Dissecting chromatin and lamin contributions to nuclear structure and function

 描述（由申请人提供）：细胞核是一种细胞器，它必须正确地转换或抵抗生物物理力以决定基因组的空间组织，而基因组的空间组织又决定了细胞的表达谱。最初的力实验表明，核纤层是一个主要的结构和电阻组成部分的核，并位于内核膜。与核纤层蛋白不同，尚不清楚系在核周边和核纤层蛋白上的异染色质或一般染色质是否有助于核的机械或结构性质。核纤层蛋白和异染色质的突变或改变的数量伴随着许多重大人类疾病的异常核形态。这些疾病包括基于核纤层蛋白突变的核纤层蛋白病，其集中在引起恒定机械应变的细胞或组织中，例如心血管系统或肌肉。目前还不清楚这些疾病或其他疾病中的突变核纤层蛋白和改变的染色质如何各自促成核力学和结构的改变。我建议使用显微切割，显微操作，和纳米级的力测量结合活细胞成像探测小鼠和人类细胞核的物理特性。首先，我建议通过耗尽或改变每个组分（染色质或核纤层蛋白）并分析力和结构响应来提供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会导致细胞核更具顺应性，这一点通过显微操作进行了量化。其次，我计划使用患有过早老化核纤层蛋白病的Hutchinson-Gilford早衰综合征（HGPS）患者的细胞，这些患者在核纤层蛋白A中有一种常见的突变，称为progerin，以研究具有核泡的细胞核物理性质的变化。核泡是核纤层蛋白病、癌症和衰老常见的表型。在所有这些病理中，细胞核变得毁容，具有远离细胞核延伸的多个叶。这种表型背后的机制尚不清楚。我将确定突变核纤层蛋白A早老蛋白的积累和异染色质的减少对具有核泡的细胞核的物理性质的贡献。通过分析每种成分的贡献以及它们之间的相互作用，我的目标是揭示与人类疾病相关的核特性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.