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

项目摘要：炎症性肠病患者肠上皮屏障的破坏 疾病(IBD)涉及上皮紧密连接(TJ)和黏附连接(AJ)的分解，以及 由于上皮细胞过度死亡而形成的粘膜伤口。肠屏障(上皮)的修复 修复)是实现疾病缓解的关键，它涉及粘膜伤口愈合和AJ/TJ 重新组装。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， 上皮性肌动球蛋白细胞骨架。非传统肌球蛋白18A(Myo18A)是一个独特的PDZ结构域 含有作为肌动蛋白细丝及其运动、非肌肉的基本调节器的支架蛋白 肌球蛋白II(NM II)。已知Myo18A控制神经元和肌肉的形态发生，以及肿瘤的生长 和传播，但它在肠道中的功能以前没有被研究过。我们的初步数据 提示Myo18A在正常人肠上皮细胞中大量表达，是一种新的 此外，Myo18A的缺失会导致各种功能缺陷，包括 上皮屏障的破坏、伤口愈合的减弱和3-D上皮的损伤 形态发生。重要的是，我们观察到Myo18A在肠道中的表达显著下调 溃疡性结肠炎患者的肠黏膜上皮。这一令人振奋的初步数据为 以下创新假设：Myo18A是一种新的肠上皮屏障调节器 完整和恢复，以及它的耗尽促进肠漏并抑制IBD的粘膜愈合 病人。 这一假说将在以下目的中得到验证：(1)确定Myo18A在调节 肠上皮细胞迁移与ECM黏附；(2)阐明Myo18A依赖的机制 上皮屏障的组装和心尖-基底细胞极性的建立；3)研究 Myo18A在体内调节肠上皮屏障的破坏和重建中的作用目标将是 通过利用体外肠上皮细胞单层、体外结肠有机物质 从IBD黏膜和体内小鼠上皮损伤和修复模型中产生。Myo18A Will的作用 通过结合功能(渗透性测量、伤口愈合)、生化检查进行检查 (免疫印迹、洗涤剂分级)、成像(FRET生物传感器、超分辨率显微镜)和 遗传方法(CRISPR/Cas9基因编辑、缺失突变体的过度表达、基因敲除小鼠)。 意义：这项拟议的研究将为调节肠道上皮细胞的机制提供新的见解 炎症期间的损伤和恢复。它还将确定新的治疗目标，以防止崩溃和 加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.