Rap1 - Krit1 Regulation of Endothelial Permeability

描述(由申请人提供)： 项目概述：应聘者的直接职业计划是通过几年的指导项目工作提升到助理项目科学家，以获得转基因小鼠方法学/遗传学方面的技能。长期目标：获得独立的RO1基金和心血管生物学独立研究的教员职位。这项提议旨在确定促进内皮屏障完整性的信号分子。内皮细胞-细胞连接的破坏加上收缩应力纤维的形成增加了血管的通透性。血管内皮细胞屏障功能失调在心血管病理中导致血管渗漏。Rap1 GTP酶稳定内皮细胞连接。KRIT1和CCM2突变会导致“脑海绵状畸形”疾病，其特征是血管内皮细胞异常。初步研究表明，KRIT1是一种具有β-整合素结合位点的RAP1效应器，并证实KRIT1和CCM2靶向于内皮连接，与稳定皮质肌动蛋白和降低通透性有关。假设：KRIT1是一个RAP1效应器，募集ICAP1调节β-整合素和/或CCM2，通过Rho-GTP酶调节f-肌动蛋白介导的屏障功能。具体目的是：1)检测KRIT1的定位和功能是否受ICAP1和CCM2结合的影响。2)评价KRIT1 PERM结构域与β-整合素在调节连接稳定性中的相互作用。3)研究Rac-和Rho-GTPase与KRIT1在调节与屏障功能相关的肌动蛋白细胞骨架中的相互作用。培养细胞中的分子相互作用将通过体外蛋白结合分析和免疫共沉淀，使用siRNA耗尽和突变结合结构域蛋白的异位表达来测试。渗透性将通过使用β-整合素、KRIT1和CCM2突变小鼠的肺血管渗漏分析在体内进行测试；并在体外使用Tran Swell过滤器中的动物培养的内皮细胞进行测试。分子定位将使用共聚焦免疫荧光显微镜成像，加上实时显微成像，以可视化标记的异位蛋白和荧光Rho-GTP酶生物传感器的动态定位。相关性：这个项目将增加对血管通透性，特别是CCM疾病的了解。它将有可能识别用于病理性渗漏的药物治疗和CCM2疾病的基因治疗的靶分子。(摘要结束)

DESCRIPTION (provided by applicant): PROJECT SUMMARY: Candidate's immediate career plans are advancement to Asst Project Scientist with several years' mentored project work to gain skills in transgenic mouse methodology/genetics. Long-term goal: acquire independent RO1 funding and a faculty position with independent research in cardiovascular biology. This proposal aims to identify signaling molecules promoting endothelial barrier integrity. Endothelial cell-cell junction disruption plus contractile stress fiber formation increase vessel permeability. Dysregulation of endothelial barrier function contributes to vessel leak in cardiovascular pathology. Rap1 GTPase stabilizes endothelial junctions. KRIT1 and CCM2 mutations produce "Cerebral Cavernous Malformations" disease characterized by vascular lesions with abnormal endothelium. Preliminary work identified KRIT1 as a Rap1 effectors having beta-integrin binding site, and demonstrated KRIT1 and CCM2 targeting to endothelial junctions that was associated with stabilized cortical actin and decreased permeability. HYPOTHESIS: KRIT1 is a Rap1 effector recruiting ICAP1 modulating beta-integrins and/or CCM2, regulating f-actin mediated barrier function through Rho-GTPases. SPECIFIC AIMS are to: 1) test whether KRIT1 localization and function are mediated by ICAP1 and CCM2 binding. 2) Evaluate KRIT1 PERM domain interactions with beta-integrins in regulating junctional stability. 3) Examine Rac- and Rho-GTPase interactions with KRIT1 in modulating actin cytoskeleton associated with barrier function. Molecular interactions in cultured cells will be tested with in vitro protein binding assays and co-immunoprecipitations, using siRNA depletion and ectopic expression of mutated binding domain proteins. Permeability will be tested in vivo with pulmonary vascular leak assays using beta-integrin, KRIT1, and CCM2 mutant mice; and in vitro using the animal's cultured endothelial cells in Tran swell filters. Molecular localization will be imaged using confocal immunofluorescence microscopy, plus real-time micro imaging to visualize kinetic localization of labeled ectopic proteins and fluorescent Rho-GTPase biosensors. RELEVANCE: This project will increase understanding of vascular permeability in general and CCM disease in particular. It will potentially identify target molecules for pharmacological treatment of pathological leak, and genetic treatment of CCM2 disease. (End of Abstract)