Unconventional myosins and the regulation of gut barrier integrity and restitution during inflammation

项目总结：炎症性肠病患者肠上皮屏障的破坏 炎症性肠病（IBD）涉及上皮紧密连接（TJ）和粘附连接（AJ）的分解，沿着 由于过度的上皮细胞死亡而形成粘膜伤口。恢复肠道屏障（上皮 恢复）对于实现疾病的缓解至关重要，并且其涉及粘膜伤口愈合和AJ/TJ 重组这两个过程都是由共同的分子事件介导的，最值得注意的是， 上皮肌动球蛋白细胞骨架。非常规肌球蛋白18 A（Myo 18 A）是一种独特的PDZ结构域 含有支架蛋白，作为肌动蛋白丝及其运动的基本调节物，非肌肉 肌球蛋白II（NM II）。已知Myo 18 A控制神经元和肌肉形态发生以及肿瘤生长 和传播，但其在肠道中的功能以前没有研究过。我们的初步数据 提示Myo 18 A在正常人肠上皮中大量表达，是一种新的 此外，Myo 18 A缺失导致各种功能缺陷，包括 上皮屏障的破坏、伤口愈合的减弱和3-D上皮细胞的损伤 形态发生重要的是，我们观察到在肠内Myo 18 A表达的显著下调， 溃疡性结肠炎患者的上皮。这一令人兴奋的初步数据为以下方面提供了有力的科学前提： 以下创新假设：Myo 18 A是一种新型的肠上皮屏障调节剂 完整性和恢复，其消耗促进肠道渗漏并抑制IBD中的粘膜愈合 患者 这一假设将在以下目的中进行检验：（1）确定Myo 18 A在调节 肠上皮细胞迁移和ECM粘附;（2）阐明Myo 18 A依赖性 上皮屏障的组装和顶端-基底细胞极性的建立; 3）检查 Myo 18 A在体内调节肠上皮屏障的破坏和恢复。目标将是 通过利用体外肠上皮细胞单层、离体结肠类器官 从IBD粘膜和上皮损伤和恢复的体内鼠模型产生。Myo 18 A的角色将 通过功能性（渗透性测量、伤口愈合）、生物化学和生物化学的组合进行检查。 （免疫印迹，去污剂分级），成像（FRET生物传感器，超分辨率显微镜），和 基因（CRISPR/Cas9基因编辑，缺失突变体的过表达，敲除小鼠）方法。 意义：这项研究将对调节肠上皮细胞的机制产生新的见解。 炎症期间的损伤和恢复。它还将确定新的治疗目标，以防止崩溃， 增强消化系统疾病患者肠道屏障的修复。

PROJECT SUMMARY: Disruption of the intestinal epithelial barrier in patients with inflammatory bowel diseases (IBD) involves disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ), along with formation of mucosal wounds due to excessive epithelial cell death. Restoration of the gut barrier (epithelial restitution) is critical for achieving remission of the disease and it involves mucosal wound healing and AJ/TJ reassembly. Both processes are mediated by common molecular events, most notably, dramatic remodeling of the epithelial actomyosin cytoskeleton. An unconventional myosin 18A (Myo18A) is a unique PDZ-domain containing scaffolding protein that acts as an essential regulator of actin filaments and their motor, non-muscle myosin II (NM II). Myo18A is known to control neuronal and muscle morphogenesis, as well as tumor growth and dissemination, however its functions in the gut have not been previously investigated. Our preliminary data suggests that Myo18A is abundantly expressed in normal human intestinal epithelium and it is a novel component of epithelial junctions, Furthermore, Myo18A depletion leads to various functional defects, including disruption of the epithelial barrier, attenuation of wound healing and impairment of 3-D epithelial morphogenesis. Importantly, we observed a marked downregulation of Myo18A expression in the intestinal epithelium of ulcerative colitis patients. This exciting preliminary data provides a strong scientific premise for the following innovative hypothesis: Myo18A is a novel regulator of the intestinal epithelial barrier integrity and restitution, and its depletion promotes gut leakiness and inhibits mucosal healing in IBD patients. This hypothesis will be tested in the following Aims: (1) to determine the roles of Myo18A in the regulation of intestinal epithelial cell migration and ECM adhesion; (2) to delineate the mechanisms of Myo18A-dependent assembly of the epithelial barrier and establishment of the apico-basal cell polarity; 3) to examine the roles of Myo18A in regulating disruption and restitution of the intestinal epithelial barrier in vivo. The Aims will be accomplished by studies utilizing in vitro intestinal epithelial cell monolayers, ex vivo colonic organoids generated from IBD mucosa and in vivo murine models of epithelial injury and restitution. Roles of Myo18A will be examined by a combination of functional (permeability measurements, wound healing), biochemical (immunoblotting, detergent fractionation), imaging (FRET biosensors, super-resolution microscopy), and genetic (CRISPR/Cas9 gene editing, overexpression of deletion mutants, knockout mice) approaches. Significance: the proposed study will yield novel insights into the mechanisms that regulate intestinal epithelial injury and restitution during inflammation. It will also identify new therapeutic targets to prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases.