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1.
Cell ; 180(4): 780-795.e25, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32059781

RESUMO

The cerebral vasculature is a dense network of arteries, capillaries, and veins. Quantifying variations of the vascular organization across individuals, brain regions, or disease models is challenging. We used immunolabeling and tissue clearing to image the vascular network of adult mouse brains and developed a pipeline to segment terabyte-sized multichannel images from light sheet microscopy, enabling the construction, analysis, and visualization of vascular graphs composed of over 100 million vessel segments. We generated datasets from over 20 mouse brains, with labeled arteries, veins, and capillaries according to their anatomical regions. We characterized the organization of the vascular network across brain regions, highlighting local adaptations and functional correlates. We propose a classification of cortical regions based on the vascular topology. Finally, we analysed brain-wide rearrangements of the vasculature in animal models of congenital deafness and ischemic stroke, revealing that vascular plasticity and remodeling adopt diverging rules in different models.

2.
Elife ; 82019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30932811

RESUMO

Sound analysis by the cochlea relies on frequency tuning of mechanosensory hair cells along a tonotopic axis. To clarify the underlying biophysical mechanism, we have investigated the micromechanical properties of the hair cell's mechanoreceptive hair bundle within the apical half of the rat cochlea. We studied both inner and outer hair cells, which send nervous signals to the brain and amplify cochlear vibrations, respectively. We find that tonotopy is associated with gradients of stiffness and resting mechanical tension, with steeper gradients for outer hair cells, emphasizing the division of labor between the two hair-cell types. We demonstrate that tension in the tip links that convey force to the mechano-electrical transduction channels increases at reduced Ca2+. Finally, we reveal gradients in stiffness and tension at the level of a single tip link. We conclude that mechanical gradients of the tip-link complex may help specify the characteristic frequency of the hair cell.

3.
Annu Rev Neurosci ; 42: 67-86, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-30699050

RESUMO

The genetic approach, based on the study of inherited forms of deafness, has proven to be particularly effective for deciphering the molecular mechanisms underlying the development of the peripheral auditory system, the cochlea and its afferent auditory neurons, and how this system extracts the physical parameters of sound. Although this genetic dissection has provided little information about the central auditory system, scattered data suggest that some genes may have a critical role in both the peripheral and central auditory systems. Here, we review the genes controlling the development and function of the peripheral and central auditory systems, focusing on those with demonstrated intrinsic roles in both systems and highlighting the current underappreciation of these genes. Their encoded products are diverse, from transcription factors to ion channels, as are their roles in the central auditory system, mostly evaluated in brainstem nuclei. We examine the ontogenetic and evolutionary mechanisms that may underlie their expression at different sites.

4.
Elife ; 62017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29111973

RESUMO

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C2-domain, Ca2+-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (OtofAla515,Ala517/Ala515,Ala517) with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.


Assuntos
Células Ciliadas Auditivas/fisiologia , Fusão de Membrana , Proteínas de Membrana/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Sinapses/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Cálcio/metabolismo , Técnicas de Introdução de Genes , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Receptores de Detecção de Cálcio/genética
5.
Proc Natl Acad Sci U S A ; 114(30): 7765-7774, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28705869

RESUMO

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15- and Cdhr23-expressing interneuron precursors in Cdhr23-/- and Cdhr15-/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23- and Cdhr15-expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients.


Assuntos
Córtex Auditivo/embriologia , Proteínas Relacionadas a Caderinas/metabolismo , Caderinas/metabolismo , Interneurônios/fisiologia , Precursores de Proteínas/metabolismo , Animais , Córtex Auditivo/metabolismo , Proteínas Relacionadas a Caderinas/genética , Caderinas/genética , Polaridade Celular , Feminino , Macaca , Masculino , Mecanotransdução Celular , Camundongos , Precursores de Proteínas/genética , Receptores Acoplados a Proteínas-G/metabolismo
6.
PLoS One ; 12(4): e0175964, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28419135

RESUMO

The calyx of Held, a large axo-somatic relay synapse containing hundreds of presynaptic active zones, is possibly the largest nerve terminal in the mammalian CNS. Studying its initial growth in-vitro might provide insights into the specification of synaptic connection size in the developing brain. However, attempts to maintain calyces of Held in organotypic cultures have not been fruitful in past studies. Here, we describe an organotypic slice culture method in which calyces of Held form in-vitro. We made coronal brainstem slices with an optimized slice angle using newborn mice in which calyces have not yet formed; the presynaptic bushy cells were genetically labeled using the Math5 promoter. After six to nine days of culturing, we readily observed large Math5-positive nerve terminals in the medial nucleus of the trapezoid body (MNTB), but not in the neighboring lateral superior olive nucleus (LSO). These calyx-like synapses expressed the Ca2+- sensor Synaptotagmin-2 (Syt-2) and the Ca2+ binding protein Parvalbumin (PV), two markers of developing calyces of Held in vivo. Application of the BMP inhibitor LDN-193189 significantly inhibited the growth of calyx synapses, demonstrating the feasibility of long-term pharmacological manipulation using this organotypic culture method. These experiments provide a method for organotypic culturing of calyces of Held, and show that the formation of calyx-like synapses onto MNTB neurons can be preserved in-vitro. Furthermore, our study adds pharmacological evidence for a role of BMP-signaling in the formation of large calyx of Held synapses.


Assuntos
Axônios/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Tronco Encefálico/fisiologia , Proteínas do Tecido Nervoso/análise , Sinapses/fisiologia , Animais , Vias Auditivas , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Tronco Encefálico/citologia , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/ultraestrutura , Camundongos , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos/métodos , Parvalbuminas/análise , Regiões Promotoras Genéticas , Pirazóis/farmacologia , Pirimidinas/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Sinaptotagmina II/análise
7.
Pflugers Arch ; 467(1): 49-72, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24957570

RESUMO

The hair bundles of cochlear hair cells play a central role in the auditory mechano-electrical transduction (MET) process. The identification of MET components and of associated molecular complexes by biochemical approaches is impeded by the very small number of hair cells within the cochlea. In contrast, human and mouse genetics have proven to be particularly powerful. The study of inherited forms of deafness led to the discovery of several essential proteins of the MET machinery, which are currently used as entry points to decipher the associated molecular networks. Notably, MET relies not only on the MET machinery but also on several elements ensuring the proper sound-induced oscillation of the hair bundle or the ionic environment necessary to drive the MET current. Here, we review the most significant advances in the molecular bases of the MET process that emerged from the genetics of hearing.


Assuntos
Potenciais de Ação/genética , Células Ciliadas Auditivas/fisiologia , Audição/genética , Mecanotransdução Celular/genética , Proteínas de Membrana/genética , Proteínas Motores Moleculares/genética , Animais , Humanos , Potenciais da Membrana/genética , Pressão
8.
Neuron ; 78(5): 855-68, 2013 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-23664551

RESUMO

During the formation of neuronal circuits, axon pathfinding decisions specify the location of synapses on the correct brain side and in correct target areas. We investigated a possible link between axon midline crossing and the subsequent development of output synapses formed by these axons. Conditional knockout of Robo3 in the auditory system forced a large commissural synapse, the calyx of Held, to be exclusively formed on the wrong, ipsilateral side. Ipsilateral calyx of Held synapses showed strong transmission defects, with reduced and desynchronized transmitter release, fewer fast-releasable vesicles, and smaller and more variable presynaptic Ca(2+) currents. Transmission defects were not observed in a downstream inhibitory synapse, and some defects persisted into adulthood. These results suggest that axon midline crossing conditions functional maturation of commissural synapses, thereby minimizing the impact of mislocalized synapses on information processing. This mechanism might be relevant to human disease caused by mutations in the ROBO3 gene.


Assuntos
Axônios/fisiologia , Tronco Encefálico/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Vias Auditivas/fisiologia , Axônios/metabolismo , Biofísica , Césio/farmacologia , Cloretos/farmacologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Estimulação Elétrica , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/genética , Humanos , Técnicas In Vitro , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-fos/metabolismo , Sinapses/genética , Fatores de Tempo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
9.
Nat Neurosci ; 16(7): 856-64, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23708139

RESUMO

Large excitatory synapses with multiple active zones ensure reliable and fast information transfer at specific points in neuronal circuits. However, the mechanisms that determine synapse size in CNS circuits are largely unknown. Here we use the calyx of Held synapse, a major relay in the auditory system, to identify and study signaling pathways that specify large nerve terminal size and fast synaptic transmission. Using genome-wide screening, we identified bone morphogenetic proteins (BMPs) as candidate signaling molecules in the area of calyx synapses. Conditional deletion of BMP receptors in the auditory system of mice led to aberrations of synapse morphology and function specifically at the calyx of Held, with impaired nerve terminal growth, loss of monoinnervation and less mature transmitter release properties. Thus, BMP signaling specifies large and fast-transmitting synapses in the auditory system in a process that shares homologies with, but also extends beyond, retrograde BMP signaling at Drosophila neuromuscular synapses.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Neurônios/citologia , Transdução de Sinais/fisiologia , Sinapses/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Proteína 2 de Resposta de Crescimento Precoce/genética , Estimulação Elétrica , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/genética , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Neurônios/ultraestrutura , Rombencéfalo/citologia , Rombencéfalo/crescimento & desenvolvimento , Transdução de Sinais/genética , Sinapses/ultraestrutura
10.
Hum Mol Genet ; 21(17): 3835-44, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22678063

RESUMO

We report a consanguineous Iranian family affected by congenital profound sensorineural deafness segregating in an autosomal recessive mode. Auditory tests implicated at least a cochlear defect in these patients. We mapped the deafness, autosomal recessive (DFNB) locus involved by linkage analysis to a 4.8 Mb region at chromosome 21q22.3-qter. Exclusion of the DFNB8/10 gene TMPRSS3, located in this chromosomal interval, led us to identify a new deafness locus, DFNB98. Whole exome sequencing allowed us to identify a homozygous frame-shifting mutation (c.1726G>T+c.1728delC) in the gene TSPEAR (thrombospondin-type laminin G domain and EAR repeats). This truncating mutation (p.V576LfsX37) impeded the secretion of the encoded protein by cells transfected with the mutated gene. Alternative splicing of TSPEAR transcripts predict two protein isoforms, 522 and 669 amino acids in length, both of which would be affected by the mutation. These isoforms are composed of a thrombospondin-type laminin G (TSP) domain followed by seven tandemly organized epilepsy-associated repeats (EARs), probably forming a ß-propeller domain. Tspear is expressed in a variety of murine tissues. Only the larger Tspear transcript was found in the cochlea, and the protein was detected by immunofluorescence at the surface of the hair bundles of sensory cells. The mammalian EAR protein family includes six known members. Defects in four of them, i.e. Lgi1, Lgi2, Vlgr1 and, we show here, TSPEAR, cause disorders with auditory features: epilepsy, which can include auditory features in humans; audiogenic seizures in animals; and/or hearing impairments in humans and mice. These observations demonstrate that EAR-containing proteins are essential for the development and function of the auditory system.


Assuntos
Surdez/genética , Loci Gênicos/genética , Proteínas/química , Proteínas/genética , Sequências Repetitivas de Aminoácidos/genética , Adulto , Animais , Audiometria , Sequência de Bases , Segregação de Cromossomos/genética , Cromossomos Humanos Par 21/genética , Cóclea/metabolismo , Feminino , Mutação da Fase de Leitura/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Linhagem , Estrutura Terciária de Proteína , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto Jovem
11.
Proc Natl Acad Sci U S A ; 108(14): 5825-30, 2011 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-21436032

RESUMO

The mechanotransducer channels of auditory hair cells are gated by tip-links, oblique filaments that interconnect the stereocilia of the hair bundle. Tip-links stretch from the tips of stereocilia in the short and middle rows to the sides of neighboring, taller stereocilia. They are made of cadherin-23 and protocadherin-15, products of the Usher syndrome type 1 genes USH1D and USH1F, respectively. In this study we address the role of sans, a putative scaffold protein and product of the USH1G gene. In Ush1g(-/-) mice, the cohesion of stereocilia is disrupted, and both the amplitude and the sensitivity of the transduction currents are reduced. In Ush1g(fl/fl)Myo15-cre(+/-) mice, the loss of sans occurs postnatally and the stereocilia remain cohesive. In these mice, there is a decrease in the amplitude of the total transducer current with no loss in sensitivity, and the tips of the stereocilia in the short and middle rows lose their prolate shape, features that can be attributed to the loss of tip-links. Furthermore, stereocilia from these rows undergo a dramatic reduction in length, suggesting that the mechanotransduction machinery has a positive effect on F-actin polymerization. Sans interacts with the cytoplasmic domains of cadherin-23 and protocadherin-15 in vitro and is absent from the hair bundle in mice defective for either of the two cadherins. Because sans localizes mainly to the tips of short- and middle-row stereocilia in vivo, we conclude that it belongs to a molecular complex at the lower end of the tip-link and plays a critical role in the maintenance of this link.


Assuntos
Actinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Caderinas/metabolismo , Cílios/metabolismo , Eletrofisiologia , Imunofluorescência , Vetores Genéticos/genética , Células Ciliadas Auditivas/ultraestrutura , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/genética , Polimerização , Precursores de Proteínas/metabolismo , Transdução de Sinais/genética
12.
J Comp Neurol ; 519(2): 194-210, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21165971

RESUMO

Stereocilin is defective in a recessive form of deafness, DFNB16. We studied the distribution of stereocilin in the developing and mature mouse inner ear and analyzed the consequences of its absence in stereocilin-null (Strc(-/-)) mice that suffer hearing loss starting at postnatal day 15 (P15) and progressing until P60. Using immunofluorescence and immunogold electron microscopy, stereocilin was detected in association with two cell surface specializations specific to outer hair cells (OHCs) in the mature cochlea: the horizontal top connectors that join the apical regions of adjacent stereocilia within the hair bundle, and the attachment links that attach the tallest stereocilia to the overlying tectorial membrane. Stereocilin was also detected around the kinocilium of vestibular hair cells and immature OHCs. In Strc(-/-) mice the OHC hair bundle was structurally and functionally normal until P9. Top connectors, however, did not form and the cohesiveness of the OHC hair bundle progressively deteriorated from P10. The stereocilia were still interconnected by tip links at P14, but these progressively disappeared from P15. By P60 the stereocilia, still arranged in a V-shaped bundle, were fully disconnected from each other. Stereocilia imprints on the lower surface of the tectorial membrane were also not observed in Strc(-/-) mice, thus indicating that the tips of the tallest stereocilia failed to be embedded in this gel. We conclude that stereocilin is essential to the formation of horizontal top connectors. We propose that these links, which maintain the cohesiveness of the mature OHC hair bundle, are required for tip-link turnover.


Assuntos
Células Ciliadas Auditivas/ultraestrutura , Proteínas/metabolismo , Membrana Tectorial/ultraestrutura , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Eletrofisiologia , Cobaias , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Mecanotransdução Celular/fisiologia , Camundongos , Camundongos Knockout , Proteínas/genética
13.
J Neurosci ; 30(40): 13281-90, 2010 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-20926654

RESUMO

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca(2+) spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca(2+) efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca(2+) sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca(2+)-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1(-/-), Syt2(-/-) and Syt7(-/-) mice). We only found a defect in RRP recovery in Syt1(-/-) mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2(-/-) and Syt7(-/-) mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca(2+)-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca(2+)-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.


Assuntos
Cóclea/crescimento & desenvolvimento , Exocitose , Células Ciliadas Auditivas Internas/fisiologia , Proteínas de Membrana/fisiologia , Sinaptotagmina II/fisiologia , Sinaptotagmina I/fisiologia , Animais , Cálcio/fisiologia , Sinalização do Cálcio/genética , Senescência Celular/genética , Senescência Celular/fisiologia , Cóclea/citologia , Capacitância Elétrica , Exocitose/genética , Feminino , Células Ciliadas Auditivas Internas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Sinapses/genética , Sinapses/fisiologia , Transmissão Sináptica/genética , Sinaptotagmina I/genética , Sinaptotagmina II/genética
14.
Pflugers Arch ; 459(1): 115-30, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19756723

RESUMO

We assessed the involvement of harmonin-b, a submembranous protein containing PDZ domains, in the mechanoelectrical transduction machinery of inner ear hair cells. Harmonin-b is located in the region of the upper insertion point of the tip link that joins adjacent stereocilia from different rows and that is believed to gate transducer channel(s) located in the region of the tip link's lower insertion point. In Ush1c (dfcr-2J/dfcr-2J) mutant mice defective for harmonin-b, step deflections of the hair bundle evoked transduction currents with altered speed and extent of adaptation. In utricular hair cells, hair bundle morphology and maximal transduction currents were similar to those observed in wild-type mice, but adaptation was faster and more complete. Cochlear outer hair cells displayed reduced maximal transduction currents, which may be the consequence of moderate structural anomalies of their hair bundles. Their adaptation was slower and displayed a variable extent. The latter was positively correlated with the magnitude of the maximal transduction current, but the cells that showed the largest currents could be either hyperadaptive or hypoadaptive. To interpret our observations, we used a theoretical description of mechanoelectrical transduction based on the gating spring theory and a motor model of adaptation. Simulations could account for the characteristics of transduction currents in wild-type and mutant hair cells, both vestibular and cochlear. They led us to conclude that harmonin-b operates as an intracellular link that limits adaptation and engages adaptation motors, a dual role consistent with the scaffolding property of the protein and its binding to both actin filaments and the tip link component cadherin-23.


Assuntos
Adaptação Fisiológica , Proteínas de Transporte/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Mecanotransdução Celular/fisiologia , Potenciais de Ação/fisiologia , Animais , Proteínas do Citoesqueleto , Imunofluorescência , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Técnicas de Patch-Clamp
15.
J Neurosci ; 27(24): 6478-88, 2007 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-17567809

RESUMO

Several lines of evidence indicate that very large G-protein-coupled receptor 1 (Vlgr1) makes up the ankle links that connect the stereocilia of hair cells at their base. Here, we show that the transmembrane protein usherin, the putative transmembrane protein vezatin, and the PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain-containing submembrane protein whirlin are colocalized with Vlgr1 at the stereocilia base in developing cochlear hair cells and are absent in Vlgr1-/- mice that lack the ankle links. Direct in vitro interactions between these four proteins further support their involvement in a molecular complex associated with the ankle links and scaffolded by whirlin. In addition, the delocalization of these proteins in myosin VIIa defective mutant mice as well as the myosin VIIa tail direct interactions with vezatin, whirlin, and, we show, Vlgr1 and usherin, suggest that myosin VIIa conveys proteins of the ankle-link complex to the stereocilia. Adenylyl cyclase 6, which was found at the base of stereocilia, was both overexpressed and mislocated in Vlgr1-/- mice. In postnatal day 7 Vlgr1-/- mice, mechanoelectrical transduction currents evoked by displacements of the hair bundle toward the tallest stereocilia (i.e., in the excitatory direction) were reduced in outer but not inner hair cells. In both cell types, stimulation of the hair bundle in the opposite direction paradoxically resulted in significant transduction currents. The absence of ankle-link-mediated cohesive forces within hair bundles lacking Vlgr1 may account for the electrophysiological results. However, because some long cadherin-23 isoforms could no longer be detected in Vlgr1-/- mice shortly after birth, the loss of some apical links could be involved too. The premature disappearance of these cadherin isoforms in the Vlgr1-/- mutant argues in favor of a signaling function of the ankle links in hair bundle differentiation.


Assuntos
Cóclea/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Ciliadas Auditivas/metabolismo , Adenilil Ciclases/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Quelantes/farmacologia , Cílios/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Embrião de Mamíferos , Proteínas da Matriz Extracelular/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura/métodos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Receptores Acoplados a Proteínas-G/deficiência , Subtilisina/farmacologia
16.
Hum Mol Genet ; 14(24): 3921-32, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16301217

RESUMO

Usher syndrome type IIa (USH2A) combines moderate to severe congenital hearing impairment and retinitis pigmentosa. It is the most common genetic form of USH. USH2A encodes usherin, which was previously defined as a basement membrane protein. A much larger USH2A transcript predicted to encode a transmembrane (TM) isoform was recently reported. Here, we address the role of TM usherin in the inner ear. Analysis of the usherin alternative transcripts in the murine inner ear revealed the existence of several predicted TM usherin isoforms with modular ectodomains of different lengths. In addition, we identified in the usherin cytoplasmic region a predicted 24 amino acid peptide, derived from a newly defined exon that is predominantly expressed in the inner ear but not in the retina. In mouse and rat inner ears, we show that TM usherin is present at the base of the differentiating stereocilia, which make up the mechanosensitive hair bundles receptive to sound. The usherin immunolabeling is transient in the hair bundles of cochlear hair cells (HCs), but persists in mature hair bundles of vestibular HCs. Several lines of evidence support the involvement of TM usherin in the composition of the ankle links, a subset of filamentous lateral links connecting stereocilia at the base. By co-immunoprecipitation and in vitro binding assays, we establish that the usherin cytodomain can bind to whirlin and harmonin, two PDZ domain-containing proteins that are defective in genetic forms of isolated deafness and USH type I, respectively. These PDZ proteins are suitable to provide the anchoring of interstereocilia lateral links to the F-actin core of stereocilia. Our results strongly suggest that congenital deafness in USH type I and type II shares similar pathogenic mechanisms, i.e. the disruption of hair bundle links-mediated adhesion forces that are essential for the proper organization of growing hair bundles.


Assuntos
Orelha Interna/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células Ciliadas Auditivas/metabolismo , Síndromes de Usher/fisiopatologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Orelha Interna/citologia , Células Ciliadas Auditivas/patologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
17.
J Cell Sci ; 118(Pt 13): 2891-9, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15976448

RESUMO

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.


Assuntos
Dineínas/metabolismo , Células Ciliadas Auditivas Internas/química , Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Animais , Linhagem Celular , Dineínas/genética , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Miosina Tipo I , Miosinas/genética
18.
Hum Mol Genet ; 14(3): 401-10, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15590698

RESUMO

Defects in myosin XVa and the PDZ domain-containing protein, whirlin, underlie deafness in humans and mice. Hair bundles of mutant mice defective for either protein have abnormally short stereocilia. Here, we show that whirlin, like myosin XVa, is present at the very tip of each stereocilium in the developing and mature hair bundles of the cochlear and vestibular system. We found that myosin XVa SH3-MyTH4 region binds to the short isoform of whirlin (PR-PDZ3) that can rescue the stereocilia growth defect in whirlin defective mice. Moreover, the C-terminal MyTH4-FERM region of myosin XVa binds to the PDZ1 and PDZ2 domains of the long whirlin isoform. We conclude that a direct myosin XVa-whirlin interaction at the stereocilia tip is likely to control the elongation of stereocilia. Whirlin, unlike myosin XVa, is also transiently localized in the basal region of developing stereocilia in rat vestibular and cochlear hair cells until P4 and P12, respectively. Notably, whirlin also interacts with myosin VIIa that is present along the entire length of the stereocilia. Finally, we show that the transmembrane netrin-G1 ligand (NGL-1) binds to the PDZ1 and PDZ2 domains of whirlin and has an extracellular region that homophilically self-interacts in a Ca2+-dependent manner. The interaction between whirlin and NGL-1 might be involved in the stabilization of interstereociliar links.


Assuntos
Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/metabolismo , Miosinas/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Cães , Células Ciliadas Auditivas/ultraestrutura , Humanos , Proteínas de Membrana/genética , Camundongos , Miosinas/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos
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