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

项目总结 循环中的白细胞募集到感染部位在机体防御疾病中起着重要作用。 病原体。白细胞从血液到周围组织的动员发生在一个多步骤的黏附过程中。 级联，这是由几个黏附分子家族介导的。整合素淋巴细胞功能- 相关蛋白1(LFA1)通过结合表达的细胞内黏附分子(ICAM)介导牢固的附着 通过血管内皮细胞，并最终导致轮回通过血管壁。申请者是Dr。 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。目标是 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，以便它能够 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， 整合素与肌动蛋白细胞骨架的连接以及沿肌动蛋白逆行方向产生的张力 流动导致LFA1在细胞表面定向。LFA1的这种对齐依赖于底物 并与整合素与肌动蛋白逆行流向相同的方向倾斜一致。这些发现有力地证明了 支持肌动蛋白细胞骨架在细胞黏附调节中的作用。摩尔博士将测试 肌动蛋白细胞骨架通过调节整合素构象平衡和亲和力的假说 作用于整合素细胞质结构域的机械力，并作为变构效应来稳定 高亲和力、延长开放的整合素构象。这项工作将利用创新的方法来衡量 LFA1的内在亲和力和构象平衡以及使用最先进的超分辨 显微技术定量肌动蛋白细胞骨架与整合素胞浆尾部的连接并直接测量 白细胞细胞表面的整合素构象。这些实验将增进我们对 对细胞黏附和迁移的调节，为未来产生了新的和创新的方法 研究并扩展摩尔博士在蛋白质化学和纯化方面的专业知识， 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%的时间投入到这一事业中 在波士顿儿童医院，蒂莫西·斯普林格博士作为他的导师，获得了发展奖。

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.