Nuclear mechanobiology in confined migration

项目摘要 细胞迁移对于许多生理过程是必不可少的，包括发育、组织稳态， 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/离开肿瘤细胞，并使肿瘤细胞能够进入或离开肿瘤细胞。 循环，并扩散到体内的远端部位以形成转移。虽然这些事实激发了研究 几十年来，细胞迁移的一个方面，直到最近才受到关注的是物理挑战 细胞在三维（3D）环境中迁移过程中所面临的问题，以及由此产生的对细胞的影响， 结构和功能。在组织中，细胞经常在需要大量变形的紧密空间中移动 细胞核是最大最硬的细胞器相关的机械应力可导致 核膜破裂、DNA损伤和基因组组织的变化。然而，许多问题， 仍然存在，包括潜在的分子机制，功能后果，以及跨 不同的细胞系。这项建议的中心目标是确定染色质的特征性变化 组织与有限的迁移，确定分子机制负责 机械诱导的染色质组织和DNA损伤的变化，并评估 这些事件的后果。为了实现这一目标，我们开发了新的实验 能够对通过精确定义的微环境迁移的细胞进行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检测方法配对，用于全基因组分析， 一组充分表征的细胞系中3D染色质组织和基因表达的变化 代表致瘤性和非致瘤性细胞。在第一个目标中，我们将识别迁移引起的 染色质组织和基因表达的变化，决定了 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DNA损伤的分子机制，在有限的迁移， 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。 我们将把研究重点放在染色质组织改变和DNA损伤引起的最早期事件上， 我们希望在多种细胞类型中表现出比长期效应更少的变化。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 通过紧密空间的迁移对细胞功能和基因组稳定性的影响，包括激活或 抑制可进一步增强细胞迁移或调节其它细胞因子的特异性转录程序 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靶向转移性肿瘤细胞的治疗，

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.