Exploring the coupling between PIEZO1 subunits gating motions using TIRF

摘要 细胞快速感知和响应机械刺激的能力对所有生物都至关重要。在脊椎动物中， 该任务主要通过机械敏感离子通道PIEZO 1和PIEZO 2来实现。事实上， 的研究已经概述了这些通道在许多生物过程中发挥的核心作用， 感觉生理学、渗透平衡和器官发育。毫不奇怪，异常PIEZO通道 活性与许多临床病症如水肿、贫血、关节弯曲，癌症， 炎症和疼痛。这些蛋白质由三条长多肽链（亚基）组装在 中央离子传导通路（孔）。我们获奖项目的第一个具体目标包括识别和 表征通道打开时这些亚基中发生的特定构象重排（门） 它在机械刺激下会产生孔隙。我们团队和其他人最近的研究表明， 一个子单元可以协作地影响其它子单元。所要求的行政 补充是为了检验这一假设。为此，我们打算购买全内反射荧光 （TIRF）升级我们的荧光显微镜。TIRF测量将使单分子 PIEZO 1通道的荧光测量，其中每个亚基用剪切应力基因标记 灵敏的荧光探针我们新的落射荧光数据表明，这些探针发射大的荧光 当通道被流体剪切应力刺激时，与孔打开相关的信号。我们预计 每个亚基的流动介导的门控运动将伴随着像素的离散的跳跃式增加 TIRF图像中的亮度。如果该选通运动是耦合的，则这些离散跳跃被预测为在时间上是 相关的如果不是，则预测这些荧光跳跃独立地发生。如果实验成功， 将加深我们对这些基本离子通道如何打开它们的孔以响应一个基本离子通道的理解。 生理刺激

Abstract The ability of cells to rapidly sense and respond to mechanical stimuli is vital for all living beings. In vertebrates, this task is mainly achieved by mechanosensitive ion channels PIEZO1 and PIEZO2. Indeed, a growing number of studies have outlined the central role played by these channels to numerous biological processes including sensory physiology, osmotic homeostasis, and organ development. Not surprisingly, abnormal PIEZO channel activity is associated with many clinical conditions such as lymphedema, anemia, arthrogryposis, cancer, inflammation, and pain. These proteins are formed by three long polypeptide chains (subunits) assembled around a central ion conduction pathway (pore). The first specific Aim of our awarded project consists of identifying and characterizing specific conformational rearrangements taking place in these subunits as the channel opens (gates) its pore upon mechanical stimulation. Recent studies from our team and others suggest that the gating motion of one subunit may cooperatively influence that of the others. The purpose of the requested administrative supplement is to test this hypothesis. To this aim, we intend to purchase a Total Internal Reflection Fluorescence (TIRF) upgrade for our epifluroescence microscope. TIRF measurements will enable single-molecule fluorescence measurements of PIEZO1 channels in which each subunit is genetically-tagged with a shear-stress sensitive fluorescence probe. Our new epifluorescence data indicate that these probes emit large fluorescence signals that correlate with pore opening when channels are stimulated by fluid shear stress. We anticipate that the flow-mediated gating motion of each subunit will be accompanied by a discrete, jump-like increase of pixel brightness in TIRF images. If this gating motion is coupled, these discrete jumps are predicted to be temporally correlated. If not, these fluorescence jumps are predicted to occur independently. If successful, these experiments will deepen our fundamental understanding of how these essential ion channels open their pore in response to a physiological stimulus.