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1.
Biochem J ; 479(12): 1393-1405, 2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35695292

RESUMO

In cochlea, deafness-related protein PDZD7 is an indispensable component of the ankle link complex, which is critical for the maturation of inner-ear hair cell for sound perception. Ankle links, connecting the different rows of cochlear stereocilia, are essential for the staircase-like development of stereocilia. However, the molecular mechanism of how PDZD7 governs stereociliary development remains unknown. Here, we reported a novel PDZD7-binding partner, FCHSD2, identified by yeast two-hybrid screening. FCHSD2 was reported to be expressed in hair cell, where it co-operated with CDC42 and N-WASP to regulate the formation of cell protrusion. The association between FCHSD2 and PDZD7 was further confirmed in COS-7 cells. More importantly, we solved the complex structure of FCHSD2 tail with PDZD7 PDZ3 domain at 2.0 Šresolution. The crystal structure shows that PDZD7 PDZ3 adopts a typical PDZ domain topology, comprising five ß strands and two α helixes. The PDZ-binding motif of FCHSD2 tail stretches through the αB/ßB groove of PDZD7 PDZ3. Our study not only uncovers the interaction between FCHSD2 tail and PDZD7 PDZ3 at the atomic level, but also provides clues of connecting the ankle link complex with cytoskeleton dynamics for exploiting the molecular mechanism of stereociliary development.


Assuntos
Proteínas de Transporte , Surdez , Proteínas de Transporte/metabolismo , Surdez/genética , Células Ciliadas Auditivas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Domínios PDZ , Estereocílios/química , Estereocílios/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(26): e2204084119, 2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35727972

RESUMO

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2, encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein (Minar2tm1b/tm1b) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies.


Assuntos
Perda Auditiva Neurossensorial , Receptor Notch2 , Receptores de Superfície Celular , Animais , Perda Auditiva Neurossensorial/genética , Humanos , Mutação com Perda de Função , Camundongos , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Superfície Celular/genética , Estereocílios/metabolismo
3.
J Cell Biol ; 221(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35175278

RESUMO

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell's cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed-TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.


Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Estereocílios/metabolismo , Animais , Membrana Celular/metabolismo , Citoplasma/metabolismo , Células HEK293 , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Células HeLa , Perda Auditiva/patologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/química , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Estereocílios/ultraestrutura
4.
Am J Pathol ; 192(5): 805-812, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35181334

RESUMO

Collapsin response mediator protein 1 (CRMP1), also known as dihydropyrimidinase-related protein 1, participates in cytoskeleton remodeling during axonal guidance and neuronal migration. In cochlear hair cells, the assembly and maintenance of the cytoskeleton is of great interest because it is crucial for the morphogenesis and maintenance of hair cells. Previous RNA sequencing analysis found that Crmp1 is highly expressed in cochlear hair cells. However, the expression profile and functions of CRMP1 in the inner ear remain unknown. In this study, the expression and localization of CRMP1 in hair cells was investigated using immunostaining, and was shown to be highly expressed in both outer and inner hair cells. Next, the stereocilia morphology of Crmp1-deficient mice was characterized. Abolishing CRMP1 did not affect the morphogenesis of hair cells. Interestingly, scanning electron microscopy detected hair cell loss at the basal cochlear region, an area responsible for high-frequency auditory perception, in Crmp1-deficient mice. Correspondingly, an auditory brainstem response test showed that mice lacking CRMP1 had progressive hearing loss at high frequencies. In summary, these data suggest that CRMP1 is required for high-frequency auditory perception.


Assuntos
Semaforina-3A , Estereocílios , Animais , Citoesqueleto/metabolismo , Audição , Camundongos , Neurogênese , Semaforina-3A/metabolismo , Estereocílios/metabolismo
5.
Mol Biol Cell ; 33(4): br6, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35044843

RESUMO

Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 (BAIAP2L2), a membrane-binding protein required for the maintenance of mechanotransduction in hair cells, is selectively retained at the tips of transducing stereocilia. BAIAP2L2 trafficked to stereocilia tips in the absence of EPS8, but EPS8 increased the efficiency of localization. A tripartite complex of BAIAP2L2, EPS8, and MYO15A formed efficiently in vitro, and these three proteins robustly targeted to filopodia tips when coexpressed in cultured cells. Mice lacking functional transduction channels no longer concentrated BAIAP2L2 at row 2 stereocilia tips, a result that was phenocopied by blocking channels with tubocurarine in cochlear explants. Transduction channels permit Ca2+ entry into stereocilia, and we found that membrane localization of BAIAP2L2 was enhanced in the presence of Ca2+. Finally, reduction of intracellular Ca2+ in hair cells using BAPTA-AM led to a loss of BAIAP2L2 at stereocilia tips. Taken together, our results show that a MYO15A-EPS8 complex transports BAIAP2L2 to stereocilia tips, and Ca2+ entry through open channels at row 2 tips retains BAIAP2L2 there.


Assuntos
Mecanotransdução Celular , Estereocílios , Animais , Células Ciliadas Auditivas , Mecanotransdução Celular/fisiologia , Proteínas de Membrana , Camundongos
6.
Mol Neurobiol ; 59(1): 386-404, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34708331

RESUMO

Hearing loss is the most common human sensory deficit. Hearing relies on stereocilia, inserted into the cuticular plate of hair cells (HCs), where they play an important role in the perception of sound and its transmission. Although numerous genes have been associated with hearing loss, the function of many hair cell genes has yet to be elucidated. Herein, we focused on nonerythroid spectrin αII (SPTAN1), abundant in the cuticular plate, surrounding the rootlets of stereocilia and along the plasma membrane. Interestingly, mice with HC-specific Sptan1 knockout exhibited rapid deafness, abnormal formation of stereocilia and cuticular plates, and loss of HCs from middle and apical turns of the cochlea during early postnatal stages. Additionally, Sptan1 deficiency led to the decreased spreading of House Ear Institute-Organ of Corti 1 cells, and induced abnormal formation of focal adhesions and integrin signaling in mouse HCs. Altogether, our findings highlight SPTAN1 as a critical molecule for HC stereocilia morphology and auditory function via regulation of focal adhesion signaling.


Assuntos
Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células Ciliadas Auditivas/metabolismo , Perda Auditiva/metabolismo , Proteínas dos Microfilamentos/metabolismo , Estereocílios/metabolismo , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Linhagem Celular , Forma Celular/fisiologia , Células Ciliadas Auditivas/patologia , Audição/fisiologia , Perda Auditiva/patologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética
7.
Hum Genet ; 141(3-4): 363-382, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34232383

RESUMO

Stereocilia protrude up to 100 µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of stereocilia components that disturb this "dynamic equilibrium" in stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy.


Assuntos
Surdez , Perda Auditiva Neurossensorial , Actinas/genética , Animais , Surdez/genética , Surdez/metabolismo , Forminas , Cabelo/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Humanos , Mecanotransdução Celular/genética , Camundongos , Proteínas dos Microfilamentos/genética , Estereocílios/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
8.
Life Sci Alliance ; 5(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34815294

RESUMO

Failure in the structural maintenance of the hair cell stereocilia bundle and ribbon synapse causes hearing loss. Here, we have studied how ER stress elicits hair cell pathology, using mouse models with inactivation of Manf (mesencephalic astrocyte-derived neurotrophic factor), encoding an ER-homeostasis-promoting protein. From hearing onset, Manf deficiency caused disarray of the outer hair cell stereocilia bundle and reduced cochlear sound amplification capability throughout the tonotopic axis. In high-frequency outer hair cells, the pathology ended in molecular changes in the stereocilia taper region and in strong stereocilia fusion. In high-frequency inner hair cells, Manf deficiency degraded ribbon synapses. The altered phenotype strongly depended on the mouse genetic background. Altogether, the failure in the ER homeostasis maintenance induced early-onset stereociliopathy and synaptopathy and accelerated the effect of genetic causes driving age-related hearing loss. Correspondingly, MANF mutation in a human patient induced severe sensorineural hearing loss from a young age onward. Thus, we present MANF as a novel protein and ER stress as a mechanism that regulate auditory hair cell maintenance in both mice and humans.


Assuntos
Cóclea/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Fatores de Crescimento Neural/genética , Estereocílios/metabolismo , Sinapses/metabolismo , Suscetibilidade a Doenças , Homeostase , Fatores de Crescimento Neural/metabolismo
9.
Elife ; 102021 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-34964715

RESUMO

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the 'tip link.' Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location, and conformational states of intact tip links, on stereocilia, remains unknown. Here, we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.


Assuntos
Proteínas Relacionadas a Caderinas/química , Precursores de Proteínas/química , Estereocílios/ultraestrutura , Animais , Proteínas Relacionadas a Caderinas/ultraestrutura , Microscopia Crioeletrônica , Camundongos , Conformação Molecular , Estrutura Molecular , Precursores de Proteínas/ultraestrutura
10.
Sci Rep ; 11(1): 23855, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34903829

RESUMO

ATP-utilizing enzymes play key roles in hair bundles, the mechanically sensitive organelles of sensory hair cells in the inner ear. We used a fluorescent ATP analog, EDA-ATP-Cy3 (Cy3-ATP), to label ATP-binding proteins in two different preparations of unfixed hair-cell stereocilia of the mouse. In the first preparation, we lightly permeabilized dissected cochleas, then labeled them with Cy3-ATP. Hair cells and their stereocilia remained intact, and stereocilia tips in rows 1 and 2 were labeled particularly strongly with Cy3-ATP. In many cases, vanadate (Vi) traps nucleotides at the active site of myosin isoforms and presents nucleotide dissociation. Co-application with Vi enhanced the tip labeling, which is consistent with myosin isoforms being responsible. By contrast, the actin polymerization inhibitors latrunculin A and cytochalasin D had no effect, suggesting that actin turnover at stereocilia tips was not involved. Cy3-ATP labeling was substantially reduced-but did not disappear altogether-in mutant cochleas lacking MYO15A; by contrast, labeling remained robust in cochleas lacking MYO7A. In the second preparation, used to quantify Cy3-ATP labeling, we labeled vestibular stereocilia that had been adsorbed to glass, which demonstrated that tip labeling was higher in longer stereocilia. We found that tip signal was reduced by ~ 50% in Myo15ash2/sh2 stereocilia as compared to Myo15ash2/+stereocilia. These results suggest that MYO15A accounts for a substantial fraction of the Cy3-ATP tip labeling in vestibular hair cells, and so this novel preparation could be utilized to examine the control of MYO15A ATPase activity in situ.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Células Ciliadas Auditivas/metabolismo , Indóis/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Citocalasina D/farmacologia , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Miosinas/metabolismo , Estereocílios/metabolismo , Estereocílios/ultraestrutura , Tiazolidinas/farmacologia , Vanadatos/farmacologia
11.
Hear Res ; 412: 108372, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34775267

RESUMO

The inner hair cells in the mammalian cochlea transduce mechanical signals to electrical signals that provide input to the auditory nerve. The spatial-temporal displacement of the inner hair cell stereocilia (IHCsc) relative to basilar membrane (BM) displacement is central to characterizing the transduction process. This study specifically focuses on measuring displacement of the stereocilia hair bundles in the radial dimensions where they are most sensitive. To simplify the mechanical response of the cochlear partition, a mechanical probe was used to drive the BM. Optical imaging was used to measure radial displacement of the inner hair cell stereocilia local to the probe in ex vivo gerbil cochleae. The mechanical probe displaced the BM in the transverse direction using sinusoidal stimuli with frequencies ranging from 10 Hz to 42.5 kHz. IHCsc displacement measurements were made in the radial dimension as a function of their longitudinal location along the length of the BM. The results were used to quantify the frequency response, longitudinal space coupling, traveling wave velocity, and wavelength of the radial displacement of the stereocilia. The measurements were centered at two best frequency locations along the BM: Proximal to the round window (first turn), and in the second turn. At both locations, frequency tuning was seen that was consistent with published place maps. At both locations, traveling waves were observed simultaneously propagating basal and apical from the probe. The velocity of the traveling waves at the center frequency (CF) of the location was higher in the first turn than in the second. As the stimulus frequency increased and approached CF for a location, the traveling wavelength decreased. Differential motion of the BM and IHCsc was observed in the second turn as the stimulus frequency increased toward CF. The longitudinal coupling measured in this study was longer than observed in previous studies. In summary the results suggest that the shape of the wave patterns present on the BM are not sufficient to characterize the displacement of the IHCsc.


Assuntos
Membrana Basilar , Células Ciliadas Auditivas Internas , Animais , Membrana Basilar/fisiologia , Cóclea/fisiologia , Gerbillinae , Estereocílios
12.
J Physiol ; 599(24): 5397-5416, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34783032

RESUMO

DFNA25 is an autosomal-dominant and progressive form of human deafness caused by mutations in the SLC17A8 gene, which encodes the vesicular glutamate transporter type 3 (VGLUT3). To resolve the mechanisms underlying DFNA25, we studied phenotypes of mice harbouring the p.A221V mutation in humans (corresponding to p.A224V in mice). Using auditory brainstem response and distortion product otoacoustic emissions, we showed progressive hearing loss with intact cochlear amplification in the VGLUT3A224V/A224V mouse. The summating potential was reduced, indicating the alteration of inner hair cell (IHC) receptor potential. Scanning electron microscopy examinations demonstrated the collapse of stereocilia bundles in IHCs, leaving those from outer hair cells unaffected. In addition, IHC ribbon synapses underwent structural and functional modifications at later stages. Using super-resolution microscopy, we observed oversized synaptic ribbons and patch-clamp membrane capacitance measurements showed an increase in the rate of the sustained releasable pool exocytosis. These results suggest that DFNA25 stems from a failure in the mechano-transduction followed by a change in synaptic transfer. The VGLUT3A224V/A224V mouse model opens the way to a deeper understanding and to a potential treatment for DFNA25. KEY POINTS: The vesicular glutamate transporter type 3 (VGLUT3) loads glutamate into the synaptic vesicles of auditory sensory cells, the inner hair cells (IHCs). The VGLUT3-p.A211V variant is associated with human deafness DFNA25. Mutant mice carrying the VGLUT3-p.A211V variant show progressive hearing loss. IHCs from mutant mice harbour distorted stereocilary bundles, which detect incoming sound stimulation, followed by oversized synaptic ribbons, which release glutamate onto the afferent nerve fibres. These results suggest that DFNA25 stems from the failure of auditory sensory cells to faithfully transduce acoustic cues into neural messages.


Assuntos
Estereocílios , Sinapses , Animais , Cóclea , Células Ciliadas Auditivas Internas , Células Ciliadas Auditivas Externas , Camundongos
13.
Sci Rep ; 11(1): 20224, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34642354

RESUMO

The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple-/- mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple-/- mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.


Assuntos
Proteínas de Transporte/genética , Cóclea/patologia , Perda Auditiva/patologia , Microtúbulos/patologia , Estereocílios/patologia , Animais , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Cóclea/citologia , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Técnicas de Inativação de Genes , Perda Auditiva/genética , Camundongos , Microscopia Eletrônica de Varredura , Microtúbulos/metabolismo , Nocodazol/farmacologia , Técnicas de Cultura de Órgãos , Estereocílios/metabolismo
14.
Hear Res ; 409: 108329, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34391192

RESUMO

The MYO7A gene encodes a motor protein with a key role in the organization of stereocilia in auditory and vestibular hair cells. Rare variants in the MYO7A (myosin VIIA) gene may cause autosomal dominant (AD) or autosomal recessive (AR) sensorineural hearing loss (SNHL) accompanied by vestibular dysfunction or retinitis pigmentosa (Usher syndrome type 1B). Familial Meniere's disease (MD) is a rare inner ear syndrome mainly characterized by low-frequency sensorineural hearing loss and episodic vertigo associated with tinnitus. Familial aggregation has been found in 6-8% of sporadic cases, and most of the reported genes were involved in single families. Thus, this study aimed to search for relevant genes not previously linked to familial MD. Through exome sequencing and segregation analysis in 62 MD families, we have found a total of 1 novel and 8 rare heterozygous variants in the MYO7A gene in 9 non-related families. Carriers of rare variants in MYO7A showed autosomal dominant or autosomal recessive SNHL in familial MD. Additionally, some novel and rare variants in other genes involved in the organization of the stereocilia links such as CDH23, PCDH15 or ADGRV1 co-segregated in the same patients. Our findings reveal a co-segregation of rare variants in the MYO7A gene and other structural myosin VIIA binding proteins involved in the tip and ankle links of the hair cell stereocilia. We suggest that recessive digenic inheritance involving these genes could affect the ultrastructure of the stereocilia links in familial MD.


Assuntos
Doença de Meniere , Miosina VIIa/genética , Células Ciliadas Vestibulares , Heterozigoto , Humanos , Doença de Meniere/genética , Mutação , Linhagem , Estereocílios , Síndromes de Usher/genética
15.
Commun Biol ; 4(1): 958, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381157

RESUMO

In vertebrate hearing organs, mechanical vibrations are converted to ionic currents through mechanoelectrical-transduction (MET) channels. Concerted stereocilia motion produces an ensemble MET current driving the hair-cell receptor potential. Mammalian cochleae are unique in that the tuning of sensory cells is determined by their mechanical environment and the mode of hair-bundle stimulation that their environment creates. However, little is known about the in situ intra-hair-bundle motions of stereocilia relative to one another, or to their environment. In this study, high-speed imaging allowed the stereocilium and cell-body motions of inner hair cells to be monitored in an ex vivo organ of Corti (OoC) mouse preparation. We have found that the OoC rotates about the base of the inner pillar cell, the hair bundle rotates about its base and lags behind the motion of the apical surface of the cell, and the individual stereocilia move semi-independently within a given hair bundle.


Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Estribo/fisiologia , Estereocílios/metabolismo , Animais , Feminino , Masculino , Camundongos
16.
Neural Plast ; 2021: 4784385, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34306060

RESUMO

Noise overexposure leads to hair cell loss, synaptic ribbon reduction, and auditory nerve deterioration, resulting in transient or permanent hearing loss depending on the exposure severity. Oxidative stress, inflammation, calcium overload, glutamate excitotoxicity, and energy metabolism disturbance are the main contributors to noise-induced hearing loss (NIHL) up to now. Gene variations are also identified as NIHL related. Glucocorticoid is the only approved medication for NIHL treatment. New pharmaceuticals targeting oxidative stress, inflammation, or noise-induced neuropathy are emerging, highlighted by the nanoparticle-based drug delivery system. Given the complexity of the pathogenesis behind NIHL, deeper and more comprehensive studies still need to be fulfilled.


Assuntos
Perda Auditiva Provocada por Ruído/etiologia , Animais , Autofagia , Cálcio/metabolismo , Ensaios Clínicos Fase II como Assunto , Reparo do DNA/genética , Drogas em Investigação/uso terapêutico , Metabolismo Energético , Junções Comunicantes , Ácido Glutâmico/fisiologia , Células Ciliadas Auditivas/patologia , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Perda Auditiva Provocada por Ruído/epidemiologia , Perda Auditiva Provocada por Ruído/genética , Humanos , Inflamação , Isoindóis/uso terapêutico , Nanopartículas , Compostos Organosselênicos/uso terapêutico , Estresse Oxidativo , Canais de Potássio/genética , Estereocílios/ultraestrutura
17.
STAR Protoc ; 2(3): 100637, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34258597

RESUMO

Fluorescence recovery after photobleaching (FRAP) has been widely used to monitor membrane properties by measuring the lateral diffusion of fluorescent particles. This protocol describes how to perform two-photon FRAP on the stereocilia of live cochlear inner hair cells using a lipophilic dye, di-3-ANEPPDHQ, to assess the stereociliary membrane diffusivity. We also detail two-photon FRAP microscope setup and calibration, as well as FRAP parameter setting and data analysis. For complete details on the use and execution of this protocol, please refer to George et al. (2020).


Assuntos
Recuperação de Fluorescência Após Fotodegradação/métodos , Células Ciliadas Auditivas/metabolismo , Estereocílios/metabolismo , Animais , Corantes/metabolismo , Difusão , Fótons , Ratos
18.
Elife ; 102021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34227465

RESUMO

Hair cells, the receptors of the inner ear, detect sounds by transducing mechanical vibrations into electrical signals. From the top surface of each hair cell protrudes a mechanical antenna, the hair bundle, which the cell uses to detect and amplify auditory stimuli, thus sharpening frequency selectivity and providing a broad dynamic range. Current methods for mechanically stimulating hair bundles are too slow to encompass the frequency range of mammalian hearing and are plagued by inconsistencies. To overcome these challenges, we have developed a method to move individual hair bundles with photonic force. This technique uses an optical fiber whose tip is tapered to a diameter of a few micrometers and endowed with a ball lens to minimize divergence of the light beam. Here we describe the fabrication, characterization, and application of this optical system and demonstrate the rapid application of photonic force to vestibular and cochlear hair cells.


The sense of hearing relies on specialized sensory cells in the inner ear. Each of these hair cells converts sounds into electrical signals that the brain can interpret. The hair cell takes its name from the bundle of rod-like structures that protrude from its top surface, which resemble hairs under the microscope. The hair bundle acts as an antenna that bends in response to sound waves. When a hair bundle moves in a particular direction, it opens ion channels in the hair-cell membrane. The resulting flow of ions into the cell triggers a cascade of events that ends with an electrical signal traveling to the brain. Many experiments on hearing rely on being able to manipulate the movement of a hair bundle. Researchers typically use one of two methods to achieve this. In the first, a flexible glass fiber pushes against the hair bundle, whereas the second involves a jet of fluid directed against the cell. Neither of these techniques can move hair bundles fast enough for researchers to explore the vast range of sound frequencies that human ears can detect. What is more, both methods are prone to introducing errors into experiments. Abeytunge, Gianoli et al. have developed a new method for moving hair bundles, this time with the aid of light. When light interacts with objects it exerts a photonic force. Abeytunge, Gianoli et al. show that a tapered optical fiber with a miniscule rounded lens can focus a laser beam to deliver enough photonic force to move a hair bundle. The laser beam does not damage the hair bundle, but moves it fast enough to allow researchers to study a broader range of mammalian hearing, while avoiding the errors that have bedeviled previous methods.


Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas/fisiologia , Audição/fisiologia , Animais , Orelha Interna , Cabelo , Fenômenos Mecânicos , Fibras Ópticas , Rana catesbeiana , Ratos , Estereocílios , Vibração
19.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34228789

RESUMO

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here, we report a role for multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis in mouse. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprint proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.


Assuntos
Citoesqueleto/metabolismo , Células Ciliadas Auditivas/metabolismo , Domínios PDZ , Actinas/metabolismo , Animais , Cóclea/metabolismo , Orelha Interna/metabolismo , Perda Auditiva/metabolismo , Proteínas de Membrana , Camundongos , Estereocílios/metabolismo
20.
Genome Biol Evol ; 13(8)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34137817

RESUMO

The large spectrum of hearing sensitivity observed in primates results from the impact of environmental and behavioral pressures to optimize sound perception and localization. Although evidence of positive selection in auditory genes has been detected in mammals including in Hominoids, selection has never been investigated in other primates. We analyzed 123 genes highly expressed in the inner ear of 27 primate species and tested to what extent positive selection may have shaped these genes in the order Primates tree. We combined both site and branch-site tests to obtain a comprehensive picture of the positively selected genes (PSGs) involved in hearing sensitivity, and drew a detailed description of the most affected branches in the tree. We chose a conservative approach, and thus focused on confounding factors potentially affecting PSG signals (alignment, GC-biased gene conversion, duplications, heterogeneous sequencing qualities). Using site tests, we showed that around 12% of these genes are PSGs, an α selection value consistent with average human genome estimates (10-15%). Using branch-site tests, we showed that the primate tree is heterogeneously affected by positive selection, with the black snub-nosed monkey, the bushbaby, and the orangutan, being the most impacted branches. A large proportion of these genes is inclined to shape hair cells and stereocilia, which are involved in the mechanotransduction process, known to influence frequency perception. Adaptive selection, and more specifically recurrent adaptive evolution, could have acted in parallel on a set of genes (ADGRV1, USH2A, PCDH15, PTPRQ, and ATP8A2) involved in stereocilia growth and the whole complex of bundle links connecting them, in species across different habitats, including high altitude and nocturnal environments.


Assuntos
Mecanotransdução Celular , Estereocílios , Animais , Células Ciliadas Auditivas/fisiologia , Audição/genética , Primatas/genética
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