Cytoskeletal regulation of intestinal barrier function and response to pathogens

应聘者描述(由申请人提供)：应聘者是一名胃肠病专家，对基础科学研究有极强的承诺，对宿主-病原体相互作用背景下的上皮细胞生物学有特殊兴趣。候选人的长期职业目标是成为一名独立资助的肠道生物学基础科学研究人员，并为我们理解哺乳动物在体内平衡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性贡献。应聘者的短期职业目标是：1)精通分子、细胞和生化技术，用于在体外和体内对细胞骨架动力学进行机械研究；2)在概念上和技术上获得前沿实验设计方法的专业知识，能够以新颖的方式阐明宿主与病原体的相互作用；3)在促进免疫学、上皮生物学和微生物学专家之间创造性和高度跨学科合作方面发展专业经验；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生涯的基石。这个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(紧密连接和粘连连接)是肠道通透性的主要调节因素，必须能够快速微调其闭塞或渗漏。神经性Wiskott-Aldrich综合征蛋白(N-WASP)是肌动蛋白成核分子家族中的典型成员，被多种宿主或病原体编码的因子激活后，与Arp2/3复合体相互作用，促进肌动蛋白细丝的时空受限组装。这一过程是多种细胞过程的中心，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在调节细胞间连接维持所涉及的动态蛋白质运输事件中发挥着核心和特定的作用，而且沿着这一过程，人们越来越多地认识到病原体，如EPEC和轮状芽孢杆菌，特异性地针对N-WASP来调节肌动蛋白细胞骨架并破坏肠道屏障功能。这一行动的具体目标 研究旨在验证两个基本假设：1)N-WASP是一个效应分子，通过与Par3/Par6/aPKC极性复合体的相互作用来平衡AJC蛋白的输送和清除；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以宿主N-WASP为靶点，以促进感染过程中AJC蛋白的清除。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，该计划将使应聘者获得研究肠道生物学和细菌相互作用的经典和前沿分子和细胞生物学方法方面的专业知识，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，一个正式的研究咨询委员会已经成立，该委员会由麻省理工学院和哈佛医学院的不同研究人员组成，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。研究环境包括波士顿儿童医院的MGH胃肠科和哈佛消化疾病中心，将提供一个智力丰富、技术资源丰富和合作的氛围，将催化候选人的科学生产力。在授权期结束时， 应聘者将成为一名独立的、由R01资助的调查员。

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.