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量的大部分，并负责地球上大多数生物转化。另一方面，细菌性病原体对人类健康构成重大威胁，在人类中引起许多传染病。此外，细菌也是我们理解基本生物过程的模式生物，特别是在分子和细胞水平上。因此，了解分子如何在细菌细胞内协调和相互作用以支持生命过程是至关重要的。在细菌中，生命过程发生在<1<m3的小体积中，染色体和数千种不同的蛋白质、RNA和小分子存在于其中。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，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，因为缺乏足够的空间和时间分辨率来阐明如此小体积内分子组装的结构和动力学。因此，我们对细菌细胞组织的了解仍然很原始，远远落后于真核细胞。对于大多数细菌蛋白，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结构组织，更不知道它们在体内的动态。这种知识缺陷严重阻碍了我们对细菌功能的理解。多亏了最近在单分子检测、超分辨率成像以及pi实验室中荧光大肠杆菌文库的建设方面的发展，我们现在有了第一个高分辨率、完整的活细菌视图。通过结合这些生物成像和系统生物学工具，我们提出以单分子灵敏度定量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的、高分辨率的大肠杆菌细胞结构图谱，并分析每个基因的分子特异性。此外，我们计划描述这种结构在响应细胞状态和环境条件时的变化。这种前所未有的、具有最终灵敏度和分辨率的细菌细胞结构的全系统视图不仅将解决细菌学中广泛的问题，而且将对微生物学和生物医学研究产生广泛的影响。

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.