Role of Nucleo-cytoskeleton Interactions in Cell Migration

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。细胞核在几乎所有的动物细胞中都是通过激活机制定位的，并占据特定的位置，反映出它们所在的细胞类型的活动。细胞核和细胞骨架之间的连接是由外核膜Nesprin蛋白和内核膜SUN蛋白介导的，Nesprin蛋白直接与细胞骨架元素相互作用，SUN蛋白锚定Nesprin蛋白。在我们实验室的早期工作中，我们发现在成纤维细胞极化过程中，细胞核被主动向后移动，以便迁移到体外伤口中，这种移动涉及到沿着移动的肌动蛋白细丝与细胞核相遇的新的线性阵列。这些排列，我们称之为跨膜肌动蛋白相关核线的坦尼线，将细胞核与移动的肌动蛋白细丝捆绑在一起，并通过干扰坦恩线的治疗来防止核移动、中心体定向和抑制细胞向体外伤口的迁移。为了更多地了解这种新的膜结构及其在细胞迁移中定位细胞核的作用，我们建议进一步开发损伤的成纤维细胞单层系统，以测试核定位的亚细胞机制以及核定位在细胞迁移中的作用。在第一个目标中，我们将通过活细胞成像实验来研究TAN线组装的动力学，利用电子显微镜和超分辨光学显微镜方法了解更多关于TAN线结构的信息，并通过筛选可能有助于这种结构在核膜上组装和/或锚定的额外蛋白质来研究TAN线的组成。我们还将通过测试它们在不同环境中迁移的细胞的核定位中的作用来检验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。我们将继续进行初步研究，以确定一组单独的核膜蛋白似乎作为肌球蛋白激活的核组织者，当被破坏时，导致细胞内肌动蛋白流动的方向性改变和细胞核移动到偏心位置。将对该系统的组件进行检查，以了解核表面的肌球蛋白激活事件以及这种激活向外扩散的方式，以确保迁移细胞中肌动蛋白流动的方向性。通过进行这些研究，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。由于参与核定位通路的编码蛋白的基因突变会导致许多人类疾病，包括肌肉营养不良、心肌病、小脑性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。

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.