Understanding the Role of Actin-Bundling Protein Supervillin in the Development and the Function of the Cuticular Plate of the Mechanosensory Hair Cells in Zebrafish

了解肌动蛋白捆绑蛋白超级绒毛蛋白在斑马鱼机械感觉毛细胞角质板的发育和功能中的作用

• 批准号: 9567439
• 负责人: Ahlam Salameh
• 金额: $ 3.65万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2019-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9567439
• 关键词: Acoustics; Actin-Binding Protein; Actins; Adult; Affinity; Agreement; American; Apical; Applications Grants; Attenuated; Axon; Binding; Binding Sites; Biological; Brain; Bundling; CRISPR/Cas technology; Cell membrane; Cells; Chimera organism; Cochlea; Complex; Confocal Microscopy; Dendrites; Destinations; Development; EMS1 gene; Electron Microscopy; Equilibrium; F-Actin; Fishes; Gelsolin; Genes; Genetic; Hair; Hair Cells; Head; Head Movements; Hearing; Human; In Situ Hybridization; Inner Hair Cells; Inner Supporting Cell; Kinocilium; Knock-out; Laboratories; Labyrinth; Lateral; Link; Loudness; Maintenance; Maps; Microfilaments; Microtubules; Modeling; Molecular; Molecular Biology Techniques; Monitor; Morphology; Mus; Mutation; Myosin Type II; Neurons; Oryctolagus cuniculus; Pattern; Physiological; Pillar Cell; Play; Posture; Protein Isoforms; Proteins; RNA; Reflex action; Research; Role; Scanning; Sensory; Series; Shapes; Signal Transduction; Site; Spinal Ganglia; Stimulus; Structure; Subcellular structure; Supporting Cell; Surface; Techniques; Teenagers; Testing; Type II Hair Cell; Work; Zebrafish; base; deafness; equilibration disorder; experience; experimental study; flasks; hearing impairment; mutant; non-muscle myosin; novel; postnatal; protein crosslink; receptor; sound; transmission process; villin

The mechanosensitive hair cells of the inner ear consist of a precise array of actin-based stereocilia that insert as rootlets into a dense actin-based meshwork known as the cuticular plate Numerous studies have demonstrated that the proteins that shape the stereocilia are required for normal hearing and balance. On the other hand, little is known about the role the proteins that shape the cuticular plate play in hearing and balance. Indeed, the function that the cuticular plate itself endows to hair bundle development and maintenance remains elusive. Within the cuticular plate, ultrastructural analyses from Tilney’s group in the 1980s revealed numerous types of actin- associated proteins, which presumably control the morphology and the integrity of this unique subcellular structure. Supervillin, a 205 kD actin-crosslinking protein from the villin/gelsolin superfamily, regulates actin dynamics by interacting with non-muscle myosin II. Mutations in MYH9 gene that encodes myosin II are linked to the human hearing loss DFNA17. We showed using RNA in situ hybridization that two isoforms of supervillin; svila and svilc are robustly expressed in zebrafish hair cells of the inner ear. We also demonstrated using our novel Svila rabbit polyclonal antiserum that Svila localizes to the cuticular plate of hair cells in the inner ear of zebrafish. In mice, we showed that Svil is also expressed in the hair cells of the inner ear. In agreement, in mice, in the vestibular type I and type II hair cells, Svil localizes to the cuticular plate at both postnatal day 1 and adult stages. In mice cochlea, Svil localizes to the cuticular plate and near the apical junctional complexes of supporting cells, that surround hair cells, including the head plates of Deiters’ and outer pillar cells and the apicolateral margins of inner phalangeal cells. Here, we identify supervillin role in the development and function of the mechanosensory hair cells is to bundle actin filament networks to shape the cuticular plate. We are utilizing genetic and molecular biological techniques along with physiological experiments to test this hypothesized role in a zebrafish model. In this grant proposal we further study the role of supervillin in hair bundle formation and function.

内耳的机械敏感毛细胞由基于肌动蛋白的精确阵列组成 立体胶体将其插入rootlets中 大量研究表明，塑造立体胶质的蛋白质是必需的 正常的听力和平衡。另一方面，关于蛋白质的作用知之甚少 在听力和平衡中塑造可爱的盘子。确实，可爱盘子的功能 本身赋予了头发束的开发和维护仍然难以捉摸。在表皮内 板块是1980年代蒂尔尼小组的超微结构分析，揭示了许多类型的肌动蛋白 相关蛋白质，大概可以控制这种独特的形态和完整性 亚细胞结构。 SupperIllin，Villin/Gelsolin的205 kD肌动蛋白交联蛋白 超家族，通过与非肌肉肌球蛋白II相互作用来调节肌动蛋白动力学。突变 编码肌球蛋白II的MYH9基因与人类听力损失DFNA17有关。我们展示了 使用RNA的原位杂交，即Supervillin的两个同工型； svila和svilc很强 在内耳的斑马鱼毛细胞中表达。我们还使用小说Svila证明了 兔多克隆抗血清，Svila定位于内耳中可爱的毛细胞 斑马鱼。在小鼠中，我们表明SVIL在内耳的毛细胞中也表达。在 在小鼠中，在前庭I型和II型毛细胞中达成协议，SVIL定位于可定性 板在产后第1天和成人阶段。在小鼠耳蜗中，SVIL本地化在可爱的盘子上 在支撑细胞的顶端连接络合物附近，围绕毛细胞，包括 Deiters和Outer Pillar细胞的头部板以及内骨内侧边缘 细胞。在这里，我们确定了Supervillin在机理的发展和功能中的作用 毛细胞将捆绑肌动蛋白细丝网络塑造可爱的板。我们正在使用遗传 和分子生物学技术以及物理实验来测试 在斑马鱼模型中的假设作用。在这项赠款建议中，我们进一步研究了 头发束的形成和功能中的SupperIllin。