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

项目总结/摘要 大约15%的美国人口患有运动或功能性GI疾病（FGID），如肠易激综合征 IBS综合征。FGID的病理生理学仍然知之甚少，导致靶向治疗效果不佳。 治疗。胃肠道上皮中的肠内分泌细胞（EECs）释放信号分子， 影响FGID患者的过程，如运动和分泌。大部分FGID患者 机械感觉异常胃肠道上皮中的机械敏感性内皮细胞群表现出 与专门的感觉上皮细胞，如皮肤中的轻触感受器，在结构和功能上相似。 机械敏感的EECs感知物理力并将其转化为激素释放。因此，在本发明中， 机械敏感性内皮细胞是胃肠道上皮中的主要机械传感器，它们可能是胃肠道上皮细胞的靶点。 治疗FGD。它们的特征在于表达Piezo 2，Piezo 2是一种响应于 力Piezo 2产生启动EEC机械转导的受体电流。Piezo 2不是唯一的 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细胞力的关键 传输了解Piezo 2定位及其与其他机械敏感性 蛋白质对于理解感觉上皮机械转导是重要的。这个项目的总体目标是 一项新的研究计划是揭示机械敏感蛋白在内皮细胞中共同作用的机制， 高效的力传感器假设肌动蛋白细胞骨架在Piezo 2 + 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进行机械转导。 目的1通过超分辨率成像和共聚焦技术研究EEC中机械感觉蛋白的组装。 免疫沉淀研究。这些实验探索了这些物质之间的直接和间接相互作用。 机械传感器Piezo 2、肌动蛋白纤维和claudin-4。目的2研究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学的总体EEC机械转导 Piezo 2的研究这些实验探索了力是如何在上皮层中传递以启动细胞分裂的。 受体电流这项工作的结果有望弥合Piezo 2 + EEC的知识差距 机械转导，以及告知感觉上皮细胞中更广泛的机械传感机制。的 拟议的工作将在一个支持性的环境中进行，提供先进的工具和专家， 实现特定目标的知识，包括与细胞骨架专家的合作 生物学，以及在所提出的成像技术方面具有丰富经验的成像核心。该提案包括一个 全面的培训计划与医生科学家导师，其中PI将获得宝贵的技能， 分子力学转导在临床相关问题上的研究。沿着研究活动， 该计划还包括临床培训和跟踪活动，为PI过渡到下一阶段做好准备 成为未来的外科医生兼科学家

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.