Nuclear mechanobiology in confined migration

项目摘要 细胞迁移对许多生理过程是必不可少的，包括发育、组织动态平衡、 和伤口愈合。同时，细胞迁移使肿瘤细胞能够侵入其他组织，进入/退出 循环，并扩散到体内较远的部位，形成转移。虽然这些事实促使研究 关于几十年来的细胞迁移，最近才受到关注的一个方面是物理挑战 细胞在三维(3D)环境中迁移时面临的问题，以及由此对细胞 结构和功能。在组织中，细胞经常在需要大量变形的狭窄空间中移动。 细胞核是最大、最坚硬的细胞器。相关的机械应力可能会导致 核膜破裂、DNA损伤和基因组结构改变。然而，许多问题， 仍然存在，包括潜在的分子机制，功能后果，以及 不同的细胞系。这项建议的中心目标是确定染色质的特征变化 与受限迁移相关的组织，确定负责 机械诱导的染色质组织改变和DNA损伤，并评估 这些事件的功能后果。为了实现这一目标，我们开发了新颖的实验 能够在精确定义的微环境中迁移的细胞的扩展活细胞成像的平台 同时可视化核变形、核膜破裂、DNA损伤和染色质修饰。 这些平台将与分子生物学方法和分析配对，用于全基因组分析 一组特征良好的细胞系中3D染色质组织和基因表达的变化 既代表致癌细胞又代表非致瘤细胞。在第一个目标中，我们将确定由 染色质组织和基因表达的变化，决定了导致 改变的染色质组织，并评估改变的染色质组织的功能后果。 在第二个目标中，我们将确定在受限迁移和 确定迁移诱导的DNA损伤对细胞活力、细胞周期进程和衰老的影响。 我们将重点研究染色质结构改变和DNA损伤所导致的最早事件， 我们预计，与长期效应相比，它在多种细胞类型中表现出的差异更小。我们的最终目标是 揭示核机械生物学的一般原理，这将有助于更好地理解 在狭窄空间中迁移对细胞功能和基因组稳定性的影响，包括激活或 抑制特定的转录程序，可能进一步增强细胞迁移或调节其他 细胞功能。从这些研究中获得的见解可能有助于指导各种临床治疗方法 从伤口愈合和免疫反应到针对转移的肿瘤细胞的治疗。

Project Summary Cell migration is essential for numerous physiological processes, including development, tissue homeostasis, and wound healing. At the same time, cell migration enables tumor cells to invade other tissues, enter/exit the circulation, and spread to distant sites in the body to form metastases. While these facts have motivated research on cell migration for many decades, one aspect that has only recently received attention is the physical challenge that cells face during migration in three-dimensional (3D) environments, and the resulting impact on cellular structure and function. In tissues, cells frequently move through tight spaces that require substantial deformation of the cell nucleus, which is the largest and stiffest organelle. The associated mechanical stress can result in nuclear envelope rupture, DNA damage, and changes in genomic organization. Many questions, however, remain, including the underlying molecular mechanisms, the functional consequences, and the variability across different cell lines. The central goal of this proposal is to identify the characteristic changes in chromatin organization associated with confined migration, determine the molecular mechanisms responsible for the mechanically-induced changes in chromatin organization and DNA damage, and assess the functional consequences of these events. To achieve this goal, we have developed novel experimental platforms that enable extended live-cell imaging of cells migrating through precisely-defined microenvironments while visualizing nuclear deformation, nuclear envelope rupture, DNA damage, and chromatin modifications. These platforms will be paired with molecular biology approaches and assays for genome-wide analysis of changes in 3D chromatin organization and gene expression in a panel of well-characterized cell lines representing both tumorigenic and non-tumorigenic cells. In the first aim, we will identify migration-induced changes in chromatin organization and gene expression, determine the molecular mechanisms responsible for altered chromatin organization, and assess the functional consequences of the altered chromatin organization. In the second aim, we will identify the molecular mechanisms for DNA damage during confined migration and determine the impact of migration-induced DNA damage on cell viability, cell cycle progression, and senescence. We will focus our studies on the earliest events resulting from altered chromatin organization and DNA damage, which we expect to exhibit less variation across multiple cell types than longer-term effects. Our ultimate goal is to uncover general principles in nuclear mechanobiology that will lead to an improved understanding of the impact of migration through tight spaces on cellular function and genomic stability, including the activation or suppression of specific transcriptional programs that may further enhance cell migration or modulate other cellular functions. Insights gained from these studies may help guide therapeutic approach for a variety of clinical conditions, from wound healing and immune-responses to therapies targeting metastatic tumor cells.