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

细胞核是一种细胞器，它必须适当地抵抗或抵抗生物物理力量，以决定细胞的功能。 基因组的空间组织和控制机械转导，决定基因组结构的因素， 细胞的表达谱。核力学的两个主要贡献者是核纤层， 内核膜内的纤维丝，以及充满细胞核的染色质。核纤层蛋白的改变， 染色质致密化发生在许多主要的人类疾病中和健康细胞分化期间。无论是 在某些情况下，细胞核、细胞和组织的力学和形态学可能发生急剧变化。目前 基于疾病的核泡和基于健康分化的变化的机制基础， 核形态学和力学是未知的。我的研究发现，染色质及其组蛋白介导的 压实状态和交联决定了初始力响应（< 30%应变）和形态， 作为第二因素，在较长的变形下，对层压A的作用决定了应变硬化。我第一 我建议使用我开发的显微切割，显微操作，和纳米级的力量， 测量方法，以进一步阐明核力学在基因组组织中的作用。期间 核拉伸实验我将确定染色质如何响应核变形，通过 成像单个染色体基因座（CRISPR标记）和整体染色质纳米结构。第二，我会 研究核起泡和破裂的疾病相关表型的功能影响， 可以由基于染色质的核力学引起或抑制。我会确定核泡是否 症状或疾病的原因，通过活细胞成像和生物化学技术来分析系统性 DNA损伤，正确的转录和基因组内容物在水泡中的忠实分离。最后要 利用成熟的角质形成细胞原代细胞模型，研究细胞核形态学的基础 分化过程中的变化，祖细胞到终末细胞，以及Ras激活后体内平衡的丧失，以模拟 癌症过渡期总的来说，我的目标是发展一个独立的职业生涯，调查机械基础， 70多年来，在疾病和健康细胞分化中观察到形态学变化。

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.