Cytoskeletal regulation of intestinal barrier function and response to pathogens

肠道屏障功能的细胞骨架调节和对病原体的反应

• 批准号: 8445253
• 负责人: John Garber
• 金额: $ 16.02万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445253
• 关键词: Ablation; Actins; Adherens Junction; Advisory Committees; Affect; Apical; Award; Basic Science; Binding; Binding Sites; Biochemical; Biological; Biology; Boston; Cell Line; Cell physiology; Cells; Cellular biology; Citrobacter rodentium; Complex; Cytoskeleton; Data; Development; Diarrhea; Digestive System Disorders; Disease; Drosophila genus; Dynamin; Ectopic Expression; Environment; Epithelial; Epithelial Cells; Equilibrium; Escherichia coli EHEC; Event; Excision; Exhibits; Experimental Designs; Family; Fostering; Functional disorder; Funding; Gastroenterologist; Generations; Goals; Health; Histopathology; Homeostasis; Human; Immune; Immunology; In Vitro; Infection; Inflammation; Intercellular Junctions; Intestines; Lead; Maintenance; Mammalian Cell; Mediating; Medical Research; Membrane; Mentorship; Microbe; Microbiology; Microfilaments; Modeling; Molecular; Monomeric GTP-Binding Proteins; Mouse Cell Line; Mus; Pathogenicity; Pathway interactions; Pediatric Hospitals; Permeability; Pilot Projects; Play; Positioning Attribute; Process; Productivity; Proteins; Publications; Reagent; Regulation; Regulatory Pathway; Research; Research Personnel; Resistance; Role; Signal Pathway; Signaling Molecule; Sorting - Cell Movement; Techniques; Testing; Therapeutic; Tight Junctions; Time; Virulence Factors; Wiskott-Aldrich Syndrome; Work; base; career; career development; enteric pathogen; enteropathogenic Escherichia coli; experience; gastrointestinal; in vivo; innovation; insight; interdisciplinary collaboration; interest; intestinal epithelium; intestinal homeostasis; medical schools; member; mortality; mouse model; mutant; nexin; novel; pathogen; planetary Atmosphere; polymerization; programs; protein complex; protein transport; relating to nervous system; response; trafficking

DESCRIPTION (provided by applicant): The candidate is a gastroenterologist with an extremely strong commitment to basic science research, and a specific interest in epithelial cell biology in the context of host-pathogen interactions. The candidate's long- term career goal is to become an independently funded investigator in the basic science of intestinal biology, and to make fundamental contributions to our understanding of the mechanisms that regulate mammalian epithelial barrier integrity during homeostasis and during interaction with intestinal microbes. The candidate's short-term career goals are 1) to become proficient in molecular, cellular and biochemical techniques useful for mechanistically investigating cytoskeletal dynamics both in vitro and in vivo; 2) to acquire expertise in conceptually and technologically cutting-edge approaches to experimental design that can elucidate host- pathogen interactions in novel ways; 3) to develop professional experience in fostering creative and highly interdisciplinary collaborations between experts in immunology, epithelial biology and microbiology; 4) to produce sufficient preliminary data and publications that will serve as the basis for competitive R01 funding as the cornerstone of a career in academic medical research. The overall theme of this project is to understand how factors that regulate the actin cytoskeleton influence the stability and dynamic function of intercellular junctions within the intestinal epithelium. The apical junction complex (the tight junction and adherens junction) is the major regulator of intestinal permeability and must be capable of rapidly fine-tuning its occlusiveness or leakiness. The Neural Wiskott-Aldrich Syndrome protein (N-WASP) is the canonical member of a family of actin nucleating molecules that, upon activation by a variety of host- or pathogen-encoded factors, interact with the Arp2/3 complex and promote the spatiotemporally restricted assembly of actin filaments. This process is central to diverse cellula processes, but there is growing evidence from polarized epithelial cell lines and Drosophila that N-WASP plays a central and specific role in regulating the dynamic protein trafficking events involved in intercellular junction maintenance, and along this line, there has been increasing recognition of pathogens, such as EPEC and C. rodentium, that specifically target N-WASP to modulate the actin cytoskeleton and disrupt intestinal barrier function. The specific goals of this study are to test two fundamental hypotheses: 1) N-WASP is an effector molecule that balances both the delivery and removal of AJC proteins through an interaction with the Par3/Par6/aPKC polarity complex, and 2) AE pathogens require N-WASP to induce epithelial barrier dysfunction, and the translocated effector protein EspF targets host N-WASP to enhance removal of AJC proteins during infection. The proposed work will be pursued within the context of an intensive and formalized career development program, which will allow the candidate to acquire expertise in both classic and leading edge molecular and cell biological approaches to studying intestinal biology and bacterial interactions, and will take advantage of the highly collaborative and complementary co-mentorship of Scott Snapper (immune and epithelial biology) and John Leong (microbiology). In addition, a formal research advisory committee, consisting of a diverse group of researchers at MGH and Harvard Medical School with deep expertise in immunology, gut histopathology, and junction biology, has been established and has overseen the candidate's progress since July 2010. The research environment, which includes the MGH Gastrointestinal Unit and the Harvard Digestive Diseases Center at Children's Hospital Boston, will provide an intellectually enriching, technically resourceful and collaborative atmosphere which will catalyze the candidate's scientific productivity. At the conclusion of the award period, the candidate will be well positioned for a career as an independent, R01-funded investigator.

描述（由申请人提供）：候选人是一位胃肠病学家，非常致力于基础科学研究，并且对宿主与病原体相互作用背景下的上皮细胞生物学特别感兴趣。候选人的长期职业目标是成为肠道生物学基础科学领域的独立资助研究者，并为我们理解在稳态和与肠道微生物相互作用期间调节哺乳动物上皮屏障完整性的机制做出基础贡献。候选人的短期职业目标是 1) 精通分子、细胞和生化技术，这些技术可用于机械研究体外和体内的细胞骨架动力学； 2）获得概念和技术前沿的实验设计方法方面的专业知识，能够以新颖的方式阐明宿主-病原体相互作用； 3) 积累专业经验，促进免疫学、上皮生物学和微生物学专家之间的创造性和高度跨学科合作； 4) 提供足够的初步数据和出版物，作为竞争性 R01 资助的基础，作为学术医学研究职业的基石。该项目的总体主题是了解调节肌动蛋白细胞骨架的因素如何影响肠上皮内细胞间连接的稳定性和动态功能。顶端连接复合体（紧密连接和粘附连接）是肠道通透性的主要调节器，必须能够快速微调其闭塞性或渗漏性。神经维斯科特-奥尔德里奇综合征蛋白 (N-WASP) 是肌动蛋白成核分子家族的典型成员，在被多种宿主或病原体编码因子激活后，与 Arp2/3 复合物相互作用，并促进肌动蛋白丝的时空限制组装。这一过程是多种细胞过程的核心，但来自极化上皮细胞系和果蝇的越来越多的证据表明，N-WASP 在调节涉及细胞间连接维持的动态蛋白质运输事件中发挥着核心和特定的作用，沿着这一思路，人们越来越多地认识到病原体，例如 EPEC 和啮齿类动物，它们专门针对 N-WASP 来调节肌动蛋白细胞骨架并破坏 肠道屏障功能。本次活动的具体目标 研究的目的是测试两个基本假设：1) N-WASP 是一种效应分子，通过与 Par3/Par6/aPKC 极性复合物相互作用来平衡 AJC 蛋白的递送和去除，2) AE 病原体需要 N-WASP 来诱导上皮屏障功能障碍，而易位的效应蛋白 EspF 靶向宿主 N-WASP，以增强 AJC 蛋白的去除。 感染。拟议的工作将在密集和正式的职业发展计划的背景下进行，这将使候选人能够获得经典和前沿分子和细胞生物学方法的专业知识，以研究肠道生物学和细菌相互作用，并将利用 Scott Snapper（免疫和上皮生物学）和 John Leong（微生物学）的高度协作和互补的共同指导。此外，还成立了一个正式的研究咨询委员会，该委员会由 MGH 和哈佛医学院的多位研究人员组成，他们在免疫学、肠道组织病理学和连接生物学方面拥有深厚的专业知识，自 2010 年 7 月以来一直监督候选人的进展。研究环境包括 MGH 胃肠病科和波士顿儿童医院的哈佛消化疾病中心，将提供一个智力丰富、技术丰富、资源丰富的研究环境。 协作氛围将促进候选人的科学生产力。奖励期结束时， 该候选人将成为一名由 R01 资助的独立调查员。