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首页> 外文期刊>BMC Neuroscience >Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear development in zebrafish
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Proteomic and functional analysis of NCS-1 binding proteins reveals novel signaling pathways required for inner ear development in zebrafish

机译：NCS-1结合蛋白的蛋白质组学和功能分析揭示了斑马鱼内耳发育所需的新信号通路

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Background The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated. Results We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1 , pi4kβ, dan , and pink1 in semicircular canal formation. Conclusion Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kβ/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.
机译：背景技术半圆形管是脊椎动物内耳的前庭系统的一个细分，用作角加速度传感器。然而，对于控制这种复杂结构发展的潜在分子机制目前知之甚少。斑马鱼代表了一种特别易处理的模型系统，用于研究内耳发育。这是因为在胚胎早期发育过程中可以很容易地看到耳朵，并且可以使用正向和反向遗传技术来发现有助于适当耳朵发育的新基因。先前我们已经表明，在斑马鱼中，钙感应分子神经元钙传感器1（NCS-1）是半圆形管形成所必需的。 NCS-1在调节半规管形成中的功能尚未阐明。结果我们启动了一个多步骤功能蛋白质组学策略，以鉴定有助于斑马鱼内耳发育的神经元钙传感器1（NCS-1）结合伴侣（NBP）。通过结合文献和数据库搜索进行Y2H筛查，我们确定了10个人类NBP。斑马鱼EST和基因组数据库的BLAST搜索使我们能够克隆每个人NBP的斑马鱼直系同源物。通过调查斑马鱼NBP mRNA的表达谱，我们确定了七个在发育中的内耳表达并与ncs-1a表达谱重叠。 GST下拉实验证实了选定的NBP与NCS-1相互作用，而吗啉代介导的敲除实验证明了arf1，pi4kβ，dan和pink1在半规管形成中的重要作用。结论基于它们的功能概况，提出了Ncs-1a /Pi4kβ/ Arf1形成一个信号通路，该通路调节前庭器官发育所需的分子成分，包括Dan和Bmp4的分泌。第二组NBP由Pink1，Hint2和Slc25a25组成，注定要在线粒体中定位。我们的发现揭示了参与半规管系统发展的新型信号传导途径，并暗示了NCS-1在线粒体功能中的未知作用，因为它与几种线粒体蛋白相关。

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