Deep Super-localization Microscopy and Effectively Unbleachable Labeling for 4D Nucleomics

用于 4D 核组学的深度超定位显微镜和有效不可漂白标记

• 批准号: 9306083
• 负责人: Jan T. Liphardt
• 金额: $ 33万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9306083
• 关键词: Alpha Cell; Architecture; Area; Biochemical; Biological; Biological Testing; Biology; Caliber; Cancer Biology; Cell Nucleus; Cells; Characteristics; Chromatin; Chromatin Loop; Chromatin Structure; Communities; Complex; Custom; DNA; Disease; Down Syndrome; Engineering; Enzymes; Eukaryotic Cell; Gene Expression; Gene Expression Regulation; Genomics; Histones; Hour; Human; Human body; Image; Imaging Device; Impairment; Individual; Label; Length; Light; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of lung; Mammalian Cell; Measurement; Mediating; Metabolic Diseases; Metabolism; Microscope; Microscopy; Modification; Monitor; Nucleosome Core Particle; Optics; Performance; Process; Proteins; Public Health; Recruitment Activity; Research; Research Personnel; Resources; Rett Syndrome; Role; Site; System; Technology; Testing; Thick; Time; Validation; Variant; Work; base; cell type; design; developmental disease; fluorescence microscope; human DNA; human disease; human stem cells; light microscopy; malignant breast neoplasm; millisecond; mouse model; nanometer; nervous system disorder; programs; public health relevance; scaffold; senescence; single molecule; stem cell biology; stem cell division; temporal measurement; theories; tool; whole genome

 DESCRIPTION (provided by applicant): Mammalian cells have multiple mechanisms for regulating chromatin structure and dynamics. These mechanisms include enzymatic modification of nucleosomes, core histone variants, tethering of chromosomal sites via chromatin modifying enzymes, chromatin remodelers, chromatin architectural proteins (CAPs), and a panoply of RNAs. Abnormalities of nucleome architecture have been implicated in diseases including Down's Syndrome, metabolic diseases, neurological disorders such as Rett syndrome, and cancers of the breast, lung, and brain. Unfortunately, it has proven difficult to relate local biochemical modifications within the chromatin to the broader organization, dynamics, and function of the nucleome. The difficulty of making these connections reflects the complexity of the underlying biology and several critical gaps in our measurement capabilities. The nucleome research community currently has (1) tools with whole-genome coverage but with limited cell-to-cell and temporal resolution and (2) tools with high spatial and temporal resolutio but with limited coverage, short measurement duration, and poor performance in the `thick' cells that constitute most of the cell types in the human body. We are a team of investigators from the areas of super-localization microscopy, optical labels, stem cell and cancer biology, and chromatin theory. Together, we have developed a pair of connected technologies for 4D nucleomics. We propose to now take the next step, and refine and stringently test: (1) a new class of genetically encoded and effectively unbleachable fiducials for multiplexed labeling of chromatin components, and (2) a 4D super-localization microscope for simultaneously tracking many targets within `thick' (~10 micron diameter) nuclei. With the labels, the imaging of single molecules within living nuclei is no longer temporally limited by photo bleaching, a new capability in light microscopy. The hardware is designed for simultaneous 4D super- localization tracking of many loci in cell types such as human stem cells, a key hardware capability for nucleome biology. To facilitate the spread of the tools into the broader biological community, we will test our tools in two systems, (1) the role of the USP16 deubiquitinase enzyme in human Down's Syndrome and (2) alterations of loci dynamics in the presence of the SATB1 DNA `loopscape controller'. When combined and validated as a unit, the tools are designed to allow measurement of the nucleome's architecture and dynamics during major cellular transitions such as division, differentiation, and senescence.

 描述（由申请人提供）：哺乳动物细胞具有多种调节染色质结构和动力学的机制。这些机制包括核小体的酶促修饰、核心组蛋白变体、通过染色质修饰酶束缚染色体位点、染色质重塑剂、染色质结构蛋白 (CAP) 和一系列 RNA。核体结构的异常与唐氏综合症、代谢性疾病、雷特综合症等神经系统疾病以及乳腺癌、肺癌和脑癌等疾病有关。 不幸的是，事实证明很难将染色质内的局部生化修饰与核组的更广泛的组织、动力学和功能联系起来。建立这些联系的难度反映了基础生物学的复杂性以及我们测量能力的几个关键差距。核组研究界目前拥有（1）具有全基因组覆盖的工具，但细胞间和时间分辨率有限；（2）具有高空间和时间分辨率的工具，但覆盖范围有限，测量持续时间短，并且在构成人体大多数细胞类型的“厚”细胞中表现不佳。 我们是一个由来自超定位显微镜、光学标记、干细胞和癌症生物学以及染色质理论领域的研究人员组成的团队。我们共同开发了两种用于 4D 核组学的互联技术。我们建议现在采取下一步，并完善和严格测试：（1）一类新的基因编码且有效不可漂白的基准，用于染色质成分的多重标记，以及（2）4D超定位显微镜，用于同时跟踪“厚”（直径约10微米）细胞核内的许多目标。有了这些标签，活细胞核内单分子的成像不再受到光漂白的暂时限制，光漂白是光学显微镜的一项新功能。该硬件设计用于对人类干细胞等细胞类型中的许多位点进行同步 4D 超定位跟踪，这是核组生物学的关键硬件功能。为了促进这些工具传播到更广泛的生物界，我们将在两个系统中测试我们的工具，(1) USP16 去泛素酶在人类唐氏综合症中的作用，以及 (2) SATB1 DNA“loopscape 控制器”存在下基因座动态的改变。当作为一个单元进行组合和验证时，这些工具旨在允许在主要细胞转变（例如分裂、分化和衰老）期间测量核组的结构和动态。