Dissecting chromatin and lamin contributions to nuclear structure and function

 描述(申请人提供)：细胞核是细胞器，它必须适当地转导或抵抗生物物理力来决定基因组的空间组织，这反过来又决定细胞的表达谱。初始力实验表明，板层是原子核的主要结构和阻力成分，位于核内包膜内。与板层不同，目前尚不清楚连接到核周边和板层的异染色质，还是一般的染色质对核的机械或结构特性有贡献。在许多人类主要疾病中，层粘连蛋白和异染色质的突变或数量改变伴随着核形态的异常。这些疾病包括基于板层突变的椎板病，这些板层集中在引起持续机械应变的细胞或组织中，如心血管系统或肌肉。目前尚不清楚这些疾病或其他疾病中突变的层粘连蛋白和改变的染色质是如何导致核力学和结构改变的。我建议使用显微解剖、微操作和纳米级的力测量，并结合活细胞成像来探索老鼠和人类细胞核的物理性质。首先，我建议通过耗尽或改变每个成分(染色质或层蛋白)，并分析力和结构响应，来提供对野生型细胞核特性的全面分析。初步证据表明，染色质有助于细胞核的电阻特性：使用组蛋白脱乙酰酶(HDAC)抑制剂增加分解的常染色质的量，可以产生更顺应性的细胞核，正如显微操作所定量的那样。其次，我计划使用一种叫做孕激素的层蛋白A常见突变的早衰型椎板病(HGPS)患者的细胞来研究带有核泡的核的物理性质的变化。核泡是椎板病、癌症和衰老的常见表型。在所有这些病理中，细胞核变得畸形，并有多个叶向外延伸。这种表型背后的机制尚不清楚。我将确定突变层蛋白A孕激素的积累和异染色质减少对具有核泡的核的物理性质的贡献。通过分析每个组成部分的贡献以及它们之间的相互作用，我的目的是揭示与人类疾病相关的核性质变化背后的机制基础。

 DESCRIPTION (provided by applicant): The nucleus is the organelle which must properly transduce or resist biophysical forces to dictate the spatial organization of the genome, which in turn determines the expression profile of the cell. Initial force experiments have revealed that lamins are a major structural and resistive component of the nucleus and are located inside the inner nuclear envelope. Unlike lamins, it is unknown if heterochromatin, which is tethered to the nuclear periphery and lamins, or chromatin in general contributes to the mechanical or structural properties of nuclei. Mutation or altered amounts of lamins and heterochromatin accompany aberrant nuclear morphology in many major human diseases. These diseases include laminopathies, based in mutations in lamins, which are focused in cells or tissues that incur constant mechanical strain, such as the cardiovascular system or muscles. It is unclear how mutant lamin and altered chromatin in these diseases or other diseases each contribute to alterations in nuclear mechanics and structure. I propose to use microdissection, micromanipulation, and nanonewton-level force measurement in conjunction with live cell imaging to probe the physical properties of mouse and human nuclei. First, I propose to provide a comprehensive analysis of nuclear properties in wild-type cells by depleting or altering each component (chromatin or lamin protein) and analyzing force and structural response. Preliminary evidence suggests that chromatin contributes to the resistive properties of the nucleus: increasing the amount of decompact euchromatin using a histone deacetylase (HDAC) inhibitor results in a more compliant nucleus as quantified by micromanipulation. Second, I plan to use cells of patients with a premature aging laminopathy disease Hutchinson-Gilford progeria syndrome (HGPS) with a common mutation in lamin A called progerin to investigate changes in the physical properties of nuclei with nuclear blebs. Nuclear blebs are a phenotype common to laminopathies, cancer and aging. In all of these pathologies the nucleus becomes disfigured with multiple lobes that extend away from the nucleus. The mechanics underlying this phenotype are unknown. I will determine the contributions of accumulation of mutant lamin A progerin and decreased heterochromatin to the physical properties of nuclei with nuclear blebs. Through analyzing the contribution of each component as well as the interplay between them I aim to uncover the mechanistic basis behind changes in nuclear properties relevant to human disease.