Exploring the coupling between PIEZO1 subunits gating motions using TIRF

使用 TIRF 探索 PIEZO1 亚基之间的门控运动之间的耦合

• 批准号: 10381223
• 负责人: YUN LUO
• 金额: $ 10.85万
• 依托单位: WESTERN UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10381223
• 关键词: Administrative Supplement; Anemia; Arthrogryposis; Award; Biological Process; Cells; Clinical; Coupled; Coupling; Data; Fluorescence; Goals; Homeostasis; Inflammation; Ion Channel; Ions; Liquid substance; Lymphedema; Malignant Neoplasms; Measurement; Mechanical Stimulation; Mechanics; Mediating; Microscope; Molecular; Molecular Conformation; Motion; Pain; Pathway interactions; Physiological; Piezo ion channels; Play; Process; Proteins; Role; Sensory Physiology; Signal Transduction; Stimulus; Testing; Vertebrates; equipment acquisition; experimental study; fluorescence imaging; organ growth; polypeptide; response; shear stress; single molecule

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.

抽象的 细胞快速感知和响应机械刺激的能力对于所有生物都至关重要。在脊椎动物中， 此任务主要是通过机械敏感的离子通道Piezo1和Piezo2来实现的。确实，一个越来越多的数字 研究概述了这些渠道对许多生物学过程所起的核心作用 感觉生理，渗透稳态和器官发育。毫不奇怪，异常压电通道 活性与许多临床状况有关，例如淋巴水肿，贫血，关节炎，癌症， 炎症和疼痛。这些蛋白质由三个长多肽链（亚基）形成 中央离子传导途径（孔）。我们授予项目的第一个具体目的是确定和 表征当通道打开时在这些亚基中进行的特定构象重排（门） 它在机械刺激时的孔。我们团队和其他人的最新研究表明， 一个亚基可以合作影响其他亚基。请求的管理目的 补充是检验这一假设。为此，我们打算购买总内反射荧光 （TIRF）升级我们的表皮显微镜。 TIRF测量将使单分子能够 压电1通道的荧光测量值，其中每个亚基都用剪切压力在遗传上标记 敏感的荧光探针。我们新的落荧光数据表明，这些探针发出了大荧光 当通道被流体剪切应力刺激时，与孔口开口相关的信号。我们预计 每个亚基的流量介导的门控运动将伴随着离散的，跳高的像素的增加 TIRF图像中的亮度。如果这种门控运动是耦合的，则这些离散的跳跃被预测为暂时 相关。如果没有，预计这些荧光跳跃将独立发生。如果成功，这些实验 将加深我们对这些基本离子渠道如何打开其孔的基本理解 生理刺激。