Mechanisms of mechanotransduction in the enterochromaffin cells

 描述（由申请人提供）：这个指导职业发展奖（K 08）提案描述了一个为期五年的培训计划，为医生科学家候选人，长期目标是成为健康和疾病胃肠道（GI）中细胞和分子机械敏感性的学术权威。该候选人在博士和博士后工作期间获得了离子通道生物物理学的坚实基础。候选人现在建议巩固他的基础，并获得一套新的补充技能，在胃肠道机械敏感性的独立研究生涯。机械敏感性对于正常的胃肠道功能是重要的，并且异常的机械敏感性导致疾病。肠嗜铬细胞（EC）是机械敏感的胃肠道上皮细胞，合成和释放5-羟色胺（5-HT），调节胃肠道运动和感觉。机械力是EC细胞释放5-HT的主要刺激，但EC细胞机械感觉的分子机制尚不清楚。急性细胞机械感觉涉及机械敏感离子通道（MSC）。最近克隆的MSC PIEZO 2对于默克尔细胞机械感觉是关键的，其是皮肤上皮的机械敏感细胞，具有与EC细胞的发育和功能相似性。本申请的中心假设是PIEZO 2离子通道是将机械能转化为EC细胞释放的5-HT的主要机械传感器。我们将在2个AIM中检验中心假设。AIM 1中的实验将确定PIEZO 2在EC细胞中的定位和机械活化。AIM 2中的实验将确定PIEZO 2激活如何与单个EC细胞和肠类器官中的5-HT释放偶联，以及PIEZO 2激活如何与GI运动偶联。AIM得到了强有力的初步数据的支持，这些数据表明PIEZO 2对人类和小鼠结肠EC细胞具有特异性，机械刺激激活原代EC细胞中的PIEZO 2，导致5-HT的释放，而结肠上皮中PIEZO 2的阻断会降低运动性。为了验证中心假设，我们将使用免疫组织化学和超分辨率成像，电生理学和光遗传学与单细胞和肠类器官的机械刺激并行，通过电化学和新技术进行5-HT测量，在野生型和转基因动物中进行结肠运动的体外和体内记录。这项工作将 在马约诊所的学术环境中进行，并得到胃肠病学和肝病学部门的全力支持。候选人将得到几名顾问的支持，这些顾问是候选人认为需要培训的领域的专家。他将由一个强大的指导委员会（博士。两人都是国际权威-Farrugia博士在离子通道在胃肠道运动，和博士。 LaRusso在上皮生物学和机械感觉。作为这项工作的结果，候选人将大大提高我们对人体生理学中EC细胞机械敏感性机制的理解，目标是为治疗策略提供一个新的平台，并获得R 01应用的数据。

 DESCRIPTION (provided by applicant): This Mentored Career Development Award (K08) proposal describes a five year training program for the physician scientist candidate with a long-term goal of becoming an academic authority in cellular and molecular mechanosensitivity in the gastrointestinal (GI) tract in health and disease. The candidate acquired a strong basic science foundation in ion channel biophysics during his PhD and post-doctoral work. The candidate now proposes to solidify his foundation and acquire a set of novel complementary skills necessary for an independent research career in GI tract mechanosensitivity. Mechanosensitivity is important for normal GI tract function and abnormal mechanosensitivity leads to disease. Enterochromaffin (EC) cells are mechanosensitive GI epithelial cells that synthesize and release serotonin (5-HT), which regulates GI motility and sensation. Mechanical forces are a major stimulus for 5-HT release by the EC cell, but the molecular mechanisms of EC cell mechanosensation are unclear. Acute cellular mechanosensation involves mechanosensitive ion channels (MSCs). A recently cloned MSC PIEZO2 is critical for Merkel cell mechanosensation, which is a mechanosensitive cell of the skin epithelium with developmental and functional similarities to the EC cell. The central hypothesis of this application is that PIEZO2 ion channels are the primary mechanosensors that transduce mechanical energy into 5-HT release by the EC cells. We will test the central hypothesis in 2 AIMs. The experiments in AIM 1 will determine the localization and mechanical activation of PIEZO2 in EC cells. The experiments in AIM 2 will determine how PIEZO2 activation is coupled to 5-HT release in single EC cells and intestinal organoids and how PIEZO2 activation couples to GI motility. The AIMs are supported by strong preliminary data which show that PIEZO2 is specific to human and mouse colon EC cells, that mechanical stimuli activate PIEZO2 in primary EC cells, leading to release of 5-HT, while the block of PIEZO2 in the colon epithelium decreases motility. To test the central hypothesis, we will use immunohistochemistry and super resolution imaging, electrophysiology and optogenetics in parallel with mechanostimulation of single cells and intestinal organoids, 5-HT measurements by electrochemistry and novel techniques, novel ex vivo and in vivo recordings of colonic motility in wild type and transgenic animals. This work will be performed in an academically nurturing environment within Mayo Clinic and with full support of the Division of Gastroenterology & Hepatology. The candidate will be supported by several consultants who are experts in the areas that the candidate identified as necessary training. He will be guided by a strong mentorship committee (Drs. Gianrico Farrugia and Nicholas LaRusso). Both are international authorities - Dr. Farrugia in ion channels in GI motility, and Dr. LaRusso in epithelial biology and mechanosensation. As a result of this work, the candidate will significantly advance our understanding of EC cell mechanosensitivity mechanisms in human physiology with a goal of providing a novel platform for therapeutic strategies as well as obtain data for a R01 application.