Person: Corey, David
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Corey
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Corey, David
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Publication Heterodimeric capping protein is required for stereocilia length and width regulation(The Rockefeller University Press, 2017) Avenarius, Matthew R.; Krey, Jocelyn F.; Dumont, Rachel A.; Morgan, Clive P.; Benson, Connor B.; Vijayakumar, Sarath; Cunningham, Christopher L.; Scheffer, Deborah I.; Corey, David; Müller, Ulrich; Jones, Sherri M.; Barr-Gillespie, Peter G.Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear’s stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.Publication Genome-wide association analysis on normal hearing function identifies PCDH20 and SLC28A3 as candidates for hearing function and loss(Oxford University Press, 2015) Vuckovic, Dragana; Dawson, Sally; Scheffer, Deborah I.; Rantanen, Taina; Morgan, Anna; Di Stazio, Mariateresa; Vozzi, Diego; Nutile, Teresa; Concas, Maria P.; Biino, Ginevra; Nolan, Lisa; Bahl, Aileen; Loukola, Anu; Viljanen, Anne; Davis, Adrian; Ciullo, Marina; Corey, David; Pirastu, Mario; Gasparini, Paolo; Girotto, GiorgiaHearing loss and individual differences in normal hearing both have a substantial genetic basis. Although many new genes contributing to deafness have been identified, very little is known about genes/variants modulating the normal range of hearing ability. To fill this gap, we performed a two-stage meta-analysis on hearing thresholds (tested at 0.25, 0.5, 1, 2, 4, 8 kHz) and on pure-tone averages (low-, medium- and high-frequency thresholds grouped) in several isolated populations from Italy and Central Asia (total N = 2636). Here, we detected two genome-wide significant loci close to PCDH20 and SLC28A3 (top hits: rs78043697, P = 4.71E−10 and rs7032430, P = 2.39E−09, respectively). For both loci, we sought replication in two independent cohorts: B58C from the UK (N = 5892) and FITSA from Finland (N = 270). Both loci were successfully replicated at a nominal level of significance (P < 0.05). In order to confirm our quantitative findings, we carried out RT-PCR and reported RNA-Seq data, which showed that both genes are expressed in mouse inner ear, especially in hair cells, further suggesting them as good candidates for modulatory genes in the auditory system. Sequencing data revealed no functional variants in the coding region of PCDH20 or SLC28A3, suggesting that variation in regulatory sequences may affect expression. Overall, these results contribute to a better understanding of the complex mechanisms underlying human hearing function.Publication SHIELD: an integrative gene expression database for inner ear research(Oxford University Press, 2015) Shen, Jun; Scheffer, Déborah I.; Kwan, Kelvin Y.; Corey, DavidThe inner ear is a highly specialized mechanosensitive organ responsible for hearing and balance. Its small size and difficulty in harvesting sufficient tissue has hindered the progress of molecular studies. The protein components of mechanotransduction, the molecular biology of inner ear development and the genetic causes of many hereditary hearing and balance disorders remain largely unknown. Inner-ear gene expression data will help illuminate each of these areas. For over a decade, our laboratories and others have generated extensive sets of gene expression data for different cell types in the inner ear using various sample preparation methods and high-throughput genome-wide approaches. To facilitate the study of genes in the inner ear by efficient presentation of the accumulated data and to foster collaboration among investigators, we have developed the Shared Harvard Inner Ear Laboratory Database (SHIELD), an integrated resource that seeks to compile, organize and analyse the genomic, transcriptomic and proteomic knowledge of the inner ear. Five datasets are currently available. These datasets are combined in a relational database that integrates experimental data and annotations relevant to the inner ear. The SHIELD has a searchable web interface with two data retrieval options: viewing the gene pages online or downloading individual datasets as data tables. Each retrieved gene page shows the gene expression data and detailed gene information with hyperlinks to other online databases with up-to-date annotations. Downloadable data tables, for more convenient offline data analysis, are derived from publications and are current as of the time of publication. The SHIELD has made published and some unpublished data freely available to the public with the hope and expectation of accelerating discovery in the molecular biology of balance, hearing and deafness. Database URL: https://shield.hms.harvard.eduPublication Sorting Out a Promiscuous Superfamily: Towards Cadherin Connectomics(Elsevier BV, 2014) Sotomayor, Marcos; Gaudet, Rachelle; Corey, DavidMembers of the cadherin superfamily of proteins are involved in diverse biological processes such as morphogenesis, sound transduction, and neuronal connectivity. Key to cadherin function is their extracellular domain containing cadherin repeats, which can mediate interactions involved in adhesion and cell signaling. Recent cellular, biochemical, and structural studies have revealed that physical interaction among cadherins is more complex than originally thought. Here we review work on new cadherin complexes and discuss how the classification of the mammalian family can be used to search for additional cadherin-interacting partners. We also highlight some of the challenges in cadherin research; namely, the characterization of a cadherin connectome in biochemical and structural terms, as well as the elucidation of molecular mechanisms underlying the functional diversity of nonclassical cadherins in vivo.Publication PSIP1/LEDGF: a new gene likely involved in sensorineural progressive hearing loss(Nature Publishing Group, 2015) Girotto, Giorgia; Scheffer, Déborah I.; Morgan, Anna; Vozzi, Diego; Rubinato, Elisa; Di Stazio, Mariateresa; Muzzi, Enrico; Pensiero, Stefano; Giersch, Anne; Corey, David; Gasparini, PaoloHereditary Hearing Loss (HHL) is an extremely heterogeneous disorder. Approximately 30 out of 80 known HHL genes are associated with autosomal dominant forms. Here, we identified PSIP1/LEDGF (isoform p75) as a novel strong candidate gene involved in dominant HHL. Using exome sequencing we found a frameshift deletion (c.1554_1555del leading to p.E518Dfs*2) in an Italian pedigree affected by sensorineural mild-to-moderate HHL but also showing a variable eye phenotype (i.e. uveitis, optic neuropathy). This deletion led to a premature stop codon (p.T519X) with truncation of the last 12 amino acids. PSIP1 was recently described as a transcriptional co-activator regulated by miR-135b in vestibular hair cells of the mouse inner ear as well as a possible protector against photoreceptor degeneration. Here, we demonstrate that it is ubiquitously expressed in the mouse inner ear. The PSIP1 mutation is associated with a peculiar audiometric slope toward the high frequencies. These findings indicate that PSIP1 likely plays an important role in HHL.Publication Live-cell imaging of actin dynamics reveals mechanisms of stereocilia length regulation in the inner ear(Nature Pub. Group, 2015) Drummond, Meghan C.; Barzik, Melanie; Bird, Jonathan E.; Zhang, Duan-Sun; Lechene, Claude; Corey, David; Cunningham, Lisa L.; Friedman, Thomas B.The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips.Publication Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament severing proteins(2015) Narayanan, Praveena; Chatterton, Paul; Ikeda, Akihiro; Ikeda, Sakae; Corey, David; Ervasti, James M.; Perrin, Benjamin J.Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of cross-linked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here, we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically-gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Together, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length.Publication Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo(Nature Publishing Group, 2016) Rivera-Monroy, Jhon; Musiol, Lena; Unthan-Fechner, Kirsten; Farkas, Ákos; Clancy, Anne; Coy-Vergara, Javier; Weill, Uri; Gockel, Sarah; Lin, Shuh-Yow; Corey, David; Kohl, Tobias; Ströbel, Philipp; Schuldiner, Maya; Schwappach, Blanche; Vilardi, FabioTail-anchored (TA) proteins are post-translationally inserted into membranes. The TRC40 pathway targets TA proteins to the endoplasmic reticulum via a receptor comprised of WRB and CAML. TRC40 pathway clients have been identified using in vitro assays, however, the relevance of the TRC40 pathway in vivo remains unknown. We followed the fate of TA proteins in two tissue-specific WRB knockout mouse models and found that their dependence on the TRC40 pathway in vitro did not predict their reaction to receptor depletion in vivo. The SNARE syntaxin 5 (Stx5) was extremely sensitive to disruption of the TRC40 pathway. Screening yeast TA proteins with mammalian homologues, we show that the particular sensitivity of Stx5 is conserved, possibly due to aggregation propensity of its cytoplasmic domain. We establish that Stx5 is an autophagy target that is inefficiently membrane-targeted by alternative pathways. Our results highlight an intimate relationship between the TRC40 pathway and cellular proteostasis.Publication Hair-Cell Mechanotransduction Persists in TRP Channel Knockout Mice(Public Library of Science, 2016) Wu, Xudong; Indzhykulian, Artur; Niksch, Paul; Webber, Roxanna M.; Garcia-Gonzalez, Miguel; Watnick, Terry; Zhou, Jing; Vollrath, Melissa A.; Corey, DavidMembers of the TRP superfamily of ion channels mediate mechanosensation in some organisms, and have been suggested as candidates for the mechanotransduction channel in vertebrate hair cells. Some TRP channels can be ruled out based on lack of an inner ear phenotype in knockout animals or pore properties not similar to the hair-cell channel. Such studies have excluded Trpv4, Trpa1, Trpml3, Trpm1, Trpm3, Trpc1, Trpc3, Trpc5, and Trpc6. However, others remain reasonable candidates. We used data from an RNA-seq analysis of gene expression in hair cells as well as data on TRP channel conductance to narrow the candidate group. We then characterized mice lacking functional Trpm2, Pkd2, Pkd2l1, Pkd2l2 and Pkd1l3, using scanning electron microscopy, auditory brainstem response, permeant dye accumulation, and single-cell electrophysiology. In all of these TRP-deficient mice, and in double and triple knockouts, mechanotransduction persisted. Together with published studies, these results argue against the participation of any of the 33 mouse TRP channels in hair cell transduction.Publication Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity(Public Library of Science, 2016) Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, DavidWe describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.