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、通过染色质修饰酶的染色体位点的束缚、染色质重塑剂、染色质结构蛋白（CAP）和一系列RNA。核组结构的异常已经涉及包括唐氏综合征、代谢疾病、神经障碍如Rett综合征以及乳腺癌、肺癌和脑癌的疾病。 不幸的是，它已被证明是困难的染色质内的局部生物化学修饰的更广泛的组织，动力学和核组的功能。建立这些联系的难度反映了基础生物学的复杂性和我们测量能力的几个关键差距。核组研究界目前有（1）具有全基因组覆盖但具有有限的细胞间和时间分辨率的工具，和（2）具有高空间和时间分辨率但具有有限的覆盖范围、短测量持续时间和在构成人体中大多数细胞类型的“厚”细胞中的差性能的工具。 我们是一个来自超定位显微镜，光学标签，干细胞和癌症生物学，染色质理论等领域的研究团队。我们共同开发了一对用于4D核组学的连接技术。我们建议现在采取下一步，并完善和严格测试：（1）一类新的遗传编码和有效的不可漂白的基准，用于染色质成分的多重标记，和（2）4D超定位显微镜，用于同时跟踪“厚”（~10微米直径）核内的许多目标。有了这些标记，活细胞核内单个分子的成像不再受到光漂白的时间限制，这是光学显微镜的一种新能力。该硬件设计用于同时4D超定位跟踪细胞类型（如人类干细胞）中的许多基因座，这是核组生物学的关键硬件能力。为了促进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）USP 16去泛素化酶在人类唐氏综合征中的作用和（2）在SATB 1 DNA“loopscape标记”存在下基因座动态的改变。当作为一个单元组合和验证时，这些工具被设计成允许在主要细胞转变（如分裂、分化和衰老）期间测量核组的结构和动态。