Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520.
Department of Microbial Pathogenesis, School of Medicine, Yale University, New Haven, CT 06536.
Proc Natl Acad Sci U S A. 2017 Jun 6;114(23):6098-6103. doi: 10.1073/pnas.1705823114. Epub 2017 May 22.
Type III protein secretion machines have evolved to deliver bacterially encoded effector proteins into eukaryotic cells. Although electron microscopy has provided a detailed view of these machines in isolation or fixed samples, little is known about their organization in live bacteria. Here we report the visualization and characterization of the type III secretion machine in live bacteria by 2D and 3D single-molecule switching superresolution microscopy. This approach provided access to transient components of this machine, which previously could not be analyzed. We determined the subcellular distribution of individual machines, the stoichiometry of the different components of this machine in situ, and the spatial distribution of the substrates of this machine before secretion. Furthermore, by visualizing this machine in mutants we obtained major insights into the machine's assembly. This study bridges a major resolution gap in the visualization of this nanomachine and may serve as a paradigm for the examination of other bacterially encoded molecular machines.
III 型蛋白分泌机器已经进化到可以将细菌编码的效应蛋白输送到真核细胞中。尽管电子显微镜已经提供了这些机器在分离或固定样本中的详细视图，但对于它们在活细菌中的组织知之甚少。在这里，我们通过二维和三维单分子切换超分辨率显微镜报告了活细菌中 III 型分泌机器的可视化和表征。这种方法提供了对该机器的瞬态组件的访问，这些组件以前无法进行分析。我们确定了单个机器的亚细胞分布、该机器不同组件的原位计量以及分泌前该机器底物的空间分布。此外，通过在突变体中可视化该机器，我们获得了对机器组装的主要见解。这项研究弥合了可视化该纳米机器的一个主要分辨率差距，并可能成为检查其他细菌编码分子机器的范例。
