Investigating the role of lipid membrane in the cochlear hair cell mechanotransduction

项目摘要/摘要 机械-电转换(MET)过程允许机械信息的传输 从声音到电信号，这是耳蜗系统功能的基本步骤。这方面的失败 这一过程会导致听力损失和耳聋。了解MET的基本属性将导致更好的 对耳聋的了解，导致有针对性的治疗和治疗。Met发生在 毛束是由尖端连接介导的，细胞外蛋白连接较短的立体纤毛和相邻的较高 立体纤毛。发束向最高的立体纤毛方向的偏转增加了顶端-链节的张力和张开 位于较短的立体纤毛顶部的MET频道。尽管有大量的工作涉及 脂膜对机械敏感离子通道的调节作用，有一个有限但越来越多的数据关于 脂质调节对耳蜗毛细胞的影响。 脂质环境可通过膜力学的改变间接或直接影响通道 通过个体的脂类/蛋白质相互作用。PIP2是一种内源性磷脂，调节MET通道 性质，潜在地通过直接相互作用或通过改变膜机制间接地。伸展一下 激活的信道修改器GsMTx4在阻塞的同时降低了MET信道的静止开放概率(Po 通过降低外源性钙离子或使毛细胞去极化而引起的Po的增加，提示脂质 膜可能参与调制MET通道P0。电压和钙离子的作用可能是中介的。 通过多价离子在相邻脂类之间相互作用引起的脂类堆积的变化。我们最近的直接指示 双光子荧光恢复法一次测定单个立体纤毛的膜扩散率 光漂白后(FRAP)研究表明，立体纤毛膜对钙和电压很敏感，但 而不是胞体，MET通道Po与膜的扩散率共同变化，支持MET的假设 通道可以通过膜力学进行调制。然而，由于FRAP的空间和时间限制， 我们无法对立体纤毛膜进行局部和动态监测。 为了进一步验证这一假设并克服当前的技术限制，我将结合 用活细胞荧光寿命成像(FLIM)检测新型粘度传感器的电生理学 首次在哺乳动物耳蜗处发现了具有更高时空分辨率的膜粘度。这就做 评估立体纤毛膜粘度随电压、钙和膜的局部和时间变化 像胆固醇和PIP2这样的成分，并将这些影响与MET通道Po的变化相关联。这些研究 将加深我们对脂膜在毛细胞机械转导中的重要性的基本认识。 了解立体纤毛的机械基础中的关键成分既是生物物理学的 以及生物学上的相关性。这些新技术的开发和使用将极大地推进我的事业 作为一名独立的调查者，可能在听觉领域和其他领域有更广泛的应用。

Project Summary/Abstract The mechano-electrical transduction (MET) process allows the transduction of mechanical information from sound into electrical signals, and it is a fundamental step in cochlear system function. Failures in this process lead to hearing loss and deafness. Understanding the basic properties of MET will lead to a better understanding of deafness, leading to targeted treatments and therapies. MET takes place at the level of the hair bundle and is mediated by tip links, extracellular proteins connecting shorter stereocilia to adjacent taller stereocilia. Deflections of the hair bundle towards the tallest stereocilia row increase tip-link tension and open MET channels that reside at the top of the shorter stereocilia. Although there is a large body of work regarding lipid membrane modulation of mechanosensitive ion channels, there is a limited but growing body of data on lipid modulation of cochlear hair cell MET. The lipid environment can affect channels indirectly through changes in membrane mechanics, or directly through individual lipid/protein interactions. PIP2, an endogenous phospholipid, modulates MET channel properties, potentially through a direct interaction or indirectly by altering membrane mechanics. A stretch activated channel modifier, GsMTx4 reduces the resting open probability (Po) of MET channel while also blocking the increase in Po induced by lowering external calcium or depolarizing the hair cell, suggesting the lipid membrane may be involved in modulating MET channel Po. The effect of voltage and calcium could be mediated through changes in lipid packing due to multivalent ions interacting between adjacent lipids. Our recent direct assessment of membrane diffusivity of individual stereocilium at a time using two-photon Fluorescent Recovery after Photobleaching (FRAP) demonstrated that stereocilia membrane is sensitive to calcium and voltage but not the soma, and MET channel Po co-varies with membrane diffusivity, supporting the hypothesis that the MET channel can be modulated by membrane mechanics. However, due to spatial and temporal limitations of FRAP, we were unable to monitor stereocilia membrane locally and dynamically. To further test this hypothesis and overcome current technological limitations, I will combine electrophysiology with live-cell fluorescence lifetime imaging (FLIM) of a novel viscosity sensor to examine the membrane viscosity with improved spatio-temporal resolution for the first time in mammalian cochlea. I will assess local and temporal changes in the stereociliary membrane viscosity with voltage, calcium, and membrane components like cholesterol and PIP2 and correlate these effects to changes in MET channel Po. These studies will enhance our basic understanding of the importance of lipid membrane in hair cell mechanotransduction. Understanding the crucial components in the mechanical underpinnings of the stereocilia are both biophysically and biologically relevant. The development and use of these new technologies will greatly advance my career as an independent investigator and likely have broader applications in the auditory field and beyond.