Hair-Bundle Lipids

描述(由申请人提供)：本提案的目的是确定脂质在毛细胞机械转导中的作用。我们提出了三个具体目标。在目标1中，我们将使用质谱仪和组织化学定位来确定发束中存在哪些脂类。此外，我们还将测量聚磷脂酰肌醇的周转率，并确定合成和降解它们的酶的特性、定位和活性。这些实验是基于这样一种假设，即发束具有不同寻常的唇部成分和分布，这些特征对转导很重要。在目标2中，利用光漂白后的荧光恢复，我们将测定大块毛束膜的动力学以及单个脂类的动力学。这些实验的动机是假设束膜可能具有降低脂质流动性的区域。最后，在目标3中，我们将确定脂类如何调节转导和适应。这些实验将检验以下假设：(A)膜结合调节肌球蛋白-1c的活性，(B)肌球蛋白-1c与膜的结合改变脂质动力学，(C)适应马达和转导通道在专门的脂域相互作用，以及(D)在“门控弹簧”中测量的弹性是由于立体纤毛膜本身的特性。总之，这项提案中的实验将使我们能够确定脂类如何调节转导和适应。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the role of lipids in mechanotransduction by hair cells. We propose three specific aims. In Aim 1, we will determine which lipids are present in hair bundles using mass spectrometry and histochemical localization. In addition, we will measure the turnover of polyphosphatidylinositols and determine the identity, localization, and activities of enzymes that synthesize and degrade them. These experiments are shaped by the hypothesis that hair bundles have an unusual lip d composition and distribution and that these characteristics are important for transduction. In Aim 2, using fluorescence recovery after photobleaching, we will determine dynamics of the bulk hair-bundle membrane as well as the dynamics of individual lipid species. These experiments are motivated by the hypothesis that bundle membranes may have domains of reduced lipid mobility. Finally, in Aim 3 we will determine how lipids regulate transduction and adaptation. These experiments will test the hypotheses that (a) membrane association regulates myosin-1c activity, (b) myosin-1c binding to membranes alters lipid dynamics, (c) the adaptation motor and transduction channel interact in a specialized lipid domain, and (d) that the elasticity measured in the "gating spring" is due to properties of the stereocilia membrane itself. Together, the experiments in this proposal will allow us to determine how lipids regulate transduction and adaptation.