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.
