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一系列精确的肌动蛋白组成。 立体纤毛，以根的形式插入到被称为角质板的基于肌动蛋白的致密网络中 大量研究表明，形成立体纤毛的蛋白质是 听力和平衡感正常。另一方面，人们对这些蛋白质所起的作用知之甚少 塑造在听力和平衡中起作用的角质板。事实上，角质板的功能 发束本身的发育和维护仍然难以捉摸。在角质层内 平板，20世纪80年代Tilney研究小组的超微结构分析显示了多种类型的肌动蛋白- 相关蛋白，它们可能控制着这个独特的 亚细胞结构。超级绒毛蛋白是一种205 kD的肌动蛋白/明胶蛋白交联蛋白 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的动力学。 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 利用RNA原位杂交技术研究超绒毛蛋白的两种亚型：svila和svilc 在内耳的斑马鱼毛细胞中表达。我们还用我们的小说《斯维拉》演示了 Svila定位于内耳毛细胞角质板的兔抗血清 斑马鱼。在小鼠身上，我们发现SvIL也在内耳的毛细胞中表达。在……里面 一致认为，在小鼠的前庭I型和II型毛细胞中，SvIL定位于角质层 胎盘在出生后第1天和成体阶段。在小鼠耳蜗中，SvIL定位于角质板 靠近支持细胞的顶端连接复合体，环绕毛细胞，包括 Deiters细胞头板和外柱细胞及内指骨尖外侧缘 细胞。在这里，我们确定了超级绒毛蛋白在机械感觉的发展和功能中的作用 毛细胞是成束的肌动蛋白细丝网络，形成角质板。我们正在利用基因 以及分子生物学技术和生理实验来验证这一点 在斑马鱼模型中假想的角色。在这项赠款提案中，我们进一步研究了 超绒毛蛋白在发束形成和功能中的作用。