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

项目摘要/摘要 了解疾病中细胞功能及其功能障碍的机制需要一张详细的图片 细胞内的分子相互作用。具体地说，希望具有单分子成像工具 灵敏度、分子规模的分辨率和基因组规模的能力，使这些能够直接可视化 分子相互作用和探索不同基因和基因产物的集体行为 对细胞和组织功能的影响。我的实验室的长期研究目标是开发先进的荧光技术 符合这些要求的成像方法，并应用这些方法来阐明 具有基础和医学意义的细胞功能。 未来五年，我们建议重点开展以下两个方向的研究。 (1)了解神经元中一种新的细胞骨架结构的结构和功能。我们的NIGMS- 对超分辨率风暴成像的开发和应用的支持研究导致了 在神经元中发现周期性的膜相关细胞骨架结构，主要是在轴突中。当我们 对这一结构的一些关键组件的超微结构组织有一个基本模型，包括 肌动蛋白、幽灵蛋白和相关分子，我们预计许多额外的蛋白质成分存在于此 结构。这些组成部分及其结构组织仍有待确定。我们对此的理解 这一新结构的功能作用也远未完成。我们建议使用超分辨率 结合其他方法进行成像以确定蛋白质组分和结构组织 这种周期性的骨架结构，并探讨其在轴突形态发生和 突触发生、动作电位的产生和传播、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控的重要作用。这个 染色质的空间组织在基因组的许多基本功能中起着重要作用，如基因 表达调控、DNA复制、修复和重组。特别是，染色质的许多特征 构象与基因调控有关，如开放和闭合染色质状态，启动子- 增强子相互作用、拓扑相关结构域和染色体区域。监管不善 染色质结构与多种疾病有关。然而，我们的社会仍然存在许多差距。 了解染色质和染色体的三维(3D)组织。我们的NIGMS- 风暴成像和染色质重塑研究的支持研究使我们能够开发出一种超 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 通过发展追踪染色质链的3D路径的能力来实现染色质/染色体成像能力 并研究有关染色质组织的问题，这些问题对基因调控很重要。

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.