Structure and function of the prokaryotic cytoskeleton by electron cryotomography

描述(申请人提供)：细菌几乎无处不在，在工业和环境中扮演着重要的角色，对人类和其他有机体的健康和疾病都是重要的参与者。考虑到它们的重要性，令人惊讶的是，我们对基本的细菌细胞生物学仍然不太了解。例如，我们仍然不知道细菌如何产生和保持它们特有的形状，建立极性，组织它们的基因组，分离它们的染色体，分裂，在某些情况下还会移动。在真核生物中，所有这些任务都是由细胞骨架细丝执行的，但由于之前的成像技术未能揭示细菌中的类似结构，长期以来人们一直认为细菌不具有细胞骨架。最近，荧光显微镜显示细菌有相当大的内部秩序。在这笔赠款的第一个周期中，我们使用了另一项年轻的技术，电子冷冻断层摄影术(ECT)，产生了处于接近自然状态的完整细菌细胞的三维图像，分辨率达到“分子”(~4-6 nm)。使用ECT，我们直接显示了约20个不同细菌物种的数百个细胞骨架细丝，证明了细菌细胞骨架实际上既是通用的，也是复杂的。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来一般地识别这些细丝。这将使我们能够解决现有的光镜和电子显微镜结果之间的关键差异，并对细菌细胞骨架的结构和功能产生迫切需要的洞察。我们还提出了提高电磁成像质量的总体工作和扩大被研究的细丝数量的工作。 与公共卫生相关：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.