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（由适用提供）：哺乳动物细胞具有确定染色质结构和动力学的多种机制。这些机制包括核素的酶促修饰，核心组蛋白变异，通过染色质修饰酶的染色体位点绑扎酶，染色质重塑剂，染色质构建蛋白（CAPS）和RNAS的全下。核心结构的异常已在包括唐氏综合症，代谢疾病，神经系统疾病（例如RETT综合征）以及乳腺癌，肺和大脑等癌症等疾病中实现。不幸的是，事实证明，将染色质中的局部生物化学修饰与核的更广泛的组织，动力学和功能相关联。建立这些联系的困难反映了我们的测量能力中潜在生物学的复杂性和几个关键差距。核心研究社区目前具有（1）具有全基因组覆盖率的工具，但细胞到细胞和临时分辨率有限，以及（2）具有高空间和临时分辨率的工具，但覆盖范围有限，测量时间短，并且在构成人体大部分细胞类型的“厚''厚”细胞中性能差。我们是来自超定位显微镜，光学标签，干细胞和癌症生物学和染色质理论领域的研究人员团队。我们共同开发了一对4D核的连接技术。我们提议现在采取下一步，并进行完善并严格测试：（1）一类新的遗传编码且有效地不可漂白的基金来用于染色质成分的多路复用标记，以及（2）4D超级定位显微镜，用于简单地跟踪``厚度''（厚10微米'（〜10 micron dimomet of）核能。使用标签，活核中单分子的成像不再受光显微镜中的新能力暂时限制。该硬件设计用于同时在细胞类型（例如人类干细胞）（核生物学的关键硬件能力）中同时对许多定位的超级定位跟踪。为了促进工具传播到更广泛的生物群落中，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）在SATB1 DNA```loopscape Contarter）''中的基因座动力学改变。当组合和验证为单位时，这些工具旨在允许在主要细胞过渡（例如分裂，分化和敏感性）中测量核的结构和动态。