Elucidating molecular mechanisms mediating cellular responses to touch and pain

 描述（由申请人提供）：机械转导，或细胞将机械刺激转化为电活动的机制，是生命所有领域中保守的过程，是我们与世界互动的必要方面。然而，尽管它的普遍性和重要性，机械转导并没有很好地理解在分子水平。水蛭属水蛭，通常被称为药用水蛭，长期以来一直被用作神经生物学的模型系统，因此腹神经索的分段迭代神经节中的神经元的特征已经得到很好的建立。为了确定哪些基因可能介导这些神经节中机械敏感和非机械敏感神经元之间的差异，我和其他人对水蛭神经节中的五种不同细胞类型进行了转录组学分析。分析得出的一个主要特征是 是几个转录编码通道的差异表达，这些通道与其他系统中已显示的机械敏感性通道相似。然而，我们的系统不仅允许我们确定这些通道是否赋予机械敏感性，而且还将确定该过程的性质，因为水蛭神经节中的三种不同的机械敏感神经元对触摸表现出不同的反应。具体而言，我计划：1）通过水蛭体壁制备物和体内神经元培养物中的转录本敲低来确定这些通道的缺失对神经元功能的影响; 2）使用Helobdella（另一种水蛭和成熟的发育模型系统）描述这些基因的发育轨迹和生物相关性;和3）直接确定这些通道的子集是否通过表达完整的长度蛋白和随后的功能测定。

 DESCRIPTION (provided by applicant): Mechanotransduction, or the mechanisms by which cells convert mechanical stimuli into electrical activity, is a process conserved across all domains of life and is a necessary facet of our interaction with the world. However, despite its ubiquity and importance, mechanotransduction is not well understood at a molecular level. Leeches in the genus Hirudo, commonly known as medicinal leeches, have been long used as a model system for neurobiology and as a result the characteristics of the neurons in the segmentally iterated ganglia of the ventral nerve cord have been well established. To determine what genes might mediate the differences between the mechanosensitive and non-mechanosensitive neurons in these ganglia, I and others carried out a transcriptomic analysis of five different cell types in the Hirudo ganglion. One major signature that arose from this analysis was the differential expression of several transcripts encoding channels similar to those that have been shown to be mechanosensitive in other systems. However, our system would not only allow us to ground- truth whether these channels confer mechanosensitivity, but also would determine the nature of this process, as the three different mechanosensitive neurons in the leech ganglion exhibit different responses to touch. Specifically, I plan to: 1) Determine the effect of the loss of these channels on neuronal function by transcript knock-down in Hirudo body wall preparations and in vivo neuronal culture; 2) Describe the developmental trajectory and organismal relevance of these genes using Helobdella, another leech and well-established developmental model system; and 3) Directly determine whether a subset of these channels confers mechanosensitivity in mammalian cell culture through expression of the full-length protein and subsequent functional assays.