RESUMO
Genes specifically expressed in the inner ear are candidates to underlie hereditary nonsyndromic deafness. The gene Otog has been isolated from a mouse subtractive cDNA cochlear library. It encodes otogelin, an N-glycosylated protein that is present in the acellular membranes covering the six sensory epithelial patches of the inner ear: in the cochlea (the auditory sensory organ), the tectorial membrane (TM) over the organ of Corti; and in the vestibule (the balance sensory organ), the otoconial membranes over the utricular and saccular maculae as well as the cupulae over the cristae ampullares of the three semi-circular canals. These membranes are involved in the mechanotransduction process. Their movement, which is induced by sound in the cochlea or acceleration in the vestibule, results in the deflection of the stereocilia bundle at the apex of the sensory hair cells, which in turn opens the mechanotransduction channels located at the tip of the stereo-cilia. We sought to elucidate the role of otogelin in the auditory and vestibular functions by generating mice with a targeted disruption of Otog. In Otog-/- mice, both the vestibular and the auditory functions were impaired. Histological analysis of these mutants demonstrated that in the vestibule, otogelin is required for the anchoring of the otoconial membranes and cupulae to the neuroepithelia. In the cochlea, ultrastructural analysis of the TM indicated that otogelin is involved in the organization of its fibrillar network. Otogelin is likely to have a role in the resistance of this membrane to sound stimulation. These results support OTOG as a possible candidate gene for a human nonsyndromic form of deafness.
Assuntos
Surdez/genética , Orelha Interna/fisiopatologia , Glicoproteínas de Membrana/genética , Equilíbrio Postural/fisiologia , Membrana Tectorial/fisiopatologia , Estimulação Acústica , Animais , Mapeamento Cromossômico , Cóclea/fisiologia , Cóclea/fisiopatologia , Surdez/patologia , Surdez/fisiopatologia , Orelha Interna/patologia , Orelha Interna/fisiologia , Éxons , Biblioteca Gênica , Transtornos da Audição/genética , Transtornos da Audição/fisiopatologia , Humanos , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Postura , Reflexo/genética , Células-Tronco , Membrana Tectorial/patologia , Membrana Tectorial/ultraestrutura , TransfecçãoRESUMO
Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.
Assuntos
Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Neurônios/fisiologia , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Células Cultivadas , Temperatura Baixa , Resistência a Medicamentos , Gânglios Espinais/citologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Nocodazol/farmacologia , Células PC12 , Coelhos , Ratos , Tubulina (Proteína)/metabolismo , Tirosina/metabolismoRESUMO
The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein ISGP-2], follicle-stimulating hormone [rFSH], alpha-inhibin, anti-Müllerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, alpha-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents.