Mechanosensory Transduction in Drosophila

描述（由申请人提供）：我们多样的机械感觉系统包括听觉，平衡，触觉和本体感觉等显着感觉，以及血压和肠道拉伸等较不明显的感觉。调节这些感觉的机械感觉细胞在结构和功能上不同，但有一个共同的中心特征。与其他使用第二信使传递感觉信息的感觉信号方式不同，机械感觉是通过施加力直接打开机械门控离子通道而发生的。尽管它很重要，但我们目前还不知道这个通道的身份，除了少数例外，我们也不知道转导机制的其余部分。这项研究的长期目标是确定转导机制的分子，并了解它们如何协同工作，将机械刺激转化为电信号。为了理解机械转导，这项研究利用了一种遗传上易于处理的模式生物——果蝇的轻松和优雅。果蝇是研究机械转导的理想生物，有以下几个原因：著名的分子遗传学工具，机械感觉刚毛的电记录能力，果蝇机械感觉神经元的发育和生理与脊椎动物毛细胞的发育和生理惊人的相似。这里采用的科学方法可以分为两部分：一种分子遗传途径，用于识别参与机械感觉转导的基因；另一种电生理学方法，用于理解机械感觉反应。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。该建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的原位杂交将决定哪些细胞表达Nompc以及在这些细胞中表达Nompc的位置。NompC并不是单独起作用的，它的转导机制可能包含许多分子。为了确定与NompC相互作用的分子，因此可能包含转导机制，将使用带有部分NompC的酵母双杂交试验。将在致敏的nompC背景下进行基因增强子/抑制子筛选，以确定与nompC相互作用的新基因。其他机械感觉基因将从现有的突变体中鉴定出来，新的温度敏感突变体将产生。如果没有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进行电和机械的访问，而目前还无法实现，因此将开发一种分离的机械感觉神经元制备方法来实现这种访问。深入的电生理表征转导在这些神经元将进行全细胞，电压钳记录。最后，为了了解NompC通道的门控和渗透等生物物理特性，我们将在表达NompC通道的异源细胞上进行电生理实验。本应用程序中提出的实验代表了全面了解果蝇机械转导的下一步。由于它们的转导和发育途径非常相似，来自果蝇机械感觉神经元的信息可以作为进一步理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.