Mapping the organization of the peripheral nuclear compartment

绘制周围核区室的组织图

• 批准号: 9765209
• 负责人: Larry R. Gerace
• 金额: $ 58.09万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765209
• 关键词: 3-Dimensional; Address; Adipocytes; Biochemical; CRISPR/Cas technology; Cell Differentiation process; Cell Line; Cell Lineage; Cell Nucleus; Cells; Chemicals; Chromatin; Chromosome Structures; Chromosomes; Co-Immunoprecipitations; Crosslinker; Cultured Cells; Formulation; Future; Gene Expression; Genetic Transcription; Heterochromatin; Image; Light; Maps; Mass Spectrum Analysis; Mesenchymal Stem Cells; Methodology; Methods; Modeling; Muscle Cells; Nuclear; Nuclear Envelope; Nuclear Lamin; Nuclear Lamina; Nuclear Proteins; Nuclear Structure; Pathway interactions; Peripheral; Play; Polymers; Property; Protein Dynamics; Proteins; Proteomics; Regulation; Resolution; Role; Signal Transduction; Site; Specific qualifier value; Structure; Surface; Techniques; Work; Yeasts; adipocyte differentiation; cell type; chromatin protein; crosslink; embryonic stem cell; light microscopy; mechanical properties; protein protein interaction; public health relevance; stable isotope; stem cell differentiation; yeast two hybrid system

 DESCRIPTION (provided by applicant): Intranuclear compartments make essential contributions to chromosome structure and function. One such compartment is organized by the nuclear lamina (NL), a protein meshwork that lines the nucleoplasmic surface of the nuclear envelope (NE) in metazoan cells. The NL contains a polymeric assembly of nuclear lamins associated with a large number of transmembrane and peripheral proteins. In addition to serving as a structural framework for the NE, the NL modulates numerous signaling and transcriptional pathways, and provides a tethering and regulatory site for chromatin at the nuclear periphery. The nuclear lamina and associated peripheral heterochromatin can be viewed as comprising a distinctive nuclear compartment, herein termed the nuclear lamina/peripheral chromatin (NL/PC) compartment. Although various components of the NL have been described, the analysis has not been comprehensive. Moreover, approaches are underdeveloped for defining its compositional changes during cell differentiation, and for elucidating the protein-protein interactions that specify the NL network and its chromatin interactions. To address these fundamental issues, this project aims to develop methods to isolate sub-nuclear fractions enriched in the NL/PC compartment from mesenchymal stem cells (MSCs), and from myocytes and adipocytes differentiated from these cells. With the use of these fractions, systematic mass spectrometry (MS) methods will be developed to characterize the protein composition of the NL/PC compartment and to quantitatively chart its changes during differentiation. Chemical crosslinking (XL) combined with MS will be deployed to map the interactions of constituent proteins, and biochemical methods in tandem with super-resolution light microscopy will be used to validate these associations. From implementation of this complementary set of approaches, the project will provide a comprehensive reference map of the NL and the changes that it undergoes during differentiation of defined MSC lineages. This will potentiate the formulation of more general concepts on the functions of the NL/PC compartment, and will identify new components and nodes that can be targeted in future functional studies. This is expected to substantially advance our understanding of the role of the NL in cell-type-specific regulation of peripheral heterochromatin and gene expression.

 描述（由适用提供）：核内核隔室为染色体结构和功能做出了重要贡献。一个这样的区室是由核层（NL）组织的，核层层是一种蛋白质网状，该蛋白质网将后生细胞中的核包膜（NE）编成。 NL包含与大量跨膜和外围蛋白相关的核层粘蛋白的聚合物组装。除了描述了NL的各种组成部分外，该分析还没有全面。此外，对于定义细胞分化过程中其组成变化以及阐明指定NL网络及其染色质相互作用的蛋白质 - 蛋白质相互作用的方法，方法不发达。为了解决这些基本问题，该项目旨在开发方法，以分离出富含NL/PC室中的亚核干细胞（MSC）（MSC）以及与这些细胞区别的心肌细胞和脂肪细胞的方法。通过使用这些馏分，将开发系统的质谱法（MS）方法来表征NL/PC隔室的蛋白质组成，并在分化过程中定量绘制其变化。与MS结合的化学交联（XL）将被部署以绘制组成蛋白的相互作用，并且将使用与超分辨率光学显微镜串联的生化方法来验证这些关联。从实施此完整的方法，该项目将提供NL的全面参考图及其在定义的MSC谱系分化过程中所经历的更改。这可能是关于NL/PC隔室功能的更一般概念的公式，并将确定可以在未来功能研究中针对的新组件和节点。预计这将大大促进我们对NL在细胞类型特异性调节中的作用的理解。