Role of cytoskeletal interactions with mechanogated ion channel Piezo2 in the mechanism of mechanotransduction by enteroendocrine cells

项目摘要/摘要 大约15%的美国人患有运动或功能性胃肠功能障碍(FGID)，比如肠易激 综合征(IBS)。FGIDs的病理生理机制仍然知之甚少，导致靶向性差。 治疗。胃肠道上皮中的肠内分泌细胞(EECS)释放信号分子，控制许多 影响FGID患者的过程，如运动和分泌。很大比例的FGID患者患有 机械感觉异常。胃肠道上皮中有一群机械敏感的EECs。 在结构和功能上与特殊的感觉上皮细胞相似，例如皮肤中的光触摸感受器。 机械敏感的EECS感知物理作用力，并将其转化为激素释放。因此， 机械敏感的EECs是胃肠道上皮细胞中的主要机械转导细胞，它们可能是 口蹄疫的治疗。它们的特征是表达Piezo2，这是一种响应于 武力。Piezo2产生一种受体电流，启动EEC机械转导。Piezo2并不是唯一一个 这些细胞中的机械敏感蛋白：肌动蛋白纤维和紧密连接也是上皮力的关键 变速箱。了解Piezo2的定位及其与其他机械敏感者的功能相互作用 蛋白质对于理解感觉上皮细胞的机械转导很重要。这个项目的总体目标是 建议是揭示机械敏感蛋白在EECS中共同作用的机制 高效的力传感器。假设肌动蛋白细胞骨架在Piezo2+EEC中起关键作用。 通过将通道连接到其他机械传感器并直接改变通道电流来实现机械转导。 目的1通过超分辨成像和共聚焦扫描技术研究机械感觉蛋白在EEC中的组装。 免疫沉淀研究。这些实验探索了直接和间接的相互作用 机械传感器Piezo2，肌动蛋白纤维和Claudin-4。Aim 2研究了这些机械传感器如何影响 通过跟踪力诱导的钙瞬变和电生理的整体EEC机械转导 Piezo2的研究。这些实验探索了力是如何在上皮片中传递以启动 受体电流。这项工作的结果有望弥合Piezo2+EEC中的知识差距 机械转导，以及在感觉上皮细胞中更广泛的机械传感机制。这个 拟议的工作将在提供尖端工具和专家的支持性环境中进行 实现具体目标的知识，包括与细胞骨架专家的合作 生物学，以及在建议的成像技术方面具有丰富经验的成像核心。该提案包括一项 有医生-科学家导师的全面培训计划，通过该计划，PI将获得宝贵的技能 临床相关问题的分子力学转导研究。随着研究活动的开展， 计划还包括临床培训和跟踪活动，为PI过渡到下一阶段做准备 作为一名未来的外科医生兼科学家的训练。

Project Summary/Abstract Around 15% of the US population suffers from a motility or functional GI disorder (FGID), like irritable bowel syndrome (IBS). The pathophysiology of FGIDs remains poorly understood, leading to poorly targeted treatments. Enteroendocrine cells (EECs) in the GI epithelium release signaling molecules that control many processes affected in FGID patients, like motility and secretion. A large proportion of FGID patients have abnormalities in mechanosensation. A population of mechanosensitive EECs in the GI epithelium exhibits structural and functional similarities with specialized sensory epithelia, such as light touch receptors in the skin. The mechanosensitive EECs sense physical forces and convert them into hormone release. Thus, mechanosensitive EECs are primary mechanotransducers in the GI epithelium, and they may be targets for the treatment of FGIDs. They are characterized by expression of Piezo2, an ion channel that opens in response to force. Piezo2 generates a receptor current that initiates EEC mechanotransduction. Piezo2 is not the only mechanosensitive protein in these cells: actin fibers and tight junctions are also critical in epithelial force transmission. Understanding Piezo2 localization and its functional interactions with other mechanosensitive proteins is important for understanding sensory epithelial mechanotransduction. The overall goal of this proposal is to uncover mechanisms by which mechanosensitive proteins work together in EECs to make them efficient force sensors. The hypothesis is that the actin cytoskeleton plays a critical role in Piezo2+ EEC mechanotransduction by linking the channel to other mechanosensors and directly changing channel currents. Aim 1 investigates the assembly of mechanosensory proteins in the EEC by superresolution imaging and co- immunoprecipitation studies. These experiments explore direct and indirect interactions between the mechanosensors Piezo2, actin fibers and claudin-4. Aim 2 investigates how these mechanosensors affect overall EEC mechanotransduction by tracking force-induced calcium transients and electrophysiological studies of Piezo2. These experiments explore how force is transmitted in epithelial sheets to initiate the receptor current. The results of this work are poised to bridge knowledge gaps in Piezo2+ EEC mechanotransduction, as well as inform broader mechanosensing mechanisms in sensory epithelia. The proposed work will be carried out in a supportive environment that provides cutting edge tools and expert knowledge towards achieving the specified goal, including collaborations with experts in cytoskeletal biology,and an imaging core with vast experience in the proposed imaging techniques. The proposal includes a comprehensive training plan with physician-scientist mentors, by which the PI will gain valuable skills in the study of molecular mechanotransduction on clinically relevant questions. Along with research activities, the plan also includes clinical training and shadowing activities to prepare the PI for his transition to the next stage of training as a future surgeon-scientist.