Role of Nucleo-cytoskeleton Interactions in Cell Migration

核细胞骨架相互作用在细胞迁移中的作用

• 批准号: 8730189
• 负责人: Gregg G Gundersen
• 金额: $ 36万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8730189
• 关键词: Actins; Affect; Animal Model; Animals; Area; Cardiomyopathies; Cell Nucleus; Cell Size; Cell physiology; Cells; Cellular biology; Centrosome; Cerebellar Ataxia; Coupling; Cytoskeleton; Disease; Dynein ATPase; Electron Microscopy; Elements; Ensure; Environment; Event; Fibroblasts; Focal Adhesions; Functional disorder; Genes; Genetic; Goals; Human; Image; In Vitro; Integrins; Laboratories; Lead; Learning; Life; Link; Location; Mediating; Membrane; Membrane Proteins; Microfilaments; Microtubules; Molecular; Movement; Muscular Dystrophies; Mutation; Myosin ATPase; Myosin Type II; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Outer Membrane; Organelles; Pathway interactions; Phenotype; Physiological; Play; Positioning Attribute; Protein Array; Proteins; Reading; Recycling; Resolution; Role; Signal Transduction; Site; Structure; Surface; System; Testing; Work; cell behavior; cell motility; cell type; cellular imaging; emerin; human disease; light microscopy; lissencephaly; migration; monolayer; novel; prevent; research study; response; screening; transmission process

DESCRIPTION (provided by applicant): The overall goal of this project is to understand how nucleo-cytoskeletal interactions position nuclei in migrating cells and how the positioning of nuclei affects cell migration. Nuclei are positioned by active mechanisms in virtually all animal cells and occupy specific positions that reflect the activity of the cell type they reside in. Connections between the nucleus and the cytoskeleton are mediated by outer nuclear membrane nesprin proteins, which interact directly with cytoskeletal elements, and SUN proteins in the inner nuclear membrane that anchor the nesprins. In earlier work from our laboratory, we found that the nucleus is actively moved rearward during polarization of fibroblasts for migration into in vitro wounds and that this movement involved the assembly of nesprin2 and SUN2 into novel linear arrays along moving actin filaments that encountered the nucleus. These arrays, which we termed TAN lines for transmembrane actin-associated nuclear lines, tether the nucleus to moving actin filaments and treatments that disrupt TAN lines prevent nuclear movement, centrosome orientation and inhibit migration of cells into in vitro wounds. To understand more about this novel membrane structure and its role in positioning the nucleus for cell migration, we propose to further develop the wounded fibroblast monolayer system to test subcellular mechanisms of nuclear positioning and the role of nuclear positioning in cell migration. In the first aim, we will examine the dynamics of TAN line assembly with live cell imaging experiments, learn more about the structure of TAN lines using electron microscopy and super resolution light microscopy approaches, and study the composition of TAN lines by screening for additional proteins that may contribute to the assembly and/or anchoring of this structure in the nuclear membrane. We will also examine the broader significance of TAN lines for cell migration by testing their role in nuclear positioning in cells migrating in different environments and test the specific hypothesis that TAN lines may be necessary for cells to migrate through small spaces. In the second aim, studies will focus on the question of how nuclear positioning is "read out" by migrating cells. We will pursue preliminary studies that have identified a separate set of nuclear membrane proteins that appears to act as a nuclear organizer for myosin activation and that when disrupted, results in altered directionality of actin flow in cells and movement of nuclei to eccentric positions. The components of this system will be examined to understand the myosin activation events at the nuclear surface and the manner in which this activation spreads outward to ensure directionality of actin flow in migrating cells. By pursuing these studies, we will learn more about the basic mechanisms by which the nucleus interacts with the cytoskeleton and how this contributes to the polarization of migrating cells. As mutations in genes encoding proteins involved in nuclear positioning pathways cause a number of human diseases, including muscular dystrophies, cardiomyopathies, cerebellar ataxia and lissencephaly, these studies will also provide new avenues for understanding human disease and developing strategies for their treatment.

描述（由申请人提供）：本项目的总体目标是了解核-细胞骨架相互作用如何在迁移细胞中定位核，以及核的定位如何影响细胞迁移。在几乎所有的动物细胞中，细胞核都是通过活性机制定位的，并占据反映其所在细胞类型活性的特定位置。细胞核和细胞骨架之间的连接是由外核膜nesprin蛋白介导的，其直接与细胞骨架元件相互作用，以及内核膜中的SUN蛋白，其锚nesprin。在我们实验室的早期工作中，我们发现在成纤维细胞迁移到体外伤口的极化过程中，细胞核主动向后移动，并且这种移动涉及nesprin 2和SUN 2沿着沿着移动的肌动蛋白丝组装成新颖的线性阵列，这些肌动蛋白丝遇到细胞核。这些阵列，我们称之为跨膜肌动蛋白相关核线的TAN线，将细胞核束缚在移动的肌动蛋白丝上，破坏TAN线的处理阻止细胞核移动、中心体定向并抑制细胞迁移到体外伤口中。为了更好地了解这种新型膜结构及其在定位细胞迁移的细胞核中的作用，我们建议进一步开发受伤的成纤维细胞单层系统，以测试细胞核定位的亚细胞机制和细胞迁移中细胞核定位的作用。在第一个目标中，我们将通过活细胞成像实验研究TAN线组装的动力学，使用电子显微镜和超分辨率光学显微镜方法了解更多关于TAN线的结构，并通过筛选可能有助于组装和/或锚定这种结构的其他蛋白质来研究TAN线的组成。我们还将通过测试TAN系在不同环境中迁移的细胞核定位中的作用来研究TAN系对细胞迁移的更广泛意义，并测试TAN系可能是细胞通过小空间迁移所必需的特定假设。在第二个目标中，研究将集中在迁移细胞如何“读出”核定位的问题上。我们将进行初步研究，已经确定了一个单独的一组核膜蛋白，似乎作为一个核组织者的肌球蛋白激活，当中断，结果在细胞中的肌动蛋白流动的方向性改变和运动的核偏心位置。该系统的组件将被检查，以了解在核表面的肌球蛋白激活事件和这种激活向外传播的方式，以确保迁移细胞中的肌动蛋白流的方向性。通过这些研究，我们将更多地了解细胞核与细胞骨架相互作用的基本机制，以及这如何有助于迁移细胞的极化。由于编码参与核定位途径的蛋白质的基因突变导致许多人类疾病，包括肌营养不良症、心肌病、小脑共济失调和无脑畸形，这些研究也将为理解人类疾病和开发治疗策略提供新的途径。