Analyzing the role of chromatin compaction in nuclear mechanics, structure, and function

细胞核是细胞器，它必须适当地转换或抵抗生物物理力来支配 基因组的空间组织和控制机械转导，这些因素决定了 细胞的表达配置文件。核力学的两个主要贡献者是板层，中间层 内侧核膜上排列着细丝，核内充满染色质。层板和板条的更改 染色质压缩发生在人类许多重大疾病和健康细胞分化过程中。在这两个地方 病例的核、细胞和组织的力学和形态可能会发生巨大的变化。目前， 以疾病为基础的核泡和基于健康分化的改变的机制基础 原子核的形态和力学尚不清楚。我的研究发现染色质及其组蛋白是由 压实状态和交联度决定了初始力响应(30%应变)和形态，同时还 作为层蛋白A的次要因素，决定了应变在较长变形时变硬。我第一个 建议使用我开发的显微解剖、微操作和纳米级的力 进一步阐明核力学在基因组组织中的作用的测量方法。在.期间 核拉伸实验我将确定染色质如何通过 成像单个染色体位点(CRISPR标记)和整体染色质纳米结构。第二，我会 研究核泡化和破裂的疾病相关表型对功能的影响 可由以染色质为基础的核机制引起或抑制。我会确定核气泡是否 通过活细胞成像和生化技术检测系统性疾病的症状或原因 DNA损伤，正确的转录，以及基因组内容在水泡中的忠实分离。最后，我会 利用成熟的角质形成细胞原代细胞模型研究核形态的基础 分化过程中的变化，前体到末端，以及RAS激活到模拟时的动态平衡丧失 癌症过渡。总体而言，我的目标是发展一个独立的职业生涯，研究 在疾病和健康细胞分化中观察了70多年的形态变化。

The nucleus is the organelle which must properly transduce or resist biophysical forces to dictate the spatial organization of the genome and to control mechanotransduction, factors which determine the expression profile of the cell. The two major contributors to nuclear mechanics are lamins, intermediate filaments lining the inner nuclear envelope, and chromatin, which fills the nucleus. Alteration of lamins and chromatin compaction occur in many major human diseases and during healthy cell differentiation. In both cases nuclear, cell, and tissue mechanics and morphology can change drastically. Currently, the mechanistic basis for both disease-based nuclear blebs and healthy differentiation-based changes in nuclear morphology and mechanics is unknown. My studies found that chromatin and its histone-mediated compaction state and cross-linking dictated initial force response (< 30% strain) and morphology while also contributing as a secondary factor to the lamin A dictated strain stiffening at longer deformations. I first propose to use my developed microdissection, micromanipulation, and nanonewton-level force measurement approach to further elucidate the role of nuclear mechanics in genome organization. During nuclear stretching experiments I will determine how the chromatin responds to nuclear deformation through imaging single chromosome loci (CRISPR labeling) and overall chromatin nano-structure. Second, I will investigate the functional impact of the disease-relevant phenotype of nuclear blebbing and rupture that can be caused or suppressed by chromatin-based nuclear mechanics. I will determine if nuclear blebs are a symptom or a cause of disease, via live cell imaging and biochemical techniques to assay for systemic DNA damage, proper transcription, and faithful segregation of genomic content in the bleb. Finally, I will use the well-established primary cell model of keratinocytes to investigate the basis of nuclear morphology changes during differentiation, progenitor to terminal, and loss of homeostasis upon Ras activation to mimic cancer transition. Overall, I aim to develop an independent career investigating the mechanical basis of morphology changes observed for more than 70 years in both disease and in healthy cell differentiation.