Structure and function of the prokaryotic cytoskeleton by electron cryotomography

摘要 细菌几乎无处不在，在工业和环境中扮演着至关重要的角色，而且 在人类和其他生物体的健康和疾病方面发挥着重要作用。鉴于他们的 重要的是，令人惊讶的是我们对基本细菌细胞仍然不了解 生物学。例如，我们仍然不知道细菌是如何产生和维持其 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 由细胞骨架细丝执行，但因为以前的成像技术未能 揭示了细菌中类似的结构，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已经 相当大的内部秩序。在这笔赠款的第一个周期中，我们使用了另一个年轻人 电子冷冻断层扫描(ECT)技术，以生产三维 处于接近天然状态的完整细菌细胞的图像为“分子”(~4-6 NM)分辨率。使用ECT，我们直接显示了成百上千条细胞骨架细丝 ~20种不同的细菌，证明细菌的细胞骨架实际上既是 既笼统又复杂。在这里，我们建议开发和应用新的相关光和 电子显微镜技术一般用来识别这些细丝。这应该允许 美国将解决现有光学显微镜和电子显微镜之间的关键差异 结果，并产生对 细菌细胞骨架。我们还提出了全面提高EM图像质量的工作和 扩大正在研究的细丝数量。

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.