Dissecting chromatin and lamin contributions to nuclear structure and function

剖析染色质和核纤层蛋白对核结构和功能的贡献

• 批准号: 8982675
• 负责人: Andrew Daniel Stephens
• 金额: $ 5.42万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8982675
• 关键词: Age; Aging; Antibodies; Behavior; Biochemical; Biological Assay; Bulla; Cardiovascular system; Cell Nucleus; Cells; Chromatin; Chromosomes; Confocal Microscopy; Coupled; Diabetes Mellitus; Disease; EZH2 gene; Euchromatin; Excision; Fluorescence Microscopy; Genome; Goals; Heart Diseases; Heterochromatin; Histone Deacetylase Inhibitor; Human; Image; Individual; Intermediate Filaments; Lamin B1; Lamin Type A; Lamins; Life; Lobe; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Mechanics; Metaphase; Microdissection; Microinjections; Micromanipulation; Microscopy; Molecular Profiling; Morphology; Mus; Muscle; Muscular Dystrophies; Mutation; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Lamin; Nuclear Lamina; Nuclear Structure; Organelles; Pathology; Patients; Phenotype; Premature aging syndrome; Progeria; Property; Proteins; RNA Interference; Relative (related person); Resistance; Resolution; Staining method; Stains; Stretching; Structure; Syndrome; Techniques; Testing; Tissues; Transfection; base; biophysical properties; cell type; cellular imaging; chemical genetics; fluorescence imaging; human disease; insight; interest; mutant; novel; overexpression; physical property; public health relevance; research study; response

 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.

 描述（由申请人提供）：细胞核是必须正确转导或抵抗生物物理力以决定基因组空间组织的细胞器，而基因组空间组织又决定细胞的表达谱。初始力实验表明，核纤层蛋白是细胞核的主要结构和电阻成分，位于内核膜内部。与核纤层蛋白不同，目前尚不清楚异染色质（其与核外围和核纤层蛋白相连）或染色质是否对细胞核的机械或结构特性有贡献。在许多主要人类疾病中，核纤层蛋白和异染色质的突变或数量改变伴随着核形态异常。这些疾病包括基于核纤层蛋白突变的核纤层蛋白病，其集中于产生持续机械应变的细胞或组织，例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的突变核纤层蛋白和改变的染色质如何导致核力学和结构的改变。我建议将显微解剖、显微操作和纳牛顿级力测量与活细胞成像结合使用，以探测小鼠和人类细胞核的物理特性。首先，我建议通过消耗或改变每种成分（染色质或核纤层蛋白）并分析力和结构反应，对野生型细胞的核特性进行全面分析。初步证据表明，染色质有助于细胞核的抵抗特性：使用组蛋白脱乙酰酶 (HDAC) 抑制剂增加解致密常染色质的量，可通过显微操作量化得到更顺应的细胞核。其次，我计划使用患有早衰核纤层病变疾病哈钦森-吉尔福德早衰综合症 (HGPS) 的患者的细胞来研究具有核泡的细胞核物理特性的变化，该病的核纤层蛋白 A 中存在一种称为早衰蛋白的常见突变。核泡是核纤层蛋白病、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，多个叶远离细胞核延伸。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减少对具有核泡的细胞核物理性质的贡献。通过分析每个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。