Structure and function of the prokaryotic cytoskeleton by electron cryotomography

描述（由申请人提供）：细菌几乎无处不在，在工业和环境中发挥着重要作用，是人类和其他生物体健康和疾病的重要因素。考虑到它们的重要性，令人惊讶的是，我们仍然对基本的细菌细胞生物学一无所知。例如，我们仍然不知道细菌如何产生和保持其特征形状，建立极性，组织其基因组，分离其染色体，分裂，以及在某些情况下移动。在真核生物中，所有这些任务都是由细胞骨架细丝完成的，但由于以前的成像技术未能揭示细菌中的类似结构，长期以来人们认为细菌不具有细胞骨架。最近，荧光显微镜显示细菌具有实质性的内部秩序。在第一轮资助中，我们使用了另一种年轻的技术，电子冷冻断层扫描（ECT），以接近天然状态产生完整细菌细胞的三维图像，达到“分子”（~4-6 nm）分辨率。使用ECT，我们直接可视化了约20种不同细菌物种中的数百种细胞骨架丝，证明细菌细胞骨架实际上既普遍又复杂。在这里，我们建议开发和应用新的相关光学和电子显微镜技术，以确定这些丝一般。这将使我们能够解决现有的光学和电子显微镜结果之间的关键差异，并产生急需的洞察细菌细胞骨架的结构和功能。我们还提出了工作，以提高EM图像质量一般和扩大正在研究的灯丝的数量。 公共卫生相关性：了解细菌细胞骨架的结构和功能将有助于我们了解细菌如何在健康和疾病中发挥作用，并可能在长期的未来提出新的抗生素靶点。

DESCRIPTION (provided by applicant): Bacteria are nearly ubiquitous, play vital roles in industry and the environment, and are important actors in both health and disease for humans and other organisms. Given their importance, it is surprising how much we still don't understand about basic bacterial cell biology. We still don't know, for instance, how bacteria generate and maintain their characteristic shapes, establish polarity, organize their genomes, segregate their chromosomes, divide, and in some cases move. In eukaryotes, all these tasks are performed by cytoskeletal filaments, but because previous imaging technologies failed to reveal analogous structures in bacteria, it was long thought that bacteria don't possess cytoskeletons. More recently, fluorescence microscopy has shown that bacteria have substantial internal order. In the first cycle of this grant, we used another young technology, electron cryotomography (ECT), to produce three- dimensional images of intact bacterial cells in a near-native state to "molecular" (~4-6 nm) resolution. Using ECT we directly visualized hundreds of cytoskeletal filaments in ~20 different bacterial species, proving that the bacterial cytoskeleton is in fact both general and complex. Here we propose to develop and apply new correlated light and electron microscopy techniques to identify these filaments generally. This should allow us to resolve key discrepancies between the existing light and electron microscopical results and generate much-needed insight into the structures and functions of the bacterial cytoskeleton. We also propose work to improve EM image quality generally and expand the number of filaments being studied. PUBLIC HEALTH RELEVANCE: Knowing the structure and function of the bacterial cytoskeleton will help us understand how bacteria accomplish their roles in health and disease and perhaps in the long-term future suggest new antibiotic targets.