Illuminating molecular mechanisms of cellular functions by single-molecule and super-resolution imaging

通过单分子和超分辨率成像阐明细胞功能的分子机制

• 批准号: 9474629
• 负责人: XIAOWEI ZHUANG
• 金额: $ 41.75万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9474629
• 关键词: Actins; Action Potentials; Axon; Behavior; Binding; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Structure; Chromosome Territory; Chromosomes; Cytoskeleton; DNA biosynthesis; Development; Dimensions; Disease; Enhancers; Functional disorder; Funding; Gene Expression Regulation; Generations; Genetic Recombination; Genome; Goals; Image; Imagery; Imaging Device; Laboratories; Light; Link; Medical; Membrane; Methods; Modeling; Molecular; Molecular Conformation; Morphogenesis; National Institute of General Medical Sciences; Neurons; Periodicity; Play; Proteins; Research; Research Support; Resolution; Role; Skeleton; Spectrin; Structure; Subcellular structure; Tissues; axonal degeneration; chromatin remodeling; fluorescence imaging; gene product; genome-wide; high resolution imaging; imaging approach; imaging capabilities; imaging modality; improved; interest; molecular scale; novel; promoter; repaired; single molecule; synaptogenesis

Project Summary / Abstract Understanding the mechanisms of cellular function and their dysfunction in disease requires a detailed picture of the molecular interactions in cells. In particular, it is desirable to have imaging tools with single-molecule sensitivity, molecular-scale resolution, and genome-scale capacity to allow direct visualization of these molecular interactions and to probe the collective behaviors of different genes and gene products that give rise to cell and tissue function. The long-term research goal of my laboratory is to develop advanced fluorescence imaging methods that meet these demands and to apply these methods to elucidate molecular mechanisms of cellular functions that are of both fundamental and medical significance. In the next five years, we propose to focus our NIGMS-funded research mainly in the following two directions. (1) To understand the structure and function of a novel cytoskeletal structure in neurons. Our NIGMS- supported research on the development and application of super-resolution STORM imaging has led to the discovery of a periodic membrane-associated cytoskeleton structure in neurons, primarily in axons. While we have a basic model of the ultrastructural organization of some key components of this structure, including actin, spectrin and associated molecules, we expect many additional protein components to be present in this structure. These components and their structural organization remain to be determined. Our understanding of the functional roles of this novel structure is also far from complete. We propose to use super-resolution imaging in conjunction with other methods to determine the protein components and structural organization of this periodic skeleton structure, and to investigate its functional roles in axon morphogenesis and synaptogenesis, action potential generation and propagation, axon degeneration, and other axonal functions. (2) To investigate the spatial organization of chromatin and chromosomes important for gene regulation. The spatial organization of chromatin plays an important role in many essential genome functions such as gene expression regulation, DNA replication, repair and recombination. In particular, many features of the chromatin conformation have been implicated in gene regulation, such as open and closed chromatin states, promoter- enhancer interactions, topologically-associated domains, and chromosome territories. Misregulation of chromatin structures has been implicated in a variety of diseases. However, many gaps remain in our understanding of the three-dimensional (3D) organization of chromatin and chromosomes. Our NIGMS- supported research on STORM imaging and chromatin remodeling studies allowed us to develop a super- resolution chromatin imaging approach and reveal novel chromatin organizations. We will advance our chromatin/chromosome imaging capability by developing the ability to trace the 3D path of the chromatin chain in the chromosome and to study questions on chromatin organization that are important for gene regulation.

项目摘要 /摘要 了解细胞功能的机制及其在疾病中的功能障碍需要详细的图像 细胞中的分子相互作用。特别是，希望拥有具有单分子的成像工具 敏感性，分子尺度分辨率和基因组规模的能力，以直接可视化这些能力 分子相互作用并探测产生的不同基因和基因产物的集体行为 到细胞和组织功能。我实验室的长期研究目标是开发高级荧光 满足这些需求并将这些方法应用于阐明分子机制的成像方法 具有基本意义和医学意义的细胞功能。 在接下来的五年中，我们建议将诺格姆斯资助的研究集中在以下两个方向上。 （1）了解神经元中新型细胞骨架结构的结构和功能。我们的nigms- 支持超分辨率风暴成像的开发和应用的研究导致 发现神经元中与周期性膜相关的细胞骨架结构，主要是在轴突中。当我们 具有该结构的某些关键组成部分的超微结构组织的基本模型，包括 肌动蛋白，光谱和相关分子，我们期望在此中存在许多其他蛋白质成分 结构。这些组成部分及其结构组织仍有待确定。我们对 这种新结构的功能作用也远非完整。我们建议使用超分辨率 与其他方法结合的成像，以确定蛋白质成分和结构组织 这种周期性的骨骼结构，并研究其在轴突形态发生和 突触发生，动作电位产生和传播，轴突变性和其他轴突功能。 （2）研究染色质和染色体的空间组织对基因调节很重要。这 染色质的空间组织在许多基本基因组功能（例如基因）中起重要作用 表达调控，DNA复制，修复和重组。特别是染色质的许多特征 构象与基因调节有关，例如开放和封闭的染色质状态，启动子 - 增强子相互作用，拓扑相关的域和染色体领土。误导 染色质结构与多种疾病有关。但是，我们的差距仍然存在 了解染色质和染色体的三维（3D）组织。我们的nigms- 支持暴风雨成像和染色质重塑研究的研究使我们能够开发超级 分辨率染色质成像方法并揭示新的染色质组织。我们将推进我们的 通过开发追踪染色质链3D路径的能力，染色质/染色体成像能力 在染色体中，研究对基因调节很重要的染色质组织的问题。