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

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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.