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

描述(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的生物转化。另一方面，细菌病原体对人类健康构成了重大威胁，导致了人类的许多传染病。此外，细菌也是我们了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，了解细菌细胞内的分子如何协调和相互作用以支持生命过程是至关重要的。在细菌中，生命过程发生在一小部分&lt；1&lt；m~3中，染色体和数千种不同的蛋白质、RNA和小分子驻留在这里。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精心编排的系统。然而，这些细胞的小尺寸使得探索它们的亚细胞组织变得极其困难，因为缺乏在如此小的体积内阐明分子组装的结构和动力学所需的足够的空间和时间分辨率的工具。因此，我们对细菌细胞组织的了解仍然是原始的，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，更不用说它们在体内的动态了。这种知识缺失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实验室构建荧光大肠杆菌文库方面的进展，我们现在能够对活细菌有第一个高分辨率、完整的视图。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来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图，并在活细胞中实时跟踪它们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，其中包含每个单独基因的分子特异性。此外，我们计划根据蜂窝状态和环境条件来分析此架构的变化。这种对细菌细胞结构具有终极敏感性和分辨率的史无前例的、系统范围的观点不仅将解决细菌学中的广泛问题，而且将对微生物学和生物医学研究产生广泛影响。 与公共卫生相关：在这个项目中，我们建议用单分子敏感性、纳米级空间分辨率和每个单独基因的分子特异性来确定大肠杆菌细胞结构的定量、高分辨率图谱，并使用一套生物成像和系统生物学工具来描述这种结构随环境条件的变化。这种具有终极敏感性和分辨率的系统范围内的细菌结构观点不仅将促进基础微生物学和细胞生物学的发展，还可能为细菌感染疾病提供新的治疗靶点。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在生物医学研究的其他领域具有广泛的应用。

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.