Mechanoregulation of the Leukocyte Specific Integrin LFA-1 by the Actin Cytoskeleton

项目摘要 将循环白细胞募集到感染部位在机体防御感染中起重要作用。 病原体白细胞从血液到周围组织的动员发生在多步粘附中 级联反应是由几个粘附分子家族介导的。整合素淋巴细胞功能- 相关1（LFA 1）通过结合表达的细胞内粘附分子（ICAM）介导牢固的粘附 并最终导致通过血管壁的迁移。申请人博士 特拉维斯摩尔在蒂莫西·斯普林格博士的实验室里进行了这方面的重要研究。目标 这项应用的目的是加深摩尔博士在这一重要研究领域的专业知识和发现， 发展成为一个重要的独立研究项目。摩尔博士最近领导的研究表明， 整合素连接到肌动蛋白细胞骨架上，并沿肌动蛋白逆行方向产生沿着张力 流动导致LFA 1在细胞表面上的取向。LFA 1的这种排列依赖于底物 并且与整联蛋白在与肌动蛋白逆行流动相同的方向上倾斜一致。这些发现强烈 支持肌动蛋白细胞骨架在细胞骨架调节中的作用。摩尔医生将测试 假设肌动蛋白细胞骨架调节整合素构象平衡和亲和力， 机械力作用于整联蛋白胞质结构域，并作为变构效应物稳定整联蛋白胞质结构域。 高亲和力、扩展开放整合素构象。这项工作将利用创新的方法来衡量 LFA 1的内在亲和力和构象平衡，并使用最先进的超分辨率 用显微镜定量肌动蛋白细胞骨架与整联蛋白胞质尾区的连接，并直接测量 白细胞细胞表面的整合素构象。这些实验将促进我们对 细胞增殖和迁移调节产生了用于未来的新的和创新的方法 研究，并扩大摩尔博士的蛋白质化学和纯化的专业知识， 超分辨率显微镜和计算图像分析，基因组修饰和定量 细胞表面的受体热力学摩尔博士将把100%的时间投入到这个职业生涯中 发展奖与博士蒂莫西斯普林格作为他的导师在波士顿儿童医院。

PROJECT SUMMARY Recruitment of circulating leukocytes to sites of infection plays an important role in the body's defense against pathogens. Mobilization of leukocytes from the blood to the surrounding tissue occurs in a multistep adhesion cascade, which is mediated by several adhesion molecule families. The integrin lymphocyte function- associated 1 (LFA1) mediates firm attachment by binding Intracellular Adhesion Molecules (ICAMs) expressed by the vascular endothlilium and ultimately leads to transmigration through the vessel wall. The applicant, Dr. Travis Moore has preformed important studies in this area in the laboratory of Dr. Timothy Springer. The goal of this application is to deepen Dr. Moore's expertise and findings in this important research area so that it can be developed into a significant independent research program. Dr. Moore recently lead research to show that integrin linkage to the actin cytoskeleton and the tensile force generated along the direction of retrograde actin flow resulted in the orientation of LFA1 on the cell surface. This alignment of LFA1 was substrate dependent and consistent with integrins being tilted in the same direction as retrograde actin flow. These findings strongly support a role for the actin cytoskeleton in the regulation of cellular adhesiveness. Dr. Moore will test the hypothesis that the actin cytoskeleton regulates integrin conformational equilibria and affinity through mechanical force applied to the integrin cytoplasmic domain and acts as an allosteric effector to stabilize the high-affinity, extended-open integrin conformation. This work will utilize innovative methodologies to measure the intrinsic affinities and conformational equilibria of LFA1 and use state-of-the-art super-resolution microscopy to quantitate the actin cytoskeleton linkage to the integrin cytoplasmic tail and directly measure integrin conformation on the cell surface of leukocytes. These experiments will advance our understanding of the regulation of cell adhesiveness and migration, produce novel and innovative methodologies for future research, and expand Dr. Moore's diverse background with expertise in protein chemistry and purification, super-resolution microscopy and computational image analysis, genome modification, and quantitating receptor thermodynamics on the cell surface. Dr. Moore will devote 100 % of his time to this Career Development Award with Dr. Timothy Springer as his mentor at Boston Children's Hospital.