Endothelial Cell Cytoskeletal Regulation by Cortactin

描述(由申请人提供)： 此K08应用程序的目标是为PI提供以下基本技能 成为一名成功的院士，实现自主科研 调查员身份。PI将专注于内皮，一种功能上的 动态细胞单层在血管生成和血管生成中发挥关键作用 作为血管系统和周围组织之间的半选择性屏障。 尽管它在急性肺损伤综合征中很重要，但细胞调节 内皮屏障功能，特别是屏障修复和 增强，人们对此知之甚少。血管内皮细胞(EC)研究进展 证明了该酶对肌球蛋白轻链(MLC)的磷酸化 肌球蛋白轻链激酶(MLCK)在血管诱导中起关键作用 在某些模型中的渗透率。我们实验室的研究表明，EC 表达一种新型的高分子量MLCK亚型，当酪氨酸 在与肌动蛋白结合蛋白的稳定复合体中发现了磷酸化， Cortactin，一种肌动蛋白结合蛋白，参与多个方面的 细胞骨架的组织和重排，包括Arp2/3介导的肌动蛋白 聚合反应。我们假设Cortactin扮演着一个积极的和关键的角色 屏障增强过程中内皮细胞骨架重排的作用 和迁徙。SA#1将研究皮质素在培养的肺组织中的作用 利用分子生物学技术实现EC屏障功能和迁移。SA#2 将探讨皮质肌动蛋白磷酸化在肺内皮细胞中的作用 细胞骨架调节。SA#3将描述Cortactin-EC MLCK的作用 肺内皮细胞骨架重排中的相互作用 生化、免疫组织化学和分子生物学技术。最后， SA#4将研究生物物理切应力和循环的影响 拉伸对肺内皮细胞角质形成蛋白生物学及调控的影响。穿过 这些互补的技术和课程，PI将发展新的技能 并生成关于EC屏障恢复/增强的新数据，以及 血管生成，可能会导致毁灭性的 肺水肿的后果。

DESCRIPTION (provided by applicant): The goal of this K08 application is to provide the PI with essential skills in which to be a successful academician and achieve independent scientific investigator status. The PI will focus upon the endothelium, a functionally dynamic cellular monolayer which performs critical roles in angiogenesis and as a semi-selective barrier between the vasculature and surrounding tissues. Despite its importance in acute lung injury syndromes, the cellular regulation of endothelial barrier function, especially barrier restoration and enhancement, is poorly understood. Recent work in endothelial cells (EC) demonstrates that phosphorylation of myosin light chains (MLC) by the enzyme myosin light chain kinase (MLCK) is critical to induction of vascular permeability in some models. Studies in our laboratory have revealed that EC express a novel, high molecular weight MLCK isoform that when tyrosine phosphorylated is found in stable complex with the actin-binding protein, cortactin, an actin-binding protein implicated in multiple aspects of cytoskeletal organization and rearrangement including Arp2/3-mediated actin polymerization. We hypothesize that cortactin plays an active and critical role in endothelial cell cytoskeletal rearrangement during barrier enhancement and migration. SA#1 will examine the role of cortactin in cultured pulmonary EC barrier function and migration using molecular biology techniques. SA#2 will explore the role of cortactin phosphorylation in pulmonary EC cytoskeletal regulation. SA#3 will characterize the role of cortactin-EC MLCK interaction in pulmonary endothelial cytoskeletal rearrangement through biochemical, immunohistochemical, and molecular biology techniques. Finally, SA#4 will investigate the effect of biophysical shear stress and cyclical stretch on pulmonary endothelial cortactin biology and regulation. Through these complementary techniques and coursework, the PI will develop new skills and generate novel data regarding EC barrier restoration/enhancement, and angiogenesis which may potentially lead to new therapies for the devastating consequences of pulmonary edema.