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至关重要的作用，并且是人类和其他生物体健康和疾病的重要参与者。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知之甚少。例如，我们仍然不知道细菌是如何产生和保持它们的特征形状，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，因此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有大量的内部秩序。在这项资助的第一个周期中，我们使用了另一种年轻的技术，电子冷冻断层扫描（ECT），以“分子”分辨率（~4-6纳米）在接近天然状态下生成完整细菌细胞的三维图像。利用ECT，我们直接看到了大约20种不同细菌的数百个细胞骨架细丝，证明了细菌的细胞骨架实际上是一般的和复杂的。在此，我们建议开发和应用新的相关光镜和电子显微镜技术来识别这些细丝。这将使我们能够解决现有光学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急需的见解。我们还提出了提高EM图像质量和扩大所研究细丝数量的工作。

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.