Zubair M Ahmed - One of the best experts on this subject based on the ideXlab platform.
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Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells
PLoS biology, 2013Co-Authors: Artur A. Indzhykulian, Thomas B Friedman, Zubair M Ahmed, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Inna A. Belyantseva, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca2+-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.
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Formation of a transient PCDH15–PCDH15 tip link and its subsequent replacement with PCDH15–CDH23 link.
2013Co-Authors: Artur A. Indzhykulian, Thomas B Friedman, Zubair M Ahmed, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Inna A. Belyantseva, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:The model assumes that BAPTA disrupts PCDH15–CDH23 bonds [39] and MET channels are bound to or located near the lower end of the tip link [3]. The transduction channel becomes nonfunctional after tip link disruption. Therefore, its location immediately after BAPTA treatment is unknown, although illustrated as present at the tip of stereocilium. Alternatively, MET channels may migrate away from the tip of a stereocilium as a complex with PCDH15 molecules. Harmonin-based complexes linking CDH23 to the cytoskeleton [41] are shown as blue circles near the upper end of the tip link.
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Regenerating stereocilia links appear at the tips but not at the bottom of stereocilia.
2013Co-Authors: Artur A. Indzhykulian, Thomas B Friedman, Zubair M Ahmed, Ruben Stepanyan, Anastasiia Nelina, Kateri J. Spinelli, Inna A. Belyantseva, Peter G. Barr-gillespie, Gregory I. FrolenkovAbstract:(A–B) Conventional (secondary electron) SEM images of IHC stereocilia before (A) and immediately after (B) link disruption. Dashed rectangles indicate the areas magnified in insets. Arrows point to the tip links. (C) Distribution of the links along the height of stereocilia (0%, bottom; 100%, top) in the third (shortest) row at different stages of link recovery. (D) Backscatter SEM image of IHC bundle immuno-labeled with anti-PDCH15 antibody, HL5614 (10 nm gold particles seen as white dots). (E) The same as in (D), but primary antibody was omitted. (F) Percentage of immuno-gold particles observed on links of second and third row stereocilia in IHCs at two different dilutions of HL5614. (G) Representative images of HL5614 labeling in third row stereocilia immediately and 20 min after BAPTA treatment. (H) Cumulative distribution of PCDH15 immuno-gold particles on third row stereocilium in control and during link recovery. For each time point, 50–70 stereocilia images were scaled to a common template (dashed line) and the location of every gold particle was shown by a semitransparent grey circle. (I) Distribution of PCDH15 immuno-gold particles along the height of third row stereocilia. Data in panels (C), (F), and (I) are shown as mean ± SE. Age of the cells: P3–4 plus 2–3 days in vitro (P3–4+2–3 div).
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Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome
Human Genetics, 2008Co-Authors: Zubair M Ahmed, Saima Anwar, Polina P. Belyantseva, Hanka Venselaar, Muhammad Qasim, Saima Riazuddin, Thomas B FriedmanAbstract:Mutations of PCDH15 , encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be composed of 35 exons and encodes a variety of isoforms with 3–11 ectodomains (ECs), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations, we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1, we identified homozygous mutant alleles (one missense, one splice site, three nonsense and two deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date, in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F , which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15 , mutations in the introns or regulatory elements of PCDH15 , or an additional locus for type I USH at chromosome 10q21.1.
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Ames Waltzer deaf mice have reduced electroretinogram amplitudes and complex alternative splicing of PCDH15 transcripts.
Investigative ophthalmology & visual science, 2006Co-Authors: Ricky J. L. Haywood-watson, Zubair M Ahmed, Lori L. Hampton, James F. Battey, Sten Kjellstrom, Ronald A. Bush, Yuichiro Takada, Paul A. Sieving, Thomas B FriedmanAbstract:Purpose Mutations of PCDH15, the gene encoding protocadherin 15, cause either nonsyndromic deafness DFNB23 or Usher syndrome type 1F (USH1F) in humans and deafness with balance problems in Ames waltzer (av) mice. Persons with USH1 usually begin to exhibit signs of retinitis pigmentosa (RP) in early adolescence, but av mice are reported to have functional retinas. In this study, the auditory, visual and molecular biological phenotype of PCDH15av-5J and PCDH15av-Jfb mice is characterized, and their usefulness as animal models of USH1 is evaluated. Methods Hearing thresholds of mice between 6 and 10 weeks of age were measured by auditory brain stem response (ABR). Immunohistochemistry and histology were used to examine the effect of homozygosity of PCDH15av-5J on stereocilia bundles of inner ear hair cells and on the photoreceptor cells of the retina. Scotopic and photopic Ganzfeld ERGs were recorded from homozygous PCDH15av-5J and PCDH15av-Jfb mice at different ages. Heterozygous littermates served as control subjects. Measurements of the width of the outer nuclear layer (ONL) and the length of rod photoreceptor outer segment (ROS) were made. Results Homozygous PCDH15av-5J mice have profound hearing loss and disorganized stereocilia bundles of inner ear hair cells. Compared with heterozygous littermates, homozygous PCDH15av-5J and PCDH15av-Jfb mutant mice had scotopic ERG amplitudes consistently reduced by approximately 40% at all light intensities. The b-to-a-wave ratio confirmed that the a- and b-waves were reduced proportionally in homozygous mutant mice. Histologic measurements of retinal sections revealed no significant differences in either the ONL width or the ROS length as a function of genotype. The protocadherin 15 labeling pattern with antisera PB303 in the retina of both heterozygous and homozygous PCDH15av-5J mice was indistinguishable from the wild type. Wild-type PCDH15 have many alternatively spliced isoforms. A novel isoform was found in the retina of homozygous PCDH15av-5J mice, which appears to circumvent the effect of the mutant allele (IVS14-2A-->G), which causes skipping of exon 14, a shift in the translation reading frame and a premature stop codon in exon 15. Conclusions PCDH15(av-5J) and PCDH15(av-Jfb) mice do not faithfully mimic the RP found in USH1 due to mutations of PCDH15, but have significantly attenuated ERG function in the absence of histologic change. The decline in ERG amplitude with a preserved b-to-a-wave ratio suggests a role for PCDH15 in retinal function and/or generation of the ERG potentials. Understanding the molecular mechanism by which av mice circumvent degeneration of the retina might offer insights into potential therapies for USH1.
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Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome
Human Genetics, 2008Co-Authors: Zubair M Ahmed, Saima Anwar, Polina P. Belyantseva, Hanka Venselaar, Muhammad Qasim, Saima Riazuddin, Thomas B FriedmanAbstract:Mutations of PCDH15 , encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be composed of 35 exons and encodes a variety of isoforms with 3–11 ectodomains (ECs), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations, we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1, we identified homozygous mutant alleles (one missense, one splice site, three nonsense and two deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date, in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F , which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15 , mutations in the introns or regulatory elements of PCDH15 , or an additional locus for type I USH at chromosome 10q21.1.
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PCDH15 is expressed in the neurosensory epithelium of the eye and ear and mutant alleles are responsible for both USH1F and DFNB23
Human molecular genetics, 2003Co-Authors: Zubair M Ahmed, Saima Riazuddin, S L Bernstein, Andrew J Griffith, Paul A. Sieving, Jamil Ahmad, Yan Guo, Muhammad Farooq Sabar, Riazuddin, Thomas B FriedmanAbstract:Recessive splice site and nonsense mutations of PCDH15, encoding protocadherin 15, are known to cause deafness and retinitis pigmentosa in Usher syndrome type 1F (USH1F). Here we report that non-syndromic recessive hearing loss (DFNB23) is caused by missense mutations of PCDH15. This suggests a genotype-phenotype correlation in which hypomorphic alleles cause non-syndromic hearing loss, while more severe mutations of this gene result in USH1F. We localized protocadherin 15 to inner ear hair cell stereocilia, and to retinal photoreceptors by immunocytochemistry. Our results further strengthen the importance of protocadherin 15 in the morphogenesis and cohesion of stereocilia bundles and retinal photoreceptor cell maintenance or function.
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mutations of the protocadherin gene PCDH15 cause usher syndrome type 1f
American Journal of Human Genetics, 2001Co-Authors: Zubair M Ahmed, Thomas B Friedman, Saima Riazuddin, S L Bernstein, Zahoor Ahmed, Shaheen N Khan, Andrew J Griffith, Robert J Morell, Edward R WilcoxAbstract:Human chromosome 10q21-22 harbors USH1F in a region of conserved synteny to mouse chromosome 10. This region of mouse chromosome 10 contains PCDH15, encoding a protocadherin gene that is mutated in ames waltzer and causes deafness and vestibular dysfunction. Here we report two mutations of protocadherin 15 (PCDH15) found in two families segregating Usher syndrome type 1F. A Northern blot probed with the PCDH15 cytoplasmic domain showed expression in the retina, consistent with its pathogenetic role in the retinitis pigmentosa associated with USH1F.