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1.
Cell ; 141(5): 786-98, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20510926

RESUMO

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.


Assuntos
Citoesqueleto de Actina/metabolismo , Surdez/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Células Ciliadas Auditivas Internas/citologia , Humanos , Mecanotransdução Celular , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular
2.
Proc Natl Acad Sci U S A ; 119(26): e2115190119, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35737845

RESUMO

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.


Assuntos
Citoesqueleto de Actina , Surdez , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Cóclea/metabolismo , Surdez/metabolismo , Células Ciliadas Auditivas/metabolismo , Mamíferos/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Órgão Espiral , Isoformas de Proteínas/metabolismo
3.
Nature ; 563(7733): 696-700, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30464345

RESUMO

The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transcriptoma/genética , Animais , Sequência de Bases , Biomarcadores/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
Nat Chem Biol ; 17(8): 878-887, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34045745

RESUMO

In ovoid-shaped, Gram-positive bacteria, MapZ guides FtsZ-ring positioning at cell equators. The cell wall of the ovococcus Streptococcus mutans contains peptidoglycan decorated with serotype c carbohydrates (SCCs). In the present study, we identify the major cell separation autolysin AtlA as an SCC-binding protein. AtlA binding to SCC is attenuated by the glycerol phosphate (GroP) modification. Using fluorescently labeled AtlA constructs, we mapped SCC distribution on the streptococcal surface, revealing enrichment of GroP-deficient immature SCCs at the cell poles and equators. The immature SCCs co-localize with MapZ at the equatorial rings throughout the cell cycle. In GroP-deficient mutants, AtlA is mislocalized, resulting in dysregulated cellular autolysis. These mutants display morphological abnormalities associated with MapZ mislocalization, leading to FtsZ-ring misplacement. Altogether, our data support a model in which maturation of a cell wall polysaccharide provides the molecular cues for the recruitment of cell division machinery, ensuring proper daughter cell separation and FtsZ-ring positioning.


Assuntos
Parede Celular/metabolismo , Polissacarídeos/metabolismo , Streptococcus mutans/metabolismo , Divisão Celular , Parede Celular/química , Polissacarídeos/química , Streptococcus mutans/citologia
5.
Adv Exp Med Biol ; 1239: 317-330, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451864

RESUMO

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.


Assuntos
Surdez , Audição , Miosinas , Animais , Surdez/genética , Audição/genética , Humanos , Mutação , Miosinas/genética
6.
Biophys J ; 110(10): 2252-65, 2016 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-27224490

RESUMO

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.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Varredura por Sonda/métodos , Adulto , Animais , Células Cultivadas , Meios de Cultura , Desenho de Equipamento , Feminino , Células HeLa , Humanos , Imageamento Tridimensional/instrumentação , Masculino , Camundongos , Micromanipulação/instrumentação , Micromanipulação/métodos , Microscopia Eletrônica de Varredura , Microscopia de Varredura por Sonda/instrumentação , Nanotecnologia , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos , Ratos Sprague-Dawley
7.
PLoS Biol ; 11(6): e1001583, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23776407

RESUMO

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.


Assuntos
Células Ciliadas Auditivas/fisiologia , Mecanotransdução Celular , Regeneração/fisiologia , Animais , Animais Recém-Nascidos , Proteínas Relacionadas a Caderinas , Caderinas/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Células Ciliadas Auditivas Internas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Precursores de Proteínas/metabolismo , Estereocílios/fisiologia , Estereocílios/ultraestrutura
8.
Physiol Genomics ; 45(21): 987-9, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24022220

RESUMO

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.


Assuntos
Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , Estereocílios/metabolismo , Síndromes de Usher/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto , Orelha Interna/patologia , Orelha Interna/fisiopatologia , Células Ciliadas Auditivas/patologia , Humanos , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Estereocílios/patologia , Síndromes de Usher/genética , Síndromes de Usher/fisiopatologia
9.
mBio ; 14(5): e0158923, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37681946

RESUMO

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.


Assuntos
Metapneumovirus , Humanos , Idoso , Linhagem Celular , Citoesqueleto , Corpos de Inclusão , Vírion
10.
bioRxiv ; 2023 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-37461484

RESUMO

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.

11.
Nat Commun ; 14(1): 3871, 2023 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-37391431

RESUMO

TRPA1 channels are expressed in nociceptive neurons, where they detect noxious stimuli, and in the mammalian cochlea, where their function is unknown. Here we show that TRPA1 activation in the supporting non-sensory Hensen's cells of the mouse cochlea causes prolonged Ca2+ responses, which propagate across the organ of Corti and cause long-lasting contractions of pillar and Deiters' cells. Caged Ca2+ experiments demonstrated that, similar to Deiters' cells, pillar cells also possess Ca2+-dependent contractile machinery. TRPA1 channels are activated by endogenous products of oxidative stress and extracellular ATP. Since both these stimuli are present in vivo after acoustic trauma, TRPA1 activation after noise may affect cochlear sensitivity through supporting cell contractions. Consistently, TRPA1 deficiency results in larger but less prolonged noise-induced temporary shift of hearing thresholds, accompanied by permanent changes of latency of the auditory brainstem responses. We conclude that TRPA1 contributes to the regulation of cochlear sensitivity after acoustic trauma.


Assuntos
Perda Auditiva Provocada por Ruído , Canal de Cátion TRPA1 , Animais , Camundongos , Cóclea , Células Epiteliais , Potenciais Evocados Auditivos do Tronco Encefálico , Células Labirínticas de Suporte , Canal de Cátion TRPA1/genética
12.
Cell Stem Cell ; 30(7): 950-961.e7, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37419105

RESUMO

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.


Assuntos
Proteínas Hedgehog , Células-Tronco Pluripotentes , Humanos , Proteínas Hedgehog/metabolismo , Cóclea , Células Ciliadas Auditivas Internas , Organoides , Diferenciação Celular/fisiologia
13.
Nat Methods ; 6(4): 279-81, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19252505

RESUMO

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.


Assuntos
Células Cultivadas/ultraestrutura , Microscopia de Varredura por Sonda/instrumentação , Microscopia de Varredura por Sonda/métodos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Animais , Condutividade Elétrica , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/instrumentação , Aumento da Imagem/métodos , Íons , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
14.
Nat Cell Biol ; 7(2): 148-56, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15654330

RESUMO

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 (Whrn(wi)) 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 Myo15a(sh2) 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.


Assuntos
Cílios/ultraestrutura , Surdez/genética , Células Ciliadas Auditivas Internas/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Miosinas/fisiologia , Actinas/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Fluorescência Verde , Células Ciliadas Auditivas Internas/ultraestrutura , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteínas Motores Moleculares/metabolismo
15.
Proc Natl Acad Sci U S A ; 106(24): 9703-8, 2009 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-19497859

RESUMO

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.


Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Actinas/genética , Animais , Perda Auditiva/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência
16.
J Vis Exp ; (167)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33554973

RESUMO

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.


Assuntos
Células Ciliadas Auditivas/fisiologia , Mamíferos/fisiologia , Nanopartículas/química , Estereocílios/fisiologia , Animais , Artefatos , Calibragem , Células Ciliadas Auditivas/ultraestrutura , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia , Microscopia de Força Atômica , Nanopartículas/ultraestrutura , Ratos , Padrões de Referência , Estereocílios/ultraestrutura , Vibração
17.
J Neurosci ; 29(13): 4023-34, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19339598

RESUMO

In inner ear hair cells, activation of mechanotransduction channels is followed by extremely rapid deactivation that depends on the influx of Ca(2+) through these channels. Although the molecular mechanisms of this "fast" adaptation are largely unknown, the predominant models assume Ca(2+) sensitivity as an intrinsic property of yet unidentified mechanotransduction channels. Here, we examined mechanotransduction in the hair cells of young postnatal shaker 2 mice (Myo15(sh2/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 Myo15(sh2/sh2) mice lack myosin-XVa and are abnormally short, these cells have dramatically different hair bundle morphology. Myo15(sh2/sh2) outer hair cells retain a staircase arrangement of the abnormally short stereocilia and prominent tip links. Myo15(sh2/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 Myo15(sh2/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 Ca(2+). In Myo15(sh2/sh2) inner hair cells, fast adaptation is disrupted and the transduction current is insensitive to extracellular Ca(2+). We conclude that the Ca(2+) sensitivity of the mechanotransduction channels and the fast adaptation require a structural environment that is dependent on myosin-XVa and is disrupted in Myo15(sh2/sh2) inner hair cells, but not in Myo15(sh2/sh2) outer hair cells.


Assuntos
Adaptação Fisiológica/fisiologia , Cálcio/farmacologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/fisiologia , Mecanotransdução Celular/fisiologia , Miosinas/fisiologia , Órgão Espiral/citologia , Adaptação Fisiológica/genética , Animais , Animais Recém-Nascidos , Fenômenos Biofísicos , Biofísica/métodos , Relação Dose-Resposta a Droga , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/fisiologia , Células Ciliadas Auditivas Externas/ultraestrutura , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Varredura/métodos , Mutação/fisiologia , Miosinas/genética , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodos , Estimulação Física/métodos
18.
J Assoc Res Otolaryngol ; 21(2): 121-135, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152769

RESUMO

Mammalian hair cells develop their mechanosensory bundles through consecutive phases of stereocilia elongation, thickening, and retraction of supernumerary stereocilia. Many molecules involved in stereocilia elongation have been identified, including myosin-XVa. Significantly less is known about molecular mechanisms of stereocilia thickening and retraction. Here, we used scanning electron microscopy (SEM) to quantify postnatal changes in number and diameters of the auditory hair cell stereocilia in shaker-2 mice (Myo15sh2) that lack both "long" and "short" isoforms of myosin-XVa, and in mice lacking only the "long" myosin-XVa isoform (Myo15∆N). Previously, we observed large mechanotransduction current in young postnatal inner (IHC) and outer (OHC) hair cells of both these strains. Stereocilia counts showed nearly identical developmental retraction of supernumerary stereocilia in control heterozygous, Myo15sh2/sh2, and Myo15∆N/∆N mice, suggesting that this retraction is largely unaffected by myosin-XVa deficiency. However, myosin-XVa deficiency does affect stereocilia diameters. In control, the first (tallest) and second row stereocilia grow in diameter simultaneously. However, the third row stereocilia in IHCs grow only until postnatal day 1-2 and then become thinner. In OHCs, they also grow slower than taller stereocilia, forming a stereocilia diameter gradation within a hair bundle. The sh2 mutation disrupts this gradation and makes all stereocilia nearly identical in thickness in both IHCs and OHCs, with only subtle residual diameter differences. All Myo15sh2/sh2 stereocilia grow postnatally including the third row, which is not a part of normal development. Serial sections with focused ion beam (FIB)-SEM confirmed that diameter changes of Myo15sh2/sh2 IHC and OHC stereocilia resulted from corresponding changes of their actin cores. In contrast to Myo15sh2/sh2, Myo15∆N/∆N hair cells develop prominent stereocilia diameter gradation. Thus, besides building the staircase, the short isoform of myosin-XVa is essential for controlling the diameter of the third row stereocilia and formation of the stereocilia diameter gradation in a hair bundle.


Assuntos
Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/ultraestrutura , Miosinas/fisiologia , Estereocílios/fisiologia , Actinas/metabolismo , Animais , Camundongos , Camundongos Knockout , Isoformas de Proteínas , Estereocílios/ultraestrutura
19.
Methods Mol Biol ; 493: 381-99, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18839360

RESUMO

Regulation of important cellular functions via signaling pathways is a fundamental property of the cell. Intracellular Ca(2+) is probably a best known second messenger in cell biology. In mechanosensory cells of the inner ear, the hair cells, intracellular Ca(2+) participates in a variety of functions including mechano-electrical transduction, synaptic transmission, and efferent regulation of the outer hair cells, one of two types of hair cells in the mammalian cochlea. The outer hair cells are responsible for the amplification of sound-induced vibrations within the cochlea, which determines the sensitivity of mammalian hearing. Besides Ca(2+), another intracellular ion, Cl(-) may have very specific function in the same outer hair cells. Intracellular Cl(-) is required for the motor function of prestin, a unique plasma membrane molecular motor of these cells. The goal of this article is to review practical aspects of the techniques suitable for imaging of Ca(2+) and Cl(-) in live mammalian cochlear hair cells.


Assuntos
Cóclea/metabolismo , Dermoscopia/métodos , Células Ciliadas Auditivas/metabolismo , Íons/metabolismo , Animais , Cálcio/metabolismo , Cloretos/metabolismo , Cóclea/citologia , Dermoscopia/instrumentação , Humanos
20.
Hear Res ; 376: 47-57, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30638948

RESUMO

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.


Assuntos
Células Ciliadas Auditivas/fisiologia , Células Ciliadas Auditivas/ultraestrutura , Estereocílios/fisiologia , Actinas/química , Actinas/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Diferenciação Celular , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Humanos , Mamíferos , Mecanotransdução Celular/fisiologia , Regeneração , Estereocílios/ultraestrutura
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