113 results on '"Frolenkov, Gregory I."'
Search Results
2. Generating high-fidelity cochlear organoids from human pluripotent stem cells
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Moore, Stephen T., Nakamura, Takashi, Nie, Jing, Solivais, Alexander J., Aristizábal-Ramírez, Isabel, Ueda, Yoshitomo, Manikandan, Mayakannan, Reddy, V. Shweta, Romano, Daniel R., Hoffman, John R., Perrin, Benjamin J., Nelson, Rick F., Frolenkov, Gregory I., Chuva de Sousa Lopes, Susana M., and Hashino, Eri
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- 2023
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3. Modification of cell wall polysaccharide guides cell division in Streptococcus mutans
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Zamakhaeva, Svetlana, Chaton, Catherine T., Rush, Jeffrey S., Ajay Castro, Sowmya, Kenner, Cameron W., Yarawsky, Alexander E., Herr, Andrew B., van Sorge, Nina M., Dorfmueller, Helge C., Frolenkov, Gregory I., Korotkov, Konstantin V., and Korotkova, Natalia
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- 2021
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4. Myosin-XVa Controls Both Staircase Architecture and Diameter Gradation of Stereocilia Rows in the Auditory Hair Cell Bundles
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Hadi, Shadan, Alexander, Andrew J., Vélez-Ortega, A. Catalina, and Frolenkov, Gregory I.
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- 2020
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5. Helios is a key transcriptional regulator of outer hair cell maturation
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Chessum, Lauren, Matern, Maggie S., Kelly, Michael C., Johnson, Stuart L., Ogawa, Yoko, Milon, Beatrice, McMurray, Mark, Driver, Elizabeth C., Parker, Andrew, Song, Yang, Codner, Gemma, Esapa, Christopher T., Prescott, Jack, Trent, Graham, Wells, Sara, Dragich, Abigail K., Frolenkov, Gregory I., Kelley, Matthew W., Marcotti, Walter, Brown, Steve D. M., Elkon, Ran, Bowl, Michael R., and Hertzano, Ronna
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- 2018
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6. Dynamic Assembly of Surface Structures in Living Cells
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Gorelik, Julia, Shevchuk, Andrew I., Frolenkov, Gregory I., Diakonov, Ivan A., Lab, Max J., Kros, Corné J., Richardson, Guy P., Vodyanoy, Igor, Klenerman, David, and Korchev, Yuri E.
- Published
- 2003
7. Auditory Hair Cell Stereocilia: Balancing Stability to Gain Sensitivity?
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Vélez-Ortega, A. Catalina and Frolenkov, Gregory I.
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- 2017
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8. γ-Actin Is Required for Cytoskeletal Maintenance but Not Development
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Belyantseva, Inna A., Perrin, Benjamin J., Sonnemann, Kevin J., Zhu, Mei, Stepanyan, Ruben, McGee, JoAnn, Frolenkov, Gregory I., Walsh, Edward J., Friderici, Karen H., Friedman, Thomas B., Ervasti, James M., and Frieden, Carl
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- 2009
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9. Tricellulin deficiency affects tight junction architecture and cochlear hair cells
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Nayak, Gowri, Lee, Sue I., Yousaf, Rizwan, Edelmann, Stephanie E., Trincot, Claire, Van Itallie, Christina M., Sinha, Ghanshyam P., Rafeeq, Maria, Jones, Sherri M., Belyantseva, Inna A., Anderson, James M., Forge, Andrew, Frolenkov, Gregory I., and Riazuddin, Saima
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Hair cells (Mechanoreceptors) -- Physiological aspects -- Research ,Cell junctions -- Physiological aspects -- Research ,Epithelial cells -- Physiological aspects -- Research ,Hearing loss -- Research ,Junctional complexes (Epithelium) -- Physiological aspects -- Research ,Health care industry - Abstract
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., Introduction Epithelial cells outline the lumen and surfaces of organs in the body and most commonly act as barriers between two different physiological environments. The barrier property of epithelial cells [...]
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- 2013
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10. Unbalanced bidirectional radial stiffness gradients within the organ of Corti promoted by TRIOBP.
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Babahosseini, Hesam, Belyantseva, Inna A., Yousaf, Rizwan, Tona, Risa, Hadi, Shadan, Inagaki, Sayaka, Wilson, Elizabeth, Shin-ichiro Kitajiri, Frolenkov, Gregory I., Friedman, Thomas B., and Cartagena-Rivera, Alexander X.
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CORTI'S organ ,HAIR cells ,ATOMIC force microscopy ,CELLULAR mechanics ,FOCUSED ion beams - Abstract
Hearing depends on intricate morphologies and mechanical properties of diverse inner ear cell types. The individual contributions of various inner ear cell types into mechanical properties of the organ of Corti and the mechanisms of their integration are yet largely unknown. Using sub-100-nm spatial resolution atomic force microscopy (AFM), we mapped the Young's modulus (stiffness) of the apical surface of the different cells of the freshly dissected P5-P6 cochlear epithelium from wild-type and mice lacking either Trio and F-actin binding protein (TRIOBP) isoforms 4 and 5 or isoform 5 only. Variants of TRIOBP are associated with deafness in human and in Triobp mutant mouse models. Remarkably, nanoscale AFM mapping revealed unrecognized bidirectional radial stiffness gradients of different magnitudes and opposite orientations between rows of wild-type supporting cells and sensory hair cells. Moreover, the observed bidirectional radial stiffness gradients are unbalanced, with sensory cells being stiffer overall compared to neighboring supporting cells. Deafness-associated TRIOBP deficiencies significantly disrupted the magnitude and orientation of these bidirectional radial stiffness gradients. In addition, serial sectioning with focused ion beam and backscatter scanning electron microscopy shows that a TRIOBP deficiency results in ultrastructural changes of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, correlating with messenger RNA and protein expression levels and AFM stiffness measurements that exposed a softening of the apical surface of the sensory epithelium in mutant mice. Altogether, this additional complexity in the mechanical properties of the sensory epithelium is hypothesized to be an essential contributor to frequency selectivity and sensitivity of mammalian hearing. [ABSTRACT FROM AUTHOR]
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- 2022
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11. Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48
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Riazuddin, Saima, Belyantseva, Inna A, Giese, Arnaud P J, Lee, Kwanghyuk, Indzhykulian, Artur A, Nandamuri, Sri Pratima, Yousaf, Rizwan, Sinha, Ghanshyam P, Lee, Sue, Terrell, David, Hegde, Rashmi S, Ali, Rana A, Anwar, Saima, Andrade-Elizondo, Paula B, Sirmaci, Asli, Parise, Leslie V, Basit, Sulman, Wali, Abdul, Ayub, Muhammad, Ansar, Muhammad, Ahmad, Wasim, Khan, Shaheen N, Akram, Javed, Tekin, Mustafa, Riazuddin, Sheikh, Cook, Tiffany, Buschbeck, Elke K, Frolenkov, Gregory I, Leal, Suzanne M, Friedman, Thomas B, and Ahmed, Zubair M
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- 2012
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12. TRPA1-Mediated Accumulation of Aminoglycosides in Mouse Cochlear Outer Hair Cells
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Stepanyan, Ruben S., Indzhykulian, Artur A., Vélez-Ortega, A. Catalina, Boger, Erich T., Steyger, Peter S., Friedman, Thomas B., and Frolenkov, Gregory I.
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- 2011
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13. [gamma]-Actin is required for cytoskeletal maintenance but not development
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Belyantseva, Inna A., Perrin, Benjamin J., Sonnemann, Kevin J., Zhu, Mei, Stepanyan, Ruben, McGee, JoAnn, Frolenkov, Gregory I., Walsh, Edward J., Friderici, Karen H., Friedman, Thomas B., and Ervasti, James M.
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Actin -- Health aspects ,Actin -- Properties ,Cytoskeleton -- Properties ,Cytoskeleton -- Health aspects ,Cytology -- Research ,Science and technology - Abstract
[[beta].sub.cyto]-Actin and [[gamma].sub.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].sub.cyto]-actin null mice ([Actg1.sup.-/-]) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of [[beta].sub.cyto]-actin. The surprising viability and normal hearing of young [Actg1.sup.-/-] mice means that [[beta].sub.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].sub.cyto]-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, [[gamma].sub.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.sup.-/-] stereocilia similar disruptions are observed even without noise exposure. We conclude that [[gamma].sub.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. actin | cytoskeleton | hearing
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- 2009
14. Auditory mechanotransduction in the absence of functional myosin-XVa
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Stepanyan, Ruben, Belyantseva, Inna A., Griffith, Andrew J., Friedman, Thomas B., and Frolenkov, Gregory I.
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- 2006
15. Regulation of electromotility in the cochlear outer hair cell
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Frolenkov, Gregory I.
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- 2006
16. Expression of prestin, a membrane motor protein, in the mammalian auditory and vestibular periphery
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Adler, Henry J., Belyantseva, Inna A., Merritt, Raymond C., Jr., Frolenkov, Gregory I., Dougherty, Gerard W., and Kachar, Bechara
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- 2003
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17. Building and repairing the stereocilia cytoskeleton in mammalian auditory hair cells.
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Vélez-Ortega, A. Catalina and Frolenkov, Gregory I.
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HAIR cells , *BUILDING repair , *CYTOSKELETON , *LIFE spans , *COCHLEA - Abstract
Abstract Despite all recent achievements in identification of the molecules that are essential for the structure and mechanosensory function of stereocilia bundles in the auditory hair cells of mammalian species, we still have only a rudimentary understanding of the mechanisms of stereocilia formation, maintenance, and repair. Important molecular differences distinguishing mammalian auditory hair cells from hair cells of other types and species have been recently revealed. In addition, we are beginning to solve the puzzle of the apparent life-long stability of the stereocilia bundles in these cells. New data link the stability of the cytoskeleton in the mammalian auditory stereocilia with the normal activity of mechanotransduction channels. These data suggest new ideas on how a terminally-differentiated non-regenerating hair cell in the mammalian cochlea may repair and tune its stereocilia bundle throughout the life span of the organism. Highlights • Differentially expressed proteins regulate the hair bundle staircase architecture. • The stability of the stereocilia actin core depends on intrastereocilia Ca2+ levels. • Changes in intrastereocilia Ca2+ levels may trigger signals for stereocilia repair. • Ca2+-sensitive proteins within stereocilia could be potential therapeutic targets. [ABSTRACT FROM AUTHOR]
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- 2019
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18. Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.
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Vélez-Ortega, A. Catalina and Frolenkov, Gregory I.
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- 2016
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19. CIB2 interacts with TMC1 and TMC2 and is essential for mechanotransduction in auditory hair cells.
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Giese, Arnaud P. J., Yi-Quan Tang, Sinha, Ghanshyam P., Bowl, Michael R., Goldring, Adam C., Parker, Andrew, Freeman, Mary J., Brown, Steve D. M., Riazuddin, Saima, Fettiplace, Robert, Schafer, William R., Frolenkov, Gregory I., and Ahmed, Zubair M.
- Abstract
Inner ear hair cells detect sound through deflection of stereocilia, the microvilli-like projections that are arranged in rows of graded heights. Calcium and integrin-binding protein 2 is essential for hearing and localizes to stereocilia, but its exact function is unknown. Here, we have characterized two mutant mouse lines, one lacking calcium and integrin-binding protein 2 and one carrying a human deafness-related Cib2 mutation, and show that both are deaf and exhibit no mechanotransduction in auditory hair cells, despite the presence of tip links that gate the mechanotransducer channels. In addition, mechanotransducing shorter row stereocilia overgrow in hair cell bundles of both Cib2 mutants. Furthermore, we report that calcium and integrin-binding protein 2 binds to the components of the hair cell mechanotransduction complex, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 mutations. We conclude that calcium and integrin-binding protein 2 is required for normal operation of the mechanotransducer channels and is involved in limiting the growth of transducing stereocilia. [ABSTRACT FROM AUTHOR]
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- 2017
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20. Mechanotransduction current is essential for stability of the transducing stereocilia in mammalian auditory hair cells.
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Lez-Ortega, A Catalina Vé, Freeman, Mary J., Indzhykulian, Artur A., Grossheim, Jonathan M., and Frolenkov, Gregory I.
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- 2017
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21. A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family.
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Patel, Kunjan, Giese, Arnaud P., Grossheim, J. M., Hegde, Rashima S., Delio, Maria, Samanich, Joy, Riazuddin, Saima, Frolenkov, Gregory I., Cai, Jinlu, Ahmed, Zubair M., and Morrow, Bernice E.
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GENETIC mutation ,C-terminal binding proteins ,HEARING disorders ,HEALTH of Hispanic Americans ,ENVIRONMENTAL health - Abstract
Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca
2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing. [ABSTRACT FROM AUTHOR]- Published
- 2015
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22. The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.
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Qing Fang, Indzhykulian, Artur A., Mustapha, Mirna, Riordan, Gavin P., Dolan, David F., Friedman, Thomas B., Belyantseva, Inna A., Frolenkov, Gregory I., Camper, Sally A., and Bird, Jonathan E.
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MYOSIN ,HEARING ,MECHANOTRANSDUCTION (Cytology) - Abstract
The article discusses a study which shows the significant role of myosin-15 isoforms in the maintenance of hair cell stereocilia assembly and critical for the sense of hearing.
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- 2015
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23. Ion Imaging in the Cochlear Hair Cells.
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Frolenkov, Gregory I.
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- 2009
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24. Myosin XVA.
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Ridley, Anne, Frampton, Jon, Coluccio, Lynne M., Boger, Erich T., Frolenkov, Gregory I., Friedman, Thomas B., and Belyantseva, Inna A.
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Mutations of MYO15A are associated with deafness in humans. In the shaker 2 mouse (Myo15ash2), a missense mutation in the motor domain of myosin XVa causes deafness and circling behavior, and is morphologically characterized by abnormally short stereocilia bundles on the apical surface of inner ear hair cells that also lack the characteristic staircase architecture. The precise architecture of the stereocilia bundle is considered essential for normal hearing in vertebrates. Abnormally short stereocilia also result from a mutation of whirlin, a scaffolding protein that contains three PDZ domains and is a protein partner of myosin XVa. In inner ear hair cells, endogenous myosin XVa and whirlin are both present at the tips of developing and mature tereocilia, overlapping with the site of actin polymerization. Transfection of GFP-whirlin into Whrnwi hair cells or GFP-myosin XVa into Myo15ash2 hair cells reinitiates stereocilia elongation and restores the wild-type architecture to the hair bundle. This elongation process occurs when myosin XVa interacts through its C-terminal class 1 PDZ-ligand with the third PDZ domain of whirlin and delivers whirlin to the tips of stereocilia. Thus, the programmed elongation of stereocilia to a predetermined height depends on the function of myosin XVa. Stereocilia tips are also the sites of certain components of the mechanotransduction complex. Electrophysiological analysis of hair-cell mechanotransduction in Myo15ash2 and Whrnwi mice shows that targeting and function of the mechanotransduction complex in young postnatal mice does not depend upon programmed stereocilia elongation and staircase formation of the hair bundle mediated by the myosin XVa-whirlin complex. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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25. Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells.
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Indzhykulian, Artur A., Stepanyan, Ruben, Nelina, Anastasiia, Spinelli, Kateri J., Ahmed, Zubair M., Belyantseva, Inna A., Friedman, Thomas B., Barr-Gillespie, Peter G., and Frolenkov, Gregory I.
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HAIR cells ,CORTI'S organ ,ACETIC acid ,FATTY acids ,GENETIC transduction - Abstract
Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear. [ABSTRACT FROM AUTHOR]
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- 2013
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26. Fibroblast Growth Factor Receptor 3 regulates microtubule formation and cell surface mechanical properties in the developing organ of Corti.
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Szarama, Katherine B., Stepanyan, Ruben, Petralia, Ronald S., Gavara, Nuria, Frolenkov, Gregory I., Kelley, Matthew W., and Chadwick, Richard S.
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FIBROBLAST growth factors ,CORTI'S organ ,COCHLEA ,HEARING ,MICROTUBULES - Abstract
Fibroblast growth factor (Fgf) signaling is involved in the exquisite cellular patterning of the developing cochlea, and is necessary for proper hearing function. our previous data indicate that Fgf signaling disrupts actin, which impacts the surface stiffness of sensory outer hair cells (ohCs) and non-sensory supporting pillar cells (PCs) in the organ of Corti. here, we used Atomic Force microscopy (AFm) to measure the impact of loss of function of Fgf-receptor 3, on cytoskeletal formation and cell surface mechanical properties. We find a 50% decrease in both ohC and PC surface stiffness, and a substantial disruption in microtubule formation in PCs. moreover, we find no change in ohC electromotility of Fgfr3- deficient mice. to further understand the regulation by Fgf-signaling on microtubule formation, we treated wild-type cochlear explants with Fgf-receptor agonist Fgf2, or antagonist Su5402, and find that both treatments lead to a significant reduction in β-tubulin isotypes I and ii. to identify downstream transcriptional targets of Fgf-signaling, we used QPCr arrays to probe 84 cytoskeletal regulators. of the 5 genes significantly upregulated following treatment, Clasp2, Mapre2 and Mark2 impact microtubule formation. We conclude that microtubule formation is a major downstream effector of Fgf-receptor 3, and suggest this pathway impacts the formation of fluid spaces in the organ of Corti. [ABSTRACT FROM AUTHOR]
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- 2012
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27. Fast Adaptation and Ca2+ Sensitivity of theMechanotransducer Require Myosin-XVa in Inner But Not Outer Cochlear Hair Cells.
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Stepanyan, Ruben and Frolenkov, Gregory I.
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NEUROBIOLOGY , *TRANSDUCERS , *CHANNELS (Structural members) , *HAIR cells , *COCHLEA , *MYOSIN , *LABORATORY mice - Abstract
In inner ear hair cells, activation of mechanotransduction channels is followed by extremely rapid deactivation that depends on the influx of Ca2+ through these channels. Although the molecular mechanisms of this "fast" adaptation are largely unknown, the predominant models assume Ca22+ sensitivity as an intrinsic property of yet unidentified mechanotransduction channels. Here, we examined mechanotransduction in the hair cells of young postnatal shaker 2 mice (Myo15sh2/sh2). These mice have no functional myosin-XVa, which is critical for normal growth of mechanosensory stereocilia of hair cells. Although stereocilia of both inner and outer hair cells of Myo15sh2/sh2 mice lack myosin-XVa and are abnormally short, these cells have dramatically different hair bundle morphology. Myo15sh2/sh2 outer hair cells retain a staircase arrangement of the abnormally short stereocilia and prominent tip links. Myo15sh2/sh2 inner hair cells do not have obliquely oriented tip links, and their mechanosensitivity is mediated exclusively by "top-to-top" links between equally short stereocilia. In both inner and outer hair cells of Myo15sh2/sh2 mice, we found mechanotransduction responses with a normal "wild-type" amplitude and speed of activation. Surprisingly, only outer hair cells exhibit fast adaptation and sensitivity to extracellular Ca22+. In Myo15sh2/sh2 inner hair cells, fast adaptation is disrupted and the transduction current is insensitive to extracellular Ca22+.We conclude that the Ca22+ sensitivity of the mechanotransduction channels and the fast adaptation require a structural environment that is dependent on myosin-XVa and is disrupted in Myo15sh2/sh2 inner hair cells, but not in Myo15sh2/sh2 outer hair cells. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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28. The Tip-Link Antigen, a Protein Associated with the Transduction Complex of Sensory Hair Cells, Is Protocadherin-15.
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Ahmed, Zubair M., Goodyear, Richard, Riazuddin, Saima, Lagziel, Ayala, Legan, P. Kevin, Behra, Martine, Burgess, Shawn M., Lilley, Kathryn S., Wilcox, Edward R., Riazuddin, Sheikh, Griffith, Andrew J., Frolenkov, Gregory I., Belyantseva, Inna A., Richardson, Guy P., and Friedman, Thomas B.
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ANTIGENS ,IMMUNITY ,PROTEINS ,GENETIC transduction ,MICROBIAL genetics ,CADHERINS - Abstract
Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formation. [ABSTRACT FROM AUTHOR]
- Published
- 2006
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29. Myosin-XVa is required for tip localization of whirlin and differential elongation of hair-cell stereocilia.
- Author
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Belyantseva, Inna A., Boger, Erich T., Naz, Sadaf, Frolenkov, Gregory I., Sellers, James R., Ahmed, Zubair M., Griffith, Andrew J., and Friedman, Thomas B.
- Subjects
GREEN fluorescent protein ,HAIR cells ,CELLS ,RODENTS ,HEARING impaired ,MYOSIN - Abstract
Stereocilia are microvilli-derived mechanosensory organelles that are arranged in rows of graded heights on the apical surface of inner-ear hair cells. The 'staircase'-like architecture of stereocilia bundles is necessary to detect sound and head movement, and is achieved through differential elongation of the actin core of each stereocilium to a predetermined length. Abnormally short stereocilia bundles that have a diminished staircase are characteristic of the shaker 2 (Myo15a
sh2 ) and whirler (Whrnwi ) strains of deaf mice. We show that myosin-XVa is a motor protein that, in vivo, interacts with the third PDZ domain of whirlin through its carboxy-terminal PDZ-ligand. Myosin-XVa then delivers whirlin to the tips of stereocilia. Moreover, if green fluorescent protein (GFP)-Myo15a is transfected into hair cells of Myo15ash2 mice, the wild-type pattern of hair bundles is restored by recruitment of endogenous whirlin to the tips of stereocilia. The interaction of myosin-XVa and whirlin is therefore a key event in hair-bundle morphogenesis. [ABSTRACT FROM AUTHOR]- Published
- 2005
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30. Genetic insights into the morphogenesis of inner ear hair cells.
- Author
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Frolenkov, Gregory I., Belyantseva, Inna A., Friedman, Thomas B., and Griffith, Andrew J.
- Subjects
- *
MORPHOGENESIS , *INNER ear , *HAIR cells , *DEAFNESS , *MAMMALS , *GENETICS - Abstract
The mammalian inner ear is a sensory organ that has specialized hair cells that detect sound, as well as orientation and movement of the head. The 'hair' bundle on the apical surface of these cells is a mechanosensitive organelle that consists of precisely organized actin-filled projections known as stereocilia. Alterations in hair-bundle morphogenesis can result in hearing loss, balance defects or both. Positional cloning of genes that underlie hereditary hearing loss, coupled with the characterization of corresponding mouse models, is revealing how hair cells have adapted the molecular mechanisms of intracellular motility and intercellular adhesion for the morphogenesis of their apical surfaces. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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31. Action of 2,3-butanedione monoxime on capacitance and electromotility of guinea-pig cochlear outer hair cells.
- Author
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Frolenkov, Gregory I., Mammano, Fabio, and Kachar, Bechara
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- 2001
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32. Ipsilateral Suppression of Transient Evoked Otoacoustic Emission: Role of the Medial Olivocochlear System.
- Author
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Tavartkiladze, George A., Frolenkov, Gregory I., and Artamasov, Serge V.
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- 1996
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33. Usher proteins in inner ear structure and function.
- Author
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Ahmed, Zubair M., Frolenkov, Gregory I., and Riazuddin, Saima
- Subjects
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INNER ear physiology , *USHER'S syndrome , *HEARING , *VISION , *HAIR cells , *ANIMAL models in research , *PATIENTS - Abstract
00135.2013.--Usher syndrome (USH) is a neurosensory disorder affecting both hearing and vision in humans. Linkage studies of families of USH patients, studies in animals, and characterization of purified proteins have provided insight into the molecular mechanisms of hearing. To date, 11 USH proteins have been identified, and evidence suggests that all of them are crucial for the function of the mechanosensory cells of the inner ear, the hair cells. Most USH proteins are localized to the stereocilia of the hair cells, where mechano-electrical transduction (MET) of sound-induced vibrations occurs. Therefore, elucidation of the functions of USH proteins in the stereocilia is a prerequisite to understanding the exact mechanisms of MET. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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34. Nanoscale live-cell imaging using hopping probe ion conductance microscopy.
- Author
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Novak, Pavel, Chao Li, Shevchuk, Andrew I., Stepanyan, Ruben, Caldwell, Matthew, Hughes, Simon, Smart, Trevor G., Gorelik, Julia, Ostanin, Victor P., Lab, Max J., Moss, Guy W. J., Frolenkov, Gregory I., Klenerman, David, and Korchev, Yuri E.
- Subjects
MICROSCOPY ,OPTICS ,NANOELECTROMECHANICAL systems ,MEDICAL microscopy ,HAIR cells - Abstract
We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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- View/download PDF
35. Correction: A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family.
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Patel, Kunjan, Giese, Arnaud P., Grossheim, J. M., Hegde, Rashmi S., Delio, Maria, Samanich, Joy, Riazuddin, Saima, Frolenkov, Gregory I., Cai, Jinlu, Ahmed, Zubair M., and Morrow, Bernice E.
- Subjects
PUBLISHED errata ,BIOLOGICAL periodicals ,PERIODICAL publishing ,PERIODICAL articles ,PUBLISHING ,PUBLISHED articles ,PUBLICATIONS - Published
- 2015
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- View/download PDF
36. RFX transcription factors are essential for hearing in mice.
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Elkon, Ran, Milon, Beatrice, Morrison, Laura, Shah, Manan, Vijayakumar, Sarath, Racherla, Manoj, Leitch, Carmen C., Silipino, Lorna, Hadi, Shadan, Weiss-Gayet, Michèle, Barras, Emmanuèle, Schmid, Christoph D., Ait-Lounis, Aouatef, Barnes, Ashley, Song, Yang, Eisenman, David J., Eliyahu, Efrat, Frolenkov, Gregory I., Strome, Scott E., and Durand, Bénédicte
- Subjects
DEAFNESS ,HAIR cells ,MECHANORECEPTORS ,SENSORY receptors ,KNOCKOUT mice - Abstract
Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
37. Corrigendum: Nanoscale live-cell imaging using hopping probe ion conductance microscopy.
- Author
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Novak, Pavel, Li, Chao, Shevchuk, Andrew I, Stepanyan, Ruben, Caldwell, Matthew, Hughes, Simon, Smart, Trevor G, Gorelik, Julia, Ostanin, Victor P, Lab, Max J, Moss, Guy W J, Frolenkov, Gregory I, Klenerman, David, and Korchev, Yuri E
- Subjects
MICROSCOPY - Abstract
A correction to the article "Nanoscale Live-Cell Imaging Using Hopping Probe Ion Conductance Microscopy" that was published in the March 1, 2009 issue is presented.
- Published
- 2009
- Full Text
- View/download PDF
38. The use of scanning ion conductance microscopy to image A6 cells
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Gorelik, Julia, Zhang, Yanjun, Shevchuk, Andrew I., Frolenkov, Gregory I., Sánchez, Daniel, Lab, Max J., Vodyanoy, Igor, Edwards, Christopher R.W., Klenerman, David, and Korchev, Yuri E.
- Subjects
- *
MICROSCOPY , *ALDOSTERONE , *ION-permeable membranes , *MINERALOCORTICOIDS - Abstract
Background: Continuous high spatial resolution observations of living A6 cells would greatly aid the elucidation of the relationship between structure and function and facilitate the study of major physiological processes such as the mechanism of action of aldosterone. Unfortunately, observing the micro-structural and functional changes in the membrane of living cells is still a formidable challenge for a microscopist. Method: Scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe, has been shown to be suitable for imaging non-conducting surfaces bathed in electrolytes. A specialized version of this microscopy has been developed by our group and has been applied to image live cells at high-resolution for the first time. This method can also be used in conjunction with patch clamping to study both anatomy and function and identify ion channels in single cells. Results: This new microscopy provides high-resolution images of living renal cells which are comparable with those obtained by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Continuous 24 h observations under normal physiological conditions showed how A6 kidney epithelial cells changed their height, volume, and reshaped their borders. The changes in cell area correlated with the density of microvilli on the surface. Surface microvilli density ranged from 0.5 μm-2 for extended cells to 2.5 μm2 for shrunk cells. Patch clamping of individual cells enabled anatomy and function to be correlated. Conclusions: Scanning ion conductance microscopy provides unique information about living cells that helps to understand cellular function. It has the potential to become a powerful tool for research on living renal cells. [Copyright &y& Elsevier]
- Published
- 2004
- Full Text
- View/download PDF
39. Imaging analysis reveals budding of filamentous human metapneumovirus virions and direct transfer of inclusion bodies through intercellular extensions.
- Author
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El Najjar F, Castillo SR, Moncman CL, Wu C-Y, Isla E, Velez Ortega AC, Frolenkov GI, Cifuentes-Munoz N, and Dutch RE
- Subjects
- Humans, Aged, Cell Line, Cytoskeleton, Inclusion Bodies, Virion, Metapneumovirus
- Abstract
Importance: Human metapneumovirus is an important respiratory pathogen that causes significant morbidity and mortality, particularly in the very young, the elderly, and the immunosuppressed. However, the molecular details of how this virus spreads to new target cells are unclear. This work provides important new information on the formation of filamentous structures that are consistent with virus particles and adds critical new insight into the structure of extensions between cells that form during infection. In addition, it demonstrates for the first time the movement of viral replication centers through these intercellular extensions, representing a new mode of direct cell-to-cell spread that may be applicable to other viral systems., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
- Full Text
- View/download PDF
40. Calcium and Integrin-binding protein 2 (CIB2) controls force sensitivity of the mechanotransducer channels in cochlear outer hair cells.
- Author
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Aristizábal-Ramírez I, Dragich AK, Giese APJ, Sofia Zuluaga-Osorio K, Watkins J, Davies GK, Hadi SE, Riazuddin S, Vander Kooi CW, Ahmed ZM, and Frolenkov GI
- Abstract
Calcium and Integrin-Binding Protein 2 (CIB2) is an essential subunit of the mechano-electrical transduction (MET) complex in mammalian auditory hair cells. CIB2 binds to pore-forming subunits of the MET channel, TMC1/2 and is required for their transport and/or retention at the tips of mechanosensory stereocilia. Since genetic ablation of CIB2 results in complete loss of MET currents, the exact role of CIB2 in the MET complex remains elusive. Here, we generated a new mouse strain with deafness-causing p.R186W mutation in Cib2 and recorded small but still measurable MET currents in the cochlear outer hair cells. We found that R186W variant causes increase of the resting open probability of MET channels, steeper MET current dependence on hair bundle deflection (I-X curve), loss of fast adaptation, and increased leftward shifts of I-X curves upon hair cell depolarization. Combined with AlphaFold2 prediction that R186W disrupts one of the multiple interacting sites between CIB2 and TMC1/2, our data suggest that CIB2 mechanically constraints TMC1/2 conformations to ensure proper force sensitivity and dynamic range of the MET channels. Using a custom piezo-driven stiff probe deflecting the hair bundles in less than 10 µs, we also found that R186W variant slows down the activation of MET channels. This phenomenon, however, is unlikely to be due to direct effect on MET channels, since we also observed R186W-evoked disruption of the electron-dense material at the tips of mechanotransducing stereocilia and the loss of membrane-shaping BAIAP2L2 protein from the same location. We concluded that R186W variant of CIB2 disrupts force sensitivity of the MET channels and force transmission to these channels.
- Published
- 2023
- Full Text
- View/download PDF
41. Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells.
- Author
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Galeano-Naranjo C, Veléz-Ortega AC, and Frolenkov GI
- Subjects
- Animals, Artifacts, Calibration, Hair Cells, Auditory ultrastructure, Image Processing, Computer-Assisted, Mice, Microscopy, Microscopy, Atomic Force, Nanoparticles ultrastructure, Rats, Reference Standards, Stereocilia ultrastructure, Vibration, Hair Cells, Auditory physiology, Mammals physiology, Nanoparticles chemistry, Stereocilia physiology
- Abstract
Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.
- Published
- 2021
- Full Text
- View/download PDF
42. Myosins and Hearing.
- Author
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Friedman TB, Belyantseva IA, and Frolenkov GI
- Subjects
- Animals, Humans, Mutation, Deafness genetics, Hearing genetics, Myosins genetics
- Abstract
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.
- Published
- 2020
- Full Text
- View/download PDF
43. TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.
- Author
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Katsuno T, Belyantseva IA, Cartagena-Rivera AX, Ohta K, Crump SM, Petralia RS, Ono K, Tona R, Imtiaz A, Rehman A, Kiyonari H, Kaneko M, Wang YX, Abe T, Ikeya M, Fenollar-Ferrer C, Riordan GP, Wilson EA, Fitzgerald TS, Segawa K, Omori K, Ito J, Frolenkov GI, Friedman TB, and Kitajiri SI
- Subjects
- Actins physiology, Animals, Deafness etiology, Mice, Mice, Knockout, Microfilament Proteins chemistry, Microfilament Proteins deficiency, Protein Isoforms physiology, Stereocilia ultrastructure, Hearing physiology, Microfilament Proteins physiology, Stereocilia physiology
- Abstract
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.
- Published
- 2019
- Full Text
- View/download PDF
44. Mechanotransduction current is essential for stability of the transducing stereocilia in mammalian auditory hair cells.
- Author
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Vélez-Ortega AC, Freeman MJ, Indzhykulian AA, Grossheim JM, and Frolenkov GI
- Subjects
- Animals, Animals, Newborn, Calcium metabolism, Mice, Inbred C57BL, Microscopy, Electron, Scanning, Rats, Sprague-Dawley, Hair Cells, Auditory physiology, Hair Cells, Auditory ultrastructure, Mechanotransduction, Cellular, Stereocilia physiology, Stereocilia ultrastructure
- Abstract
Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca
2+ into the cell. Here, we show that this constitutive Ca2+ influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca2+ or intracellular Ca2+ buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.- Published
- 2017
- Full Text
- View/download PDF
45. Angular Approach Scanning Ion Conductance Microscopy.
- Author
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Shevchuk A, Tokar S, Gopal S, Sanchez-Alonso JL, Tarasov AI, Vélez-Ortega AC, Chiappini C, Rorsman P, Stevens MM, Gorelik J, Frolenkov GI, Klenerman D, and Korchev YE
- Subjects
- Adult, Animals, Cells, Cultured, Culture Media, Equipment Design, Female, HeLa Cells, Humans, Imaging, Three-Dimensional instrumentation, Male, Mice, Micromanipulation instrumentation, Micromanipulation methods, Microscopy, Electron, Scanning, Microscopy, Scanning Probe instrumentation, Nanotechnology, Patch-Clamp Techniques instrumentation, Patch-Clamp Techniques methods, Rats, Sprague-Dawley, Imaging, Three-Dimensional methods, Microscopy, Scanning Probe methods
- Abstract
Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
46. Novel and recurrent CIB2 variants, associated with nonsyndromic deafness, do not affect calcium buffering and localization in hair cells.
- Author
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Seco CZ, Giese AP, Shafique S, Schraders M, Oonk AM, Grossheim M, Oostrik J, Strom T, Hegde R, van Wijk E, Frolenkov GI, Azam M, Yntema HG, Free RH, Riazuddin S, Verheij JB, Admiraal RJ, Qamar R, Ahmed ZM, and Kremer H
- Subjects
- Adolescent, Adult, Animals, COS Cells, Calcium-Binding Proteins metabolism, Child, Chlorocebus aethiops, Deafness metabolism, Female, Humans, Integrin alpha2beta1 metabolism, Male, Mutation, Missense, Pedigree, Protein Binding, Calcium metabolism, Calcium-Binding Proteins genetics, Deafness genetics, Hair Cells, Auditory metabolism
- Abstract
Variants in CIB2 can underlie either Usher syndrome type I (USH1J) or nonsyndromic hearing impairment (NSHI) (DFNB48). Here, a novel homozygous missense variant c.196C>T and compound heterozygous variants, c.[97C>T];[196C>T], were found, respectively, in two unrelated families of Dutch origin. Besides, the previously reported c.272 T>C functional missense variant in CIB2 was identified in two families of Pakistani origin. The missense variants are demonstrated not to affect subcellular localization of CIB2 in vestibular hair cells in ex vivo expression experiments. Furthermore, these variants do not affect the ATP-induced calcium responses in COS-7 cells. However, based on the residues affected, the variants are suggested to alter αIIβ integrin binding. HI was nonsyndromic in all four families. However, deafness segregating with the c.272T>C variant in one Pakistani family is remarkably less severe than that in all other families with this mutation. Our results contribute to the insight in genotype-phenotype correlations of CIB2 mutations.
- Published
- 2016
- Full Text
- View/download PDF
47. Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.
- Author
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Vélez-Ortega AC and Frolenkov GI
- Subjects
- Animals, Electric Conductivity, Mice, Microscopy, Scanning Probe methods, Nanotechnology instrumentation, Rats, Cochlea ultrastructure, Microscopy, Scanning Probe instrumentation, Stereocilia ultrastructure
- Abstract
The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.
- Published
- 2016
- Full Text
- View/download PDF
48. RFX transcription factors are essential for hearing in mice.
- Author
-
Elkon R, Milon B, Morrison L, Shah M, Vijayakumar S, Racherla M, Leitch CC, Silipino L, Hadi S, Weiss-Gayet M, Barras E, Schmid CD, Ait-Lounis A, Barnes A, Song Y, Eisenman DJ, Eliyahu E, Frolenkov GI, Strome SE, Durand B, Zaghloul NA, Jones SM, Reith W, and Hertzano R
- Subjects
- Animals, Animals, Newborn, Biological Evolution, Chromatin Immunoprecipitation, Female, Gene Expression Regulation, Hair Cells, Auditory ultrastructure, Male, Mice, Inbred C57BL, Mice, Inbred ICR, Mice, Knockout, Multigene Family, Regulatory Factor X Transcription Factors, Regulatory Factor X1, Sequence Analysis, DNA, Transcriptome, Zebrafish, DNA-Binding Proteins metabolism, Hair Cells, Auditory metabolism, Hearing physiology, Transcription Factors metabolism
- Abstract
Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.
- Published
- 2015
- Full Text
- View/download PDF
49. The 133-kDa N-terminal domain enables myosin 15 to maintain mechanotransducing stereocilia and is essential for hearing.
- Author
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Fang Q, Indzhykulian AA, Mustapha M, Riordan GP, Dolan DF, Friedman TB, Belyantseva IA, Frolenkov GI, Camper SA, and Bird JE
- Subjects
- Animals, Ear, Inner physiology, Hair Cells, Auditory physiology, Mice, Mice, Knockout, Protein Binding, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Transport, Hearing, Myosins genetics, Myosins metabolism, Stereocilia metabolism, Stereocilia physiology
- Abstract
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.
- Published
- 2015
- Full Text
- View/download PDF
50. Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing.
- Author
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Kitajiri S, Sakamoto T, Belyantseva IA, Goodyear RJ, Stepanyan R, Fujiwara I, Bird JE, Riazuddin S, Riazuddin S, Ahmed ZM, Hinshaw JE, Sellers J, Bartles JR, Hammer JA 3rd, Richardson GP, Griffith AJ, Frolenkov GI, and Friedman TB
- Subjects
- Animals, Hair Cells, Auditory, Inner cytology, Humans, Mechanotransduction, Cellular, Mice, Mice, Knockout, Microfilament Proteins genetics, Molecular Sequence Data, Actin Cytoskeleton metabolism, Deafness metabolism, Hair Cells, Auditory, Inner metabolism, Microfilament Proteins metabolism
- Abstract
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., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
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