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，并负责地球上的大多数生物转化。另一方面，细菌病原体对人类健康构成重大威胁，导致人类多种传染病。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，了解分子如何在细菌细胞内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，染色体和数千种不同的蛋白质，RNA和小分子驻留在那里。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策划的系统。然而，这些细胞的小尺寸使得探测它们的亚细胞组织非常困难，这是由于缺乏具有足够的空间和时间分辨率的工具来阐明在如此小的体积内分子组装体的结构和动力学。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，更不用说它们在体内的动力学了。这种知识的缺乏严重阻碍了我们对细菌功能的理解。由于单分子检测、超分辨率成像和荧光E。在PI实验室的大肠杆菌文库中，我们现在能够拥有第一个高分辨率的活细菌整体视图。通过结合这些生物成像和系统生物学工具，我们建议量化整个E。大肠杆菌蛋白质组的单分子敏感性，绘制细胞内分布和超微结构组织的大多数E。大肠杆菌蛋白质的纳米分辨率，并跟踪他们的动态变化和相互作用在活细胞中的真实的时间。基于这些知识，我们计划构建一个定量的，高分辨率的地图E.大肠杆菌细胞结构与每个基因的分子特异性。此外，我们计划配置文件的变化，这种架构在响应细胞状态和环境条件。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的影响。 公共卫生相关性：在这个项目中，我们建议确定一个定量的，高分辨率的E。大肠杆菌的单分子灵敏度，纳米级的空间分辨率和每个单独的基因的分子特异性，并与一套生物成像和系统生物学工具，以应对环境条件下的这种架构的变化。这种具有最终灵敏度和分辨率的细菌结构的全系统视图不仅将推进基础微生物学和细胞生物学，而且还可能为细菌感染性疾病提供新的治疗靶点。新的高灵敏度，高分辨率的成像技术和蛋白质组学分析工具在这里开发的也将有广泛的应用到其他领域的生物医学研究。

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.