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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要：肠上皮屏障的破坏是一个重要的表现胃肠道疾病，包括炎症性肠病、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合结构介导的 (TJ) 和粘附连接 (AJ)。有证据表明，AJ 和 TJ 在发炎时会分解肠粘膜造成肠道屏障渗漏。了解调节机制健康肠道的连接完整性和粘膜炎症期间驱动 AJ/TJ 解体是主要因素拟议研究的目标。 TJ 和 AJ 的完整性和重塑取决于肌动球蛋白由肌动蛋白丝和特殊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整体发挥作用。重链是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 重链的作用和调控知之甚少在正常和发炎的肠上皮中。本提案的中心创新假设提出 NM II 重链（电机）是建立和维护的关键调节器健康肠道上皮屏障的破坏以及这些马达的表达/组装受损导致粘膜炎症期间屏障破坏并抑制上皮恢复。这个假设将测试目的如下： (1) 确定不同 NM II 重链在体内肠上皮屏障的维持、破坏和恢复； (2) 调查 NM II 分子伴侣 UNC-45A 参与上皮屏障完整性的调节归还; 3）分析septin细胞骨架在NM II组装和重塑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症介质并在体内使用小鼠结肠炎模型。不同的功能 NM II 重链和 NM II 靶向伴侣和脓蛋白将通过以下组合进行分析功能性（渗透性测量、伤口愈合）、生物化学（肌动蛋白共沉降、免疫印迹、去污剂分级分离）、免疫细胞化学和遗传（CRISPR/Cas9 基因编辑、天然和突变蛋白的过度表达、基因敲除小鼠）方法。意义：拟议的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并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目标，以防止崩溃并加强修复消化系统疾病患者的肠道屏障。