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.

摘要 细胞快速感知和响应机械刺激的能力对所有生物来说都是至关重要的。在脊椎动物中， 这一任务主要是通过机械敏感离子通道PIEZO1和PIEZO2来实现的。事实上，越来越多的人 的研究概述了这些通道在许多生物过程中所起的中心作用，包括 感觉生理学、渗透平衡和器官发育。毫不奇怪，异常的压电式通道 活动与许多临床症状有关，如淋巴水肿、贫血、关节肿大、癌症、 炎症和疼痛。这些蛋白质是由三条长长的多肽链(亚基)组成的 一种中心离子传导途径(气孔)。我们获奖项目的第一个具体目标是确定和 表征当通道打开时在这些亚基中发生的特定构象重排(门) 它的毛孔依赖于机械刺激。我们团队和其他人最近的研究表明，门控运动 一个亚基可能协同影响其他亚基的亚基。所请求的管理的目的 补充就是检验这一假说。为此，我们打算购买全内反射荧光仪 (TIRF)升级我们的荧光显微镜。TIRF测量将使单分子 每个亚基带有切应力基因标记的PIEZO1通道的荧光测量 灵敏的荧光探针。我们新的荧光数据表明，这些探针发出了很大的荧光 当通道受到流体剪应力刺激时，与孔洞开度相关的信号。我们预计， 每个子单元的流介导门控运动将伴随着离散的、跳跃式的像素增加 TIRF图像中的亮度。如果这种门控运动是耦合的，这些离散的跳跃被预测为在时间上 相互关联。如果不是，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通道如何打开毛孔的基本理解 生理刺激。