This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.
Research Articles: Cellular/Molecular
Functional analysis of the transmembrane and cytoplasmic domains of Pcdh15a in zebrafish hair cells
Reo Maeda*, Itallia V. Pacentine*, Timothy Erickson and Teresa Nicolson Oregon Hearing Research Center and the Vollum Institute, Oregon Health and Science University, Portland, OR 97239
DOI: 10.1523/JNEUROSCI.2216-16.2017
Received: 12 July 2016
Revised: 7 February 2017
Accepted: 10 February 2017
Published: 20 February 2017
Author Contributions: R.M., I.V.P. and T.N. designed research; R.M., I.V.P., and T.E. obtained and analyzed all data in this research; and the manuscript was written and edited by R.M., I.V.P., T.E. and T.N. All authors read and approved the final manuscript.
Conflict of Interest: The authors declare no competing financial interests.
This study was supported by the NIH (DC013572 and DC013503), the NSF (award # AVPRS0014 to I.P.), the Uehara Memorial Foundation, the Naito Foundation, and the JSPS Postdoctoral Fellowships for Research Abroad (to R.M.).
Corresponding author: Teresa Nicolson, Ph.D., To whom correspondence should be addressed. E-mail: [email protected]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.2216-16.2017
Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published.
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process.
Copyright © 2017 the authors
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sensory ͺͳ hair cells, is protocadherin-15. The Journal of neuroscience : the official ͺͳ journal of the Society for Neuroscience 26:7022-7034. ͺͳͺ Alagramam KN, Goodyear RJ, Geng R, Furness DN, van Aken AF, Marcotti W, ͺͳͻ Kros CJ, Richardson GP (2011) Mutations in protocadherin 15 and ͺʹͲ cadherin 23 affect tip links and mechanotransduction in mammalian ͺʹͳ sensory hair cells. PLoS One 6:e19183. ͺʹʹ Assad JA, Shepherd GM, Corey DP (1991) Tip-link integrity and mechanical ͺʹ͵ transduction in vertebrate hair cells. Neuron 7:985-994. ͺʹͶ Chen X, Zaro JL, Shen WC (2013) Fusion protein linkers: property, design and ͺʹͷ functionality. Adv Drug Deliv Rev 65:1357-1369. ͺʹ Einhorn Z, Trapani JG, Liu Q, Nicolson T (2012) Rabconnectin3alpha promotes ͺʹ stable activity of the H+ pump on synaptic vesicles in hair cells. The ͺʹͺ Journal of neuroscience : the official journal of the Society for ͺʹͻ Neuroscience 32:11144-11156. ͺ͵Ͳ Elledge HM, Kazmierczak P, Clark P, Joseph JS, Kolatkar A, Kuhn P, Muller U ͺ͵ͳ (2010) Structure of the N terminus of cadherin 23 reveals a new ͺ͵ʹ adhesion mechanism for a subset of cadherin superfamily members. ͺ͵͵ Proceedings of the National Academy of Sciences of the United States of ͺ͵Ͷ America 107:10708-10712. ͺ͵ͷ Erickson T, French CR, Waskiewicz AJ. Meis1 specifies positional information ͺ͵ in the retina and tectum to organize the zebrafish visual system. Neural ͺ͵ Dev. 2010;5:22. ͺ͵ͺ Ernest S, Rauch GJ, Haffter P, Geisler R, Petit C, Nicolson T (2000) Mariner is ͺ͵ͻ defective in myosin VIIA: a zebrafish model for human hereditary ͺͶͲ deafness. Human molecular genetics 9:2189-2196. ͺͶͳ Geng R, Sotomayor M, Kinder KJ, Gopal SR, Gerka-Stuyt J, Chen DH, Hardisty- ͺͶʹ Hughes RE, Ball G, Parker A, Gaudet R, Furness D, Brown SD, Corey DP, ͺͶ͵ Alagramam KN (2013) Noddy, a mouse harboring a missense mutation ͺͶͶ in protocadherin-15, reveals the impact of disrupting a critical ͵͵ ͺͶͷ interaction site between tip-link cadherins in inner ear hair cells. The ͺͶ Journal of neuroscience : the official journal of the Society for ͺͶ Neuroscience 33:4395-4404. ͺͶͺ Goodyear RJ, Richardson GP (2003) A novel antigen sensitive to calcium ͺͶͻ chelation that is associated with the tip links and kinocilial links of ͺͷͲ sensory hair bundles. The Journal of neuroscience : the official journal ͺͷͳ of the Society for Neuroscience 23:4878-4887. ͺͷʹ Goodyear RJ, Forge A, Legan PK, Richardson GP (2010) Asymmetric ͺͷ͵ distribution of cadherin 23 and protocadherin 15 in the kinocilial links ͺͷͶ of avian sensory hair cells. The Journal of comparative neurology ͺͷͷ 518:4288-4297. ͺͷ Granato M, van Eeden FJ, Schach U, Trowe T, Brand M, Furutani-Seiki M, ͺͷ Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh ͺͷͺ RN, Mullins MC, Odenthal J, Nusslein-Volhard C (1996) Genes ͺͷͻ controlling and mediating locomotion behavior of the zebrafish embryo ͺͲ and larva. Development 123:399-413. ͺͳ Hudspeth AJ (1989) How the ear's works work. Nature 341:397-404. ͺʹ Indzhykulian AA, Stepanyan R, Nelina A, Spinelli KJ, Ahmed ZM, Belyantseva ͺ͵ IA, Friedman TB, Barr-Gillespie PG, Frolenkov GI (2013) Molecular ͺͶ remodeling of tip links underlies mechanosensory regeneration in ͺͷ auditory hair cells. PLoS Biol 11:e1001583. ͺ Kazmierczak P, Sakaguchi H, Tokita J, Wilson-Kubalek EM, Milligan RA, Muller ͺ U, Kachar B (2007) Cadherin 23 and protocadherin 15 interact to form ͺͺ tip-link filaments in sensory hair cells. Nature 449:87-91. ͺͻ Kindt KS, Finch G, Nicolson T (2012) Kinocilia mediate mechanosensitivity in ͺͲ developing zebrafish hair cells. Dev Cell 23:329-341. ͺͳ Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant ͺʹ JM, Yost HJ, Kanki JP, Chien CB (2007) The Tol2kit: a multisite gateway- ͺ͵ based construction kit for Tol2 transposon transgenesis constructs. ͺͶ Developmental dynamics : an official publication of the American ͺͷ Association of Anatomists 236:3088-3099. ͺ Lelli A, Kazmierczak P, Kawashima Y, Muller U, Holt JR (2010) Development ͺ and regeneration of sensory transduction in auditory hair cells ͺͺ requires functional interaction between cadherin-23 and ͺͻ protocadherin-15. The Journal of neuroscience : the official journal of ͺͺͲ the Society for Neuroscience 30:11259-11269. ͺͺͳ Longo-Guess CM, Gagnon LH, Cook SA, Wu J, Zheng QY, Johnson KR (2005) A ͺͺʹ missense mutation in the previously undescribed gene Tmhs underlies ͺͺ͵ deafness in hurry-scurry (hscy) mice. Proceedings of the National ͺͺͶ Academy of Sciences of the United States of America 102:7894-7899. ͺͺͷ Maeda R, Kindt KS, Mo W, Morgan CP, Erickson T, Zhao H, Clemens-Grisham R, ͺͺ Barr-Gillespie PG, Nicolson T (2014) Tip-link protein protocadherin 15 ͺͺ interacts with transmembrane channel-like proteins TMC1 and TMC2. ͺͺͺ Proceedings of the National Academy of Sciences of the United States of ͺͺͻ America 111:12907-12912. ͵Ͷ ͺͻͲ Meyers JR, MacDonald RB, Duggan A, Lenzi D, Standaert DG, Corwin JT, Corey ͺͻͳ DP (2003) Lighting up the senses: FM1-43 loading of sensory cells ͺͻʹ through nonselective ion channels. The Journal of neuroscience : the ͺͻ͵ official journal of the Society for Neuroscience 23:4054-4065. ͺͻͶ Mo W, Chen F, Nechiporuk A, Nicolson T (2010) Quantification of vestibular- ͺͻͷ induced eye movements in zebrafish larvae. BMC neuroscience 11:110. ͺͻ Nicolson T, Rusch A, Friedrich RW, Granato M, Ruppersberg JP, Nusslein- ͺͻ Volhard C (1998) Genetic analysis of vertebrate sensory hair cell ͺͻͺ mechanosensation: the zebrafish circler mutants. Neuron 20:271-283. ͺͻͻ Novak P, Li C, Shevchuk AI, Stepanyan R, Caldwell M, Hughes S, Smart TG, ͻͲͲ Gorelik J, Ostanin VP, Lab MJ, Moss GWJ, Folenkov GI, Klenerman D, ͻͲͳ Korchev YE (2009) Nanoscale live-cell imaging using hopping probe ion ͻͲʹ conductance microscopy. Nature Methods 6:279-281. ͻͲ͵ Pepermans E, Michel V, Goodyear R, Bonnet C, Abdi S, Dupont T, Gherbi S, ͻͲͶ Holder M, Makrelouf M, Hardelin JP, Marlin S, Zenati A, Richardson G, ͻͲͷ Avan P, Bahloul A, Petit C (2014) The CD2 isoform of protocadherin-15 ͻͲ is an essential component of the tip-link complex in mature auditory ͻͲ hair cells. EMBO molecular medicine 6:984-992. ͻͲͺ Seiler C, Finger-Baier KC, Rinner O, Makhankov YV, Schwarz H, Neuhauss SC, ͻͲͻ Nicolson T (2005) Duplicated genes with split functions: independent ͻͳͲ roles of protocadherin15 orthologues in zebrafish hearing and vision. ͻͳͳ Development 132:615-623. ͻͳʹ Seiler C, Ben-David O, Sidi S, Hendrich O, Rusch A, Burnside B, Avraham KB, ͻͳ͵ Nicolson T (2004) Myosin VI is required for structural integrity of the ͻͳͶ apical surface of sensory hair cells in zebrafish. Developmental biology ͻͳͷ 272:328-338. ͻͳ Senften M, Schwander M, Kazmierczak P, Lillo C, Shin JB, Hasson T, Geleoc GS, ͻͳ Gillespie PG, Williams D, Holt JR, Muller U (2006) Physical and ͻͳͺ functional interaction between protocadherin 15 and myosin VIIa in ͻͳͻ mechanosensory hair cells. The Journal of neuroscience : the official ͻʹͲ journal of the Society for Neuroscience 26:2060-2071. ͻʹͳ Shabbir MI, Ahmed ZM, Khan SY, Riazuddin S, Waryah AM, Khan SN, Camps RD, ͻʹʹ Ghosh M, Kabra M, Belyantseva IA, Friedman TB, Riazuddin S (2006) ͻʹ͵ Mutations of human TMHS cause recessively inherited non-syndromic ͻʹͶ hearing loss. Journal of medical genetics 43:634-640. ͻʹͷ Siemens J, Lillo C, Dumont RA, Reynolds A, Williams DS, Gillespie PG, Muller U ͻʹ (2004) Cadherin 23 is a component of the tip link in hair-cell ͻʹ stereocilia. Nature 428:950-955. ͻʹͺ Sollner C, Rauch GJ, Siemens J, Geisler R, Schuster SC, Muller U, Nicolson T, ͻʹͻ Tubingen Screen C (2004) Mutations in cadherin 23 affect tip links in ͻ͵Ͳ zebrafish sensory hair cells. Nature 428:955-959. ͻ͵ͳ Sotomayor M, Weihofen WA, Gaudet R, Corey DP (2012) Structure of a force- ͻ͵ʹ conveying cadherin bond essential for inner-ear mechanotransduction. ͻ͵͵ Nature 492:128-132. ͻ͵Ͷ Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount ͻ͵ͷ zebrafish embryos. Nat. Protoc. 2008;3:59–69. ͵ͷ ͻ͵ Webb SW, Grillet N, Andrade LR, Xiong W, Swarthout L, Della Santina CC, ͻ͵ Kachar B, Muller U (2011) Regulation of PCDH15 function in ͻ͵ͺ mechanosensory hair cells by alternative splicing of the cytoplasmic ͻ͵ͻ domain. Development 138:1607-1617. ͻͶͲ Xiong W, Grillet N, Elledge HM, Wagner TF, Zhao B, Johnson KR, Kazmierczak ͻͶͳ P, Muller U (2012) TMHS is an integral component of the ͻͶʹ mechanotransduction machinery of cochlear hair cells. Cell 151:1283- ͻͶ͵ 1295. ͻͶͶ Zhao B, Wu Z, Grillet N, Yan L, Xiong W, Harkins-Perry S, Muller U (2014) TMIE ͻͶͷ is an essential component of the mechanotransduction machinery of ͻͶ cochlear hair cells. Neuron 84:954-967. ͻͶ ͻͶͺ ͻͶͻ ͻͷͲ ͵