Actomyosin cytoskeleton and the regulation of intestinal eipithelial barrier

肌动球蛋白细胞骨架与肠上皮屏障的调节

• 批准号: 9304208
• 负责人: Andrei Ivanovich Ivanov
• 金额: $ 5.73万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304208
• 关键词: ATP Hydrolysis; Actins; Actomyosin; Adherens Junction; Adhesives; Apical; Binding; Biochemical; CRISPR/Cas technology; Celiac Disease; Cell physiology; Colitis; Complex; Cytoskeletal Proteins; Cytoskeleton; Detergents; Development; Digestive System Disorders; Disease; Dominant-Negative Mutation; Epithelial; Epithelial Cells; Fractionation; Functional disorder; Gastrointestinal Diseases; Genes; Genetic; Goals; Health; Heat shock proteins; Immunoblotting; Impairment; In Vitro; Incidence; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Bowel Diseases; Inflammatory disease of the intestine; Injury; Intestinal Diseases; Intestinal Mucosa; Intestines; Knock-out; Knockout Mice; Light; Maintenance; Measurement; Mediating; Microfilaments; Molecular; Molecular Chaperones; Molecular Target; Morbidity - disease rate; Motor; Motor Activity; Mucositis; Mucous Membrane; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; Nonmuscle Myosin Type IIA; Pathogenesis; Patients; Permeability; Pharmacology; Phosphorylation; Physiological; Physiology; Play; Prevention; Proteins; Public Health; Recovery; Recurrent disease; Regulation; Role; Sedimentation process; Structure; System; Testing; Tight Junctions; Work; Wound Healing; cell motility; cohort; cytokine; gastrointestinal function; healing; improved; in vivo; injured; innovation; insight; intestinal epithelium; knockout gene; mortality; mouse model; mutant; new therapeutic target; non-muscle myosin; novel; overexpression; pathogen; prevent; scaffold

PROJECT SUMMARY: Disruption of the intestinal epithelial barrier is a crucial manifestation of gastrointestinal disorders including inflammatory bowel disease, celiac disease, and infectious colitis. Integrity of the epithelial barrier is mediated by specialized adhesive structures known as tight junctions (TJ) and adherens junctions (AJ). Evidence suggests that AJ and TJ become disassembled in inflamed intestinal mucosa creating a leakiness of the gut barrier. Understanding mechanisms that regulate junctional integrity in healthy gut and drive AJ/TJ disassembly during mucosal inflammation is the major goal of the proposed study. Integrity and remodeling of TJ and AJ depend on the actomyosin cytoskeleton composed of actin filaments and a specialized motor protein non-muscle myosin (NM) II. NM II works as a molecular ensemble of different heavy chains and light chains. The heavy chains are responsible for all functional activities of this motor including actin binding, ATP hydrolysis and myofilament assembly. Surprisingly, little is known about the role and regulations of NM II heavy chains in normal and inflamed intestinal epithelium. The central innovative hypothesis of this proposal proposes that NM II heavy chains (motors) are critical regulators of the establishment and maintenance of the epithelial barrier in healthy gut and that impaired expression/assembly of these motors results in barrier disruption and inhibited epithelial restitution during mucosal inflammation. This hypothesis will be tested in the following Aims: (1) to determine the roles of different NM II heavy chains in maintenance, disruption, and restitution of the intestinal epithelial barrier in vivo; (2) to investigate the involvement of NM II chaperone, UNC-45A, in the regulation of epithelial barrier integrity and restitution; 3) to analyze the roles of the septin cytoskeleton in NM II assembly and remodeling of the intestinal epithelial barrier. These aims will be accomplished using in vitro intestinal epithelial cells exposed to inflammatory mediators and in vivo using murine models of colitis. The functions of different NM II heavy chains and NM II-targeting chaperons and septins will be analyzed via a combination of functional (permeability measurements, wound healing), biochemical (actin co-sedimentation, immunoblotting, detergent fractionation), immunocytochemical, and genetic (CRISPR/Cas9 gene editing, overexpression of native and mutant proteins, knockout mice) approaches. Significance: the proposed study will yield new insights into fundamental mechanisms that regulate normal epithelial barriers and mediate intestinal mucosal injury and restitution during inflammation. Understanding these mechanisms will provide new therapeutic targets to prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases.

项目概述：肠上皮屏障的破坏是 胃肠疾病，包括炎性肠病、乳糜泻和感染性结肠炎。 上皮屏障的完整性是由称为紧密连接的特殊粘附结构介导的 (TJ)和粘附连接（AJ）。有证据表明，AJ和TJ在炎症中分解， 肠粘膜造成肠屏障的泄漏。了解调控机制 健康肠道中的连接完整性和粘膜炎症期间驱动AJ/TJ分解是主要的 拟议研究的目标。TJ和AJ的完整性和重塑依赖于肌动球蛋白 由肌动蛋白丝和一种专门的运动蛋白非肌肉肌球蛋白（NM）II组成的细胞骨架。 NM II作为不同重链和轻链的分子系综起作用。沉重的锁链 负责该马达的所有功能活动，包括肌动蛋白结合，ATP水解和 肌丝组装令人惊讶的是，人们对NM II重链的作用和调节知之甚少 在正常和发炎的肠上皮中。这项提议的核心创新假设是 提出NM II重链（马达）是建立和维持的关键调节器 以及这些马达的表达/组装受损导致 屏障破坏和粘膜炎症期间抑制上皮恢复。这一假设将 在以下目的中进行测试：（1）确定不同NM II重链在以下中的作用： 维持、破坏和恢复体内肠上皮屏障;（2）研究 NM II分子伴侣，β-45 A参与上皮屏障完整性的调节， 3）分析Septin细胞骨架在NM II组装和重构中的作用， 肠上皮屏障这些目标将使用体外肠上皮细胞来实现 暴露于炎性介质和体内使用结肠炎的鼠模型。不同的功能 NM II重链和NM II靶向分子伴侣和胞间隔蛋白将通过以下组合进行分析： 功能（渗透性测量、伤口愈合）、生化（肌动蛋白共沉淀， 免疫印迹、去污剂分级分离）、免疫细胞化学和遗传学（CRISPR/Cas9基因 编辑、天然和突变蛋白的过表达、敲除小鼠）方法。Significance：the 这项研究将对调节正常上皮细胞的基本机制产生新的见解。 屏障和介导炎症期间肠粘膜损伤和恢复。了解这些 机制将提供新的治疗靶点，以防止崩溃和加强修复的 消化道疾病患者的肠道屏障。