Mechanosensory Transduction in Drosophila

描述（由申请人提供）：我们多样化的机械感觉系统包括听觉、平衡、触觉和本体感觉等突出感觉，以及血压和肠道拉伸检测等意识较弱的感觉。介导这些感觉的机械感觉细胞在结构和功能上是不同的，但有一个共同的中心特征。与使用第二信使传递感觉信息的其他感觉信号传导方式不同，机械感觉通过施加力直接打开机械门控离子通道而发生。尽管它的重要性，我们目前不知道这个通道的身份，或者，除了少数例外，其余的转导机制。这项研究的长期目标是定义转导机制的分子，并了解它们如何协同工作，将机械刺激转化为电信号。为了理解机械传导，这项研究利用了遗传上易于处理的模式生物果蝇的简单和优雅。果蝇是研究机械传导的理想生物体，原因有几个：著名的分子遗传工具，从机械感觉刚毛进行电记录的能力，以及果蝇机械感觉神经元和脊椎动物毛细胞的发育和生理之间惊人的相似性。这里采取的科学方法可以分为两部分：分子遗传学途径，以确定参与mechanosensory转导和电生理学方法来理解mechanosensory反应的基因。许多实验利用机械感觉转导通道NompC作为进入转导机制的生化和遗传立足点。这个提议的第一步是更好地定义NompC的表达模式。针对Nompc的抗体和机械感觉器官上的原位杂交将确定哪些细胞表达NompC以及在这些细胞内表达的位置。NompC并不单独起作用，转导机制可能包括许多分子。为了鉴定与NompC相互作用并因此可能包含转导机制的分子，将使用具有NompC部分的酵母双杂交测定。将在致敏的nompC背景中进行遗传增强子/抑制子筛选，以鉴定与nompC相互作用的新基因。其他机械传感基因将从现有的突变体中鉴定出来，并产生新的温度敏感突变体。没有伴随的机械感觉反应的生物物理分析，对转导过程的理解是不完整的。因为这需要对机械感觉神经元的电和机械访问，这是目前不可用的，将开发一种隔离的机械感觉神经元制剂，将允许这种访问。将使用全细胞电压钳记录对这些神经元中的转导进行深入的电生理学表征。最后，为了了解NompC的生物物理特性，例如门控和渗透，将对表达NompC通道的异源细胞进行电生理实验。本申请中提出的实验代表了果蝇机械转导的全面图片组装的下一步。由于它们的转导和发育途径非常相似，因此果蝇机械感觉神经元的信息可以作为进一步了解脊椎动物毛细胞分子和转导的范例。

DESCRIPTION (provided by applicant): Our diverse mechanosensory system encompass the salient senses of hearing, balance, touch, and proprioception, as well as less conscious senses like the detection of blood pressure and gut stretch. The mechanosensory cells that mediate these senses are structurally and functionally dissimilar, yet share a central feature. Unlike other sensory signaling modalities, which use second messengers to relay sensory information, mechanosensation occurs by the direct opening of mechanically gated ion channels by applied forces. Despite its importance, we currently do not know the identity of this channel or, with few exceptions, the remainder of the transduction machinery. The long-term goals underlying the proposed research are to define the molecules of the transduction machinery and understand how they work in concert to transduce mechanical stimuli into electrical signals. To understand mechanotransduction, this research takes advantage of the ease and elegance of a genetically tractable model organism, Drosophila. Fruit flies make an ideal organism for research on mechanotransduction for several reasons: renowned molecular-genetic tools, the ability to electrically record from mechanosensory bristles, and surprising similarities between the development and physiology of fly mechanosensory neurons and that of vertebrate hair cells. The scientific approach taken here can be divided into two parts: a molecular-genetic path to identify the genes involved in mechanosensory transduction and an electrophysiological approach to understand mechanosensory responses. Many of the experiments utilize the mechanosensory transduction channel, NompC, as a biochemical and genetic toehold into the transduction machinery. A first step in this proposal is to better define NompC's expression pattern. Antibodies against Nompc and in situ hybridization on mechanosensory organs will determine what cells express NompC and where within those cells it is expressed. NompC does not act alone, the transduction machinery likely encompasses many molecules. To identify molecules that interact with NompC and that are therefore likely comprise the transduction machinery, yeast two-hybrid assays with portions of NompC will be used. A genetic enhancer/supressor screen will be undertaken in a sensitized nompC background to identify new genes that interact with nompC. Other mechanosensory genes will be identified from existing mutants and new temperature-sensitive mutants will be generated. An understanding of the transduction process cannot be complete without accompanying biophysical analyses of the mechanosensory response. Because this requires electrical and mechanical access to the mechanosensory neuron that is not currently available, an isolated mechanosensory neuron preparation will be developed that will allow this access. An in-depth electrophysiological characterization of transduction in these neurons will be undertaken using whole-cell, voltage-clamp recording. Finally to understand the biophysical properties of NompC, such as gating and permeation, electrophysiolgical experiments on heterologous cells expressing the NompC channel will be undertaken. The experiments proposed in this application represent the next step in the assembly of a comprehensive picture of Drosophila mechanotransduction. Because their transduction and developmental pathways are so similar, the information from Drosophila mechanosensory neurons can be used as a paradigm to further understanding of the molecules and transduction in vertebrate hair cells.