Dynamic cellular architecture of bacteria by system-wide super-resolution imaging

描述（由申请人提供）：细菌构成了世界生物质的大部分，并负责地球上的大部分生物转化。另一方面，细菌病原体对人类健康构成重大威胁，引起人类多种传染病。此外，细菌还可以作为模型生物体，帮助我们了解基本的生物过程，特别是在分子和细胞水平上。因此，了解细菌细胞内分子如何协调和相互作用以支持生命过程至关重要。在细菌中，生命过程发生在 <1<m3 的小体积中，其中存在染色体和数千种不同的蛋白质、RNA 和小分子。现在人们认识到细菌细胞不仅仅是一袋酶，而是一个高度组织和协调的系统。然而，由于缺乏具有足够空间和时间分辨率的工具来阐明如此小体积内分子组装体的结构和动力学，这些细胞的小尺寸使得探测它们的亚细胞组织变得极其困难。因此，我们对细菌细胞组织的理解仍然很原始，远远落后于真核细胞。对于大多数细菌蛋白质，我们不知道它们在细胞内的数量，也不知道它们的空间分布和超微结构组织，更不用说它们的体内动态了。这种知识缺陷严重阻碍了我们对细菌功能的理解。由于最近在单分子检测、超分辨率成像以及 PI 实验室中荧光大肠杆菌库的构建方面取得的进展，我们现在能够首次获得活细菌的高分辨率完整视图。通过结合这些生物成像和系统生物学工具，我们建议以单分子灵敏度量化整个大肠杆菌蛋白质组，以纳米分辨率绘制大多数大肠杆菌蛋白质的细胞内分布和超微结构组织，并实时跟踪它们在活细胞中的动态变化和相互作用。基于这些知识，我们计划构建一个定量、高分辨率的大肠杆菌细胞结构图，其中包含每个基因的分子特异性。此外，我们计划分析该架构针对细胞状态和环境条件的变化。这种前所未有的、具有终极灵敏度和分辨率的全系统细菌细胞结构视图不仅将解决细菌学中的广泛问题，而且还将对微生物学和生物医学研究产生广泛影响。 公共健康相关性：在这个项目中，我们建议确定大肠杆菌细胞结构的定量、高分辨率图谱，具有单分子敏感性、纳米级空间分辨率和每个单独基因的分子特异性，并利用一套生物成像和系统生物学工具来分析该结构响应环境条件的变化。这种具有终极灵敏度和分辨率的细菌结构全系统视图不仅将推进基础微生物学和细胞生物学的发展，而且还可能为基于细菌的传染病提出新的治疗靶点。这里开发的新的高灵敏度、高分辨率成像技术和蛋白质组分析工具也将广泛应用于生物医学研究的其他领域。

DESCRIPTION (provided by applicant): Bacteria constitute the majority of the world's biomass and are responsible for most bioconversion on the planet. Bacterial pathogens, on the other hand, present major threats to human health, causing numerous infectious diseases in humans. Moreover, bacteria also serve as model organisms for us to understand fundamental biological processes, especially at the molecular and cellular levels. It is thus of paramount importance to understand how molecules coordinate and interact inside bacterial cells to support life processes. In bacteria, life processes take place in a small volume of <1<m3, where the chromosome and thousands of different proteins, RNA, and small molecules reside. It is now recognized that a bacterial cell is not simply a bag of enzymes, but a highly organized and orchestrated system. However, the small sizes of these cells have made it extremely difficult to probe their sub-cellular organization, due to the lack of tools with sufficient spatial and temporal resolution required to elucidate the structure and dynamics of molecular assemblies within such a small volume. Our understanding of bacterial cellular organization is thus still primitive, lagging far behind that of eukaryotic cells. For most bacterial proteins, we do not know their quantities inside the cell, nor do we know their spatial distributions and ultra-structural organization, let alone their in vivo dynamics. This knowledge deficit has severely hampered our understanding of how bacteria function. Thanks to recent developments in single-molecule detection, super-resolution imaging, and the construction of a fluorescent E. coli library in the PIs' labs, we are now in a position to have the first high-resolution, integral view of live bacteria. By combining these bioimaging and systems biology tools, we propose to quantify the entire E. coli proteome with single-molecule sensitivity, to map the intracellular distributions and ultra-structural organization of most E. coli proteins with nanometer resolution, and to follow their dynamic changes and interactions in real time in living cells. Based on such knowledge, we plan to construct a quantitative, high- resolution map of E. coli cellular architecture with the molecular specificity of each individual gene. Furthermore, we plan to profile changes of this architecture in response to cellular states and environmental conditions. This unprecedented, system-wide view of bacterial cellular architecture with ultimate sensitivity and resolution will not only address a wide range of questions in bacteriology, but will also have a broad impact on microbiology and biomedical research. PUBLIC HEALTH RELEVANCE: In this project, we propose to determine a quantitative, high-resolution map of cellular architecture of E. coli with single-molecule sensitivity, nanometer-scale spatial resolution and molecular specificity of each individual gene, and to profile changes of this architecture in response to environmental conditions with a set of bioimaging and systems biology tools. This system-wide view of bacterial architecture with ultimate sensitivity and resolution will not only advance fundamental microbiology and cell biology, but may also suggest new therapeutic targets for bacteria-based infectious diseases. The new high-sensitivity, high-resolution imaging techniques and proteomic analysis tools developed here will also have broad applications to other areas of biomedical research.