Biophysics of Nuclear Formation and Micronucleation

项目摘要/摘要 高等真核细胞的细胞核经历了一个动态的拆解和重组过程 在开放的有丝分裂过程中。不能改造包住整组染色体的单个核 导致小的核外小体，称为微核。微核易发生不可逆转的破裂 和灾难性的DNA损伤，这在很大程度上与癌症有关。最近的研究表明 这种屏障自整合因子(BAF/BANF1)将DNA与单一物质交联，从而引导核 跨越染色体的膜。然而，需要进一步的调查才能了解详细的情况 曝气生物滤池介导核膜的生物物理作用机制。以前的研究和 初步数据导致了这项研究的假设，BAF可能形成纤维样结构，而不仅仅是简单的 二聚体，连接和国会遥远的染色体。拟议研究的目标就是调查这一点。 核包膜形成中的可能性及其意义。为此，第一个目标是开发一种 利用非洲爪哇卵提取液和微图案DNA观察核桥接的生物物理方法 并定量地剖析了BAF复合体在这一过程中的作用 进程。在下一个目标中，将对生物滤池在核形成过程中的纤维状结构的三维动力学进行研究 使用晶格光片显微镜和分子微扰技术在活细胞中进行分析。最后， BAF在提取系统和活细胞中的DNA结合和聚合动力学将被揭示 重组在一个最简单的纯化成分系统中，以进一步研究生物物理基础。 因此，这项拟议的研究将为核形成和微核提供机制上的洞察 并为这些过程的数学建模奠定了基础。此外，结果还具有 有可能扩大我们对癌症微核的理解，从而有助于开发新的 癌症治疗和诊断学。进行这项拟议的研究，申请人的目标是加深他在 数量生物学和显微镜，同时扩展了他的技能和智力的广度 到重组的无细胞系统和翻译研究的地平线。这一项目将受益于 哈佛医学院系统生物学系的跨学科研究环境 在赞助商的大力支持下。

Project Summary/Abstract The cell nucleus of higher eukaryotic cells undergoes a dynamic process of disassembly and reassembly during the open mitosis. Failing to reform a single nucleus encasing the entire set of chromosomes often results in small extranuclear bodies, referred to as micronuclei. Micronuclei are prone to irreversible rupture and catastrophic DNA damages, which has been largely implicated in cancer. Recent works have suggested that barrier-to-autointegration factor (BAF/BANF1) crosslinks DNA to a single mass, thereby guiding nuclear membranes to bridge across chromosomes. However, further investigation is necessary to understand detailed biophysical mechanism of action by which BAF mediates nuclear membranes. Previous studies and preliminary data led to the hypothesis of this study that BAF may form fiber-like structures, beyond simple dimers, that connect and congress distant chromosomes. The goal of the proposed study is to investigate this possibility and its implications in nuclear envelope formation. To this end, the first aim is to develop a biophysical assay using Xenopus egg extract and micro-patterned DNA to scrutinize the bridging of nuclear envelope across gaps between chromosomes, and quantitatively dissect the role of BAF complex during this process. In the next aim, 3D dynamics of the fiber-like structures of BAF during nuclear formation will be analyzed in living cells, using lattice light-sheet microscopy and molecular perturbation techniques. Finally, DNA binding and polymerization dynamics of BAF revealed in the extract system and living cells will be reconstituted in a simplest system of purified components to further investigate the biophysical basis. Consequently, the proposed study will offer the mechanistic insight into nuclear formation and micronucleation and establish the foundation for mathematical modeling of these processes. Furthermore, the results have the potential to expand our understanding of cancer micronuclei, thereby aiding in the development of novel cancer therapy and diagnostics. Conducting the proposed study, the applicant aims to deepen his expertise in quantitative biology and microscopy, while at the same time, expanding the breadth of his skills and intellectual horizon to reconstituted cell-free systems and translational research. This project will benefit from the highly interdisciplinary research environment of the Systems Biology department at Harvard Medical School along with the sponsor’s strong support.