Structure and function of the prokaryotic cytoskeleton by electron cryotomography

摘要 细菌几乎无处不在，在工业和环境中发挥着重要作用， 对人类和其他生物体的健康和疾病都有重要影响。鉴于其 重要是，令人惊讶的是，我们仍然对基本的细菌细胞不了解 生物学例如，我们仍然不知道细菌是如何产生和维持它们的 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，在某些情况下移动。在真核生物中，所有这些任务 细胞骨架丝进行，但由于以前的成像技术未能 揭示了细菌的类似结构，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。在第一轮拨款中，我们使用了另一个年轻的 技术，电子冷冻断层扫描（ECT），以产生三维 接近天然状态的完整细菌细胞的“分子”图像（~4-6 nm）分辨率。使用ECT，我们直接观察到数百个细胞骨架丝， ~20种不同的细菌物种，证明细菌的细胞骨架实际上是两种 一般和复杂。在这里，我们建议开发和应用新的相关光， 电子显微镜技术来识别这些细丝。这应该允许 我们解决现有的光学和电子显微镜之间的关键差异， 结果，并产生急需的洞察力的结构和功能， 细菌细胞骨架我们还提出了提高EM图像质量的工作， 扩大正在研究的细丝的数量。

Abstract 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 threedimensional 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 generallyand expand the number of filaments being studied.