Publications by authors named "Inna A Belyantseva"

49 Publications

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.

Cell Rep 2021 Feb;34(5):108708

Laboratory of Single-Molecule Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. Electronic address:

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.
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http://dx.doi.org/10.1016/j.celrep.2021.108708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904085PMC
February 2021

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin.

Curr Biol 2021 Mar 4;31(6):1141-1153.e7. Epub 2021 Jan 4.

Department of Biology, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA. Electronic address:

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.
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http://dx.doi.org/10.1016/j.cub.2020.12.006DOI Listing
March 2021

Mouse Models of Human Pathogenic Variants of Associated with Non-Syndromic Deafness DFNB86 and DFNA65 and Syndromes Involving Deafness.

Genes (Basel) 2020 Sep 24;11(10). Epub 2020 Sep 24.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, MD 20892, USA.

Human pathogenic variants of are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, expression was detected only in spiral ganglion neurons. We engineered mouse models of p.(Asp70Tyr) and p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human . Unexpectedly, no auditory dysfunction was detected in mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse and human .
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http://dx.doi.org/10.3390/genes11101122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598720PMC
September 2020

Myosins and Hearing.

Adv Exp Med Biol 2020 ;1239:317-330

Department of Physiology, University of Kentucky College of Medicine, Lexington, KY, USA.

Hearing loss is both genetically and clinically heterogeneous, and pathogenic variants of over a hundred different genes are associated with this common neurosensory disorder. A relatively large number of these "deafness genes" encode myosin super family members. The evidence that pathogenic variants of human MYO3A, MYO6, MYO7A, MYO15A, MYH14 and MYH9 are associated with deafness ranges from moderate to definitive. Additional evidence for the involvement of these six myosins for normal hearing also comes from animal models, usually mouse or zebra fish, where mutations of these genes cause hearing loss and from biochemical, physiological and cell biological studies of their roles in the inner ear. This chapter focuses on these six genes for which evidence of a causative role in deafness is substantial.
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http://dx.doi.org/10.1007/978-3-030-38062-5_13DOI Listing
August 2020

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

JCI Insight 2019 06 20;4(12). Epub 2019 Jun 20.

Department of Otolaryngology-Head and Neck Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
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http://dx.doi.org/10.1172/jci.insight.128561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629139PMC
June 2019

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Hum Mol Genet 2019 05;28(9):1530-1547

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, MD, USA.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.
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http://dx.doi.org/10.1093/hmg/ddy445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466106PMC
May 2019

Mammalian Auditory Hair Cell Bundle Stiffness Affects Frequency Tuning by Increasing Coupling along the Length of the Cochlea.

Cell Rep 2018 06;23(10):2915-2927

The Caruso Department of Otolaryngology - Head & Neck Surgery, University of Southern California, Los Angeles, CA 90033, USA. Electronic address:

The stereociliary bundles of cochlear hair cells convert mechanical vibrations into the electrical signals required for auditory sensation. While the stiffness of the bundles strongly influences mechanotransduction, its influence on the vibratory response of the cochlear partition is unclear. To assess this, we measured cochlear vibrations in mutant mice with reduced bundle stiffness or with a tectorial membrane (TM) that is detached from the sensory epithelium. We found that reducing bundle stiffness decreased the high-frequency extent and sharpened the tuning of vibratory responses obtained postmortem. Detaching the TM further reduced the high-frequency extent of the vibrations but also lowered the partition's resonant frequency. Together, these results demonstrate that the bundle's stiffness and attachment to the TM contribute to passive longitudinal coupling in the cochlea. We conclude that the stereociliary bundles and TM interact to facilitate passive-wave propagation to more apical locations, possibly enhancing active-wave amplification in vivo.
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http://dx.doi.org/10.1016/j.celrep.2018.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309882PMC
June 2018

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

Hum Mol Genet 2018 03;27(5):780-798

School of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.
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http://dx.doi.org/10.1093/hmg/ddx440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059191PMC
March 2018

Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.

Mol Ther 2017 03 21;25(3):780-791. Epub 2017 Feb 21.

Neurotology Program, National Institute on Deafness and Other Communication Disorders (NIDCD), NIH, Bethesda, MD 20892, USA; Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA. Electronic address:

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.
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http://dx.doi.org/10.1016/j.ymthe.2017.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363211PMC
March 2017

Harnessing molecular motors for nanoscale pulldown in live cells.

Mol Biol Cell 2017 Feb 8;28(3):463-475. Epub 2016 Dec 8.

Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.

Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.
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http://dx.doi.org/10.1091/mbc.E16-08-0583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341729PMC
February 2017

Helios(®) Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates.

Methods Mol Biol 2016 ;1427:3-26

Laboratory of Molecular Genetics, Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD, 20892-3729, USA.

The transfection of vertebrate inner ear hair cells has proven to be challenging. Therefore, many laboratories attempt to use and improve different transfection methods. Each method has its own advantages and disadvantages. A particular researcher's skills in addition to available equipment and the type of experiment (in vivo or in vitro) likely determine the transfection method of choice. Biolistic delivery of exogenous DNA, mRNA, or siRNA, also known as Helios(®) Gene Gun-mediated transfection, uses the mechanical energy of compressed helium gas to bombard tissue with micron- or submicron-sized DNA or RNA-coated gold particles, which can penetrate and transfect cells in vitro or in vivo. Helios(®) Gene Gun-mediated transfection has several advantages: (1) it is simple enough to learn in a short time; (2) it is designed to overcome cell barriers even as tough as plant cell membrane or stratum corneum in the epidermis; (3) it can transfect cells deep inside a tissue such as specific neurons within a brain slice; (4) it can accommodate mRNA, siRNA, or DNA practically of any size to be delivered; and (5) it works well with various cell types including non-dividing, terminally differentiated cells that are difficult to transfect, such as neurons or mammalian inner ear sensory hair cells. The latter advantage is particularly important for inner ear research. The disadvantages of this method are: (1) low efficiency of transfection due to many variables that have to be adjusted and (2) potential mechanical damage of the tissue if the biolistic shot parameters are not optimal. This chapter provides a step-by-step protocol and critical evaluation of the Bio-Rad Helios(®) Gene Gun transfection method used to deliver green fluorescent protein (GFP)-tagged full-length cDNAs of myosin 15a, whirlin, β-actin, and Clic5 into rodent hair cells of the postnatal inner ear sensory epithelia in culture.
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http://dx.doi.org/10.1007/978-1-4939-3615-1_1DOI Listing
December 2017

Autosomal-Recessive Hearing Impairment Due to Rare Missense Variants within S1PR2.

Am J Hum Genet 2016 Feb 21;98(2):331-8. Epub 2016 Jan 21.

Center for Statistical Genetics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

The sphingosine-1-phosphate receptors (S1PRs) are a well-studied class of transmembrane G protein-coupled sphingolipid receptors that mediate multiple cellular processes. However, S1PRs have not been previously reported to be involved in the genetic etiology of human traits. S1PR2 lies within the autosomal-recessive nonsyndromic hearing impairment (ARNSHI) locus DFNB68 on 19p13.2. From exome sequence data we identified two pathogenic S1PR2 variants, c.323G>C (p.Arg108Pro) and c.419A>G (p.Tyr140Cys). Each of these variants co-segregates with congenital profound hearing impairment in consanguineous Pakistani families with maximum LOD scores of 6.4 for family DEM4154 and 3.3 for family PKDF1400. Neither S1PR2 missense variant was reported among ∼120,000 chromosomes in the Exome Aggregation Consortium database, in 76 unrelated Pakistani exomes, or in 720 Pakistani control chromosomes. Both DNA variants affect highly conserved residues of S1PR2 and are predicted to be damaging by multiple bioinformatics tools. Molecular modeling predicts that these variants affect binding of sphingosine-1-phosphate (p.Arg108Pro) and G protein docking (p.Tyr140Cys). In the previously reported S1pr2(-/-) mice, stria vascularis abnormalities, organ of Corti degeneration, and profound hearing loss were observed. Additionally, hair cell defects were seen in both knockout mice and morphant zebrafish. Family PKDF1400 presents with ARNSHI, which is consistent with the lack of gross malformations in S1pr2(-/-) mice, whereas family DEM4154 has lower limb malformations in addition to hearing loss. Our findings suggest the possibility of developing therapies against hair cell damage (e.g., from ototoxic drugs) through targeted stimulation of S1PR2.
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http://dx.doi.org/10.1016/j.ajhg.2015.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746333PMC
February 2016

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.

Mol Ther 2016 Feb 26;24(1):17-25. Epub 2015 Aug 26.

Section on Sensory Cell Biology, NIDCD, National Institutes of Health, Bethesda, Maryland, USA.

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.
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http://dx.doi.org/10.1038/mt.2015.150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754541PMC
February 2016

The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.

Elife 2015 Aug 24;4. Epub 2015 Aug 24.

Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, United States.

The precise assembly of inner ear hair cell stereocilia into rows of increasing height is critical for mechanotransduction and the sense of hearing. Yet, how the lengths of actin-based stereocilia are regulated remains poorly understood. Mutations of the molecular motor myosin 15 stunt stereocilia growth and cause deafness. We found that hair cells express two isoforms of myosin 15 that differ by inclusion of an 133-kDa N-terminal domain, and that these isoforms can selectively traffic to different stereocilia rows. Using an isoform-specific knockout mouse, we show that hair cells expressing only the small isoform remarkably develop normal stereocilia bundles. However, a critical subset of stereocilia with active mechanotransducer channels subsequently retracts. The larger isoform with the 133-kDa N-terminal domain traffics to these specialized stereocilia and prevents disassembly of their actin core. Our results show that myosin 15 isoforms can navigate between functionally distinct classes of stereocilia, and are independently required to assemble and then maintain the intricate hair bundle architecture.
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http://dx.doi.org/10.7554/eLife.08627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592939PMC
August 2015

ILDR1 null mice, a model of human deafness DFNB42, show structural aberrations of tricellular tight junctions and degeneration of auditory hair cells.

Hum Mol Genet 2015 Feb 12;24(3):609-24. Epub 2014 Sep 12.

National Institute on Deafness and Other Communication Disorders, Section on Human Genetics

In the mammalian inner ear, bicellular and tricellular tight junctions (tTJs) seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like (Ig-like) domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tTJs of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC (DFNB49) encoding tricellulin and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells (HCs) but have a normal endocochlear potential. ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in the inner ear sensory epithelia of ILDR1 null mice after the first postnatal week. As revealed by freeze-fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory HCs.
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http://dx.doi.org/10.1093/hmg/ddu474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291242PMC
February 2015

Tricellulin deficiency affects tight junction architecture and cochlear hair cells.

J Clin Invest 2013 Sep 27;123(9):4036-49. Epub 2013 Aug 27.

Laboratory of Molecular Genetics, Division of Pediatric Otolaryngology / Head and Neck Surgery, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio 45229, USA.

The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin.
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http://dx.doi.org/10.1172/JCI69031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754262PMC
September 2013

Molecular remodeling of tip links underlies mechanosensory regeneration in auditory hair cells.

PLoS Biol 2013 11;11(6):e1001583. Epub 2013 Jun 11.

Department of Physiology, University of Kentucky, Lexington, Kentucky, United States of America.

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 Ca(2+)-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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http://dx.doi.org/10.1371/journal.pbio.1001583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679001PMC
January 2014

A null mutation of mouse Kcna10 causes significant vestibular and mild hearing dysfunction.

Hear Res 2013 Jun 22;300:1-9. Epub 2013 Mar 22.

Section on Human Genetics, Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 5 Research Ct, 2A-19, Rockville, MD 20850, USA.

KCNA10 is a voltage gated potassium channel that is expressed in the inner ear. The localization and function of KCNA10 was studied in a mutant mouse, B6-Kcna10(TM45), in which the single protein coding exon of Kcna10 was replaced with a beta-galactosidase reporter cassette. Under the regulatory control of the endogenous Kcna10 promoter and enhancers, beta-galactosidase was expressed in hair cells of the vestibular organs and the organ of Corti. KCNA10 expression develops in opposite tonotopic gradients in the inner and outer hair cells. Kcna10(TM45) homozygotes display only a mild elevation in pure tone hearing thresholds as measured by auditory brainstem response (ABR), while heterozygotes are normal. However, Kcna10(TM45) homozygotes have absent vestibular evoked potentials (VsEPs) or elevated VsEP thresholds with prolonged peak latencies, indicating significant vestibular dysfunction despite the lack of any overt imbalance behaviors. Our results suggest that Kcna10 is expressed primarily in hair cells of the inner ear, with little evidence of expression in other organs. The Kcna10(TM45) targeted allele may be a model of human nonsyndromic vestibulopathy.
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http://dx.doi.org/10.1016/j.heares.2013.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3684051PMC
June 2013

Phenotypic variability of CLDN14 mutations causing DFNB29 hearing loss in the Pakistani population.

J Hum Genet 2013 Feb 13;58(2):102-8. Epub 2012 Dec 13.

National Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.

Human hereditary deafness at the DFNB29 locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetramembrane spanning that localizes to the apical tight junctions of organ of Corti hair cells and in many other tissues. Typically, the DFNB29 phenotype is characterized by prelingual, bilateral, sensorineural hearing loss. The goal of this study was to define the identity and frequency of CLDN14 mutations and associated inner ear phenotypes in a cohort of 800 Pakistani families segregating deafness. Hearing loss in 15 multi-generational families was found to co-segregate with CLDN14-linked STR markers. The sequence of the six exons and regions flanking the introns of CLDN14 in these 15 families revealed five likely pathogenic alleles. Two are novel missense substitutions (p.Ser87Ile and p.Ala94Val), whereas p.Arg81His, p.Val85Asp and p.Met133ArgfsX23 have been reported previously. Haplotype analyses indicate that p.Val85Asp and p.Met133ArgfsX23 are founder mutations. The p.Val85Asp accounts for ~67% of the mutant alleles of CLDN14 in our cohort. Combined with the previously reported data, CLDN14 mutations were identified in 18 of 800 Pakistani families (2.25; 95% CI, 1.4-3.5). Hearing loss in the affected individuals homozygous for CLDN14 mutations varied from moderate to profound. This phenotypic variability may be due to environmental factors (for example drug and noise exposure) and/or genetic modifiers.
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http://dx.doi.org/10.1038/jhg.2012.143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596117PMC
February 2013

Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48.

Nat Genet 2012 Nov 30;44(11):1265-71. Epub 2012 Sep 30.

Division of Pediatric Otolaryngology/Head & Neck Surgery, Cincinnati Children's Hospital Medical Center, Ohio, USA.

Sensorineural hearing loss is genetically heterogeneous. Here, we report that mutations in CIB2, which encodes a calcium- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). One mutation in CIB2 is a prevalent cause of deafness DFNB48 in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In mice, CIB2 is localized to the mechanosensory stereocilia of inner ear hair cells and to retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of calcium binding, CIB2 significantly decreased the ATP-induced calcium responses in heterologous cells, whereas mutations in deafness DFNB48 altered CIB2 effects on calcium responses. Furthermore, in zebrafish and Drosophila melanogaster, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We also show that CIB2 is a new member of the vertebrate Usher interactome.
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http://dx.doi.org/10.1038/ng.2426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501259PMC
November 2012

Actin in hair cells and hearing loss.

Hear Res 2012 Jun 13;288(1-2):89-99. Epub 2011 Dec 13.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 5 Research Court, Rockville, MD 20850, USA.

Hereditary deafness is genetically heterogeneous such that mutations of many different genes can cause hearing loss. This review focuses on the evidence and implications that several of these deafness genes encode actin-interacting proteins or actin itself. There is a growing appreciation of the contribution of the actin interactome in stereocilia development, maintenance, mechanotransduction and malfunction of the auditory system.
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http://dx.doi.org/10.1016/j.heares.2011.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403717PMC
June 2012

Functional null mutations of MSRB3 encoding methionine sulfoxide reductase are associated with human deafness DFNB74.

Am J Hum Genet 2011 Jan 23;88(1):19-29. Epub 2010 Dec 23.

Laboratory of Molecular Genetics, Division of Pediatric Otolaryngology Head & Neck Surgery, Cincinnati Children's Hospital Research Foundation, and the Department of Otolaryngology, College of Medicine, University of Cincinnati, OH, 45229 USA.

The DFNB74 locus for autosomal-recessive, nonsyndromic deafness segregating in three families was previously mapped to a 5.36 Mb interval on chromosome 12q14.2-q15. Subsequently, we ascertained five additional consanguineous families in which deafness segregated with markers at this locus and refined the critical interval to 2.31 Mb. We then sequenced the protein-coding exons of 18 genes in this interval. The affected individuals of six apparently unrelated families were homozygous for the same transversion (c.265T>G) in MSRB3, which encodes a zinc-containing methionine sulfoxide reductase B3. c.265T>G results in a substitution of glycine for cysteine (p.Cys89Gly), and this substitution cosegregates with deafness in the six DFNB74 families. This cysteine residue of MSRB3 is conserved in orthologs from yeast to humans and is involved in binding structural zinc. In vitro, p.Cys89Gly abolished zinc binding and MSRB3 enzymatic activity, indicating that p.Cys89Gly is a loss-of-function allele. The affected individuals in two other families were homozygous for a transition mutation (c.55T>C), which results in a nonsense mutation (p.Arg19X) in alternatively spliced exon 3, encoding a mitochondrial localization signal. This finding suggests that DFNB74 deafness is due to a mitochondrial dysfunction. In a cohort of 1,040 individuals (aged 53-67 years) of European ancestry, we found no association between 17 tagSNPs for MSRB3 and age-related hearing loss. Mouse Msrb3 is expressed widely. In the inner ear, it is found in the sensory epithelium of the organ of Corti and vestibular end organs as well as in cells of the spiral ganglion. Taken together, MSRB3-catalyzed reduction of methionine sulfoxides to methionine is essential for hearing.
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http://dx.doi.org/10.1016/j.ajhg.2010.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014371PMC
January 2011

Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing.

Cell 2010 May;141(5):786-98

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA.

Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets but distinct from bundles formed by espin, an actin crosslinker in stereocilia. We generated mutant Triobp mice (Triobp(Deltaex8/Deltaex8)) that are profoundly deaf. Stereocilia of Triobp(Deltaex8/Deltaex8) mice develop normally but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.
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http://dx.doi.org/10.1016/j.cell.2010.03.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879707PMC
May 2010

Targeted capture and next-generation sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79.

Am J Hum Genet 2010 Mar 18;86(3):378-88. Epub 2010 Feb 18.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA.

Targeted genome capture combined with next-generation sequencing was used to analyze 2.9 Mb of the DFNB79 interval on chromosome 9q34.3, which includes 108 candidate genes. Genomic DNA from an affected member of a consanguineous family segregating recessive, nonsyndromic hearing loss was used to make a library of fragments covering the DFNB79 linkage interval defined by genetic analyses of four pedigrees. Homozygosity for eight previously unreported variants in transcribed sequences was detected by evaluating a library of 402,554 sequencing reads and was later confirmed by Sanger sequencing. Of these variants, six were determined to be polymorphisms in the Pakistani population, and one was in a noncoding gene that was subsequently excluded genetically from the DFNB79 linkage interval. The remaining variant was a nonsense mutation in a predicted gene, C9orf75, renamed TPRN. Evaluation of the other three DFNB79-linked families identified three additional frameshift mutations, for a total of four truncating alleles of this gene. Although TPRN is expressed in many tissues, immunolocalization of the protein product in the mouse cochlea shows prominent expression in the taper region of hair cell stereocilia. Consequently, we named the protein taperin.
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http://dx.doi.org/10.1016/j.ajhg.2010.01.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2833391PMC
March 2010

Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse.

Am J Hum Genet 2010 Feb 4;86(2):148-60. Epub 2010 Feb 4.

Department of Otolaryngology, Kresge Hearing Research Institute, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.
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http://dx.doi.org/10.1016/j.ajhg.2010.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2820167PMC
February 2010

Twinfilin 2 regulates actin filament lengths in cochlear stereocilia.

J Neurosci 2009 Dec;29(48):15083-8

Department of Otolaryngology-Head & Neck Surgery, Stanford University School of Medicine, Stanford, California 94305, USA.

Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50-100 actin-filled stereocilia, which are organized in three rows in a staircase manner. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day 5 (P5) onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.
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http://dx.doi.org/10.1523/JNEUROSCI.2782-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2823077PMC
December 2009

Molecular basis of DFNB73: mutations of BSND can cause nonsyndromic deafness or Bartter syndrome.

Am J Hum Genet 2009 Aug 30;85(2):273-80. Epub 2009 Jul 30.

Section on Human Genetics, Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, NIH, Rockville, MD 20850, USA.

BSND encodes barttin, an accessory subunit of renal and inner ear chloride channels. To date, all mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness. We identified a BSND mutation (p.I12T) in four kindreds segregating nonsyndromic deafness linked to a 4.04-cM interval on chromosome 1p32.3. The functional consequences of p.I12T differ from BSND mutations that cause renal failure and deafness in Bartter syndrome type IV. p.I12T leaves chloride channel function unaffected and only interferes with chaperone function of barttin in intracellular trafficking. This study provides functional data implicating a hypomorphic allele of BSND as a cause of apparent nonsyndromic deafness. We demonstrate that BSND mutations with different functional consequences are the basis for either syndromic or nonsyndromic deafness.
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http://dx.doi.org/10.1016/j.ajhg.2009.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725234PMC
August 2009

Gamma-actin is required for cytoskeletal maintenance but not development.

Proc Natl Acad Sci U S A 2009 Jun 3;106(24):9703-8. Epub 2009 Jun 3.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Rockville, MD 20850, USA.

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.
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http://dx.doi.org/10.1073/pnas.0900221106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701000PMC
June 2009

Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans.

Nat Genet 2008 Nov 26;40(11):1335-40. Epub 2008 Oct 26.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Rockville, Maryland 20850, USA.

Many proteins necessary for sound transduction have been identified through positional cloning of genes that cause deafness. We report here that mutations of LRTOMT are associated with profound nonsyndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, detected by protein blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, new transcripts can arise through partial or complete coalescence of genes. We provide evidence that in the primate lineage LRTOMT evolved from the fusion of two neighboring ancestral genes, which exist as separate genes (Lrrc51 and Tomt) in rodents.
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http://dx.doi.org/10.1038/ng.245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404732PMC
November 2008

Helios Gene Gun-mediated transfection of the inner ear sensory epithelium.

Methods Mol Biol 2009 ;493:103-23

Laboratory of Molecular Genetics, Section on Human Genetics, NIDCD, National Institutes of Health, Rockville MD, USA.

Helios Gene Gun-mediated transfection is a biolistic method for mechanical delivery of exogenous DNA into cells in vitro or in vivo. The technique is based on bombardment of a targeted cellular surface by micron- or submicron-sized DNA-coated gold particles that are accelerated by a pressure pulse of compressed helium gas. The main advantage of Helios Gene Gun-mediated transfections is that it functions well on various cell types, including terminally differentiated cells that are difficult to transfect, such as neurons or inner ear sensory hair cells, and cells in internal cellular layers, such as neurons in organotypic brain slices. The successful delivery of mRNA, siRNA, or DNA of practically any size can be achieved using biolistic transfection. This chapter provides a detailed description and critical evaluation of the methodology used to transfect cDNA expression constructs, including green fluorescent protein (GFP) tagged full-length cDNAs of myosin XVa, whirlin, and beta-actin, into cultured inner ear sensory epithelia using the Bio-Rad Helios Gene Gun.
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http://dx.doi.org/10.1007/978-1-59745-523-7_7DOI Listing
January 2009