Structures and Dynamics in Live Bacteria Revealed by Super-Resolution Fluorescence Microscopy
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CitationWang, Wenqin. 2012. Structures and Dynamics in Live Bacteria Revealed by Super-Resolution Fluorescence Microscopy. Doctoral dissertation, Harvard University.
AbstractLight microscopy, in particular fluorescence microscopy, is a widely used imaging method in biological research due to its noninvasive nature and molecular specificity. The resolution of conventional fluorescence microscopy is limited to a few hundred nanometers by the diffraction of light, leaving many biological structures too small to be optically resolved. Stochastic Optical Reconstruction Microscopy (STORM) technique overcomes this limit by localizing single photoswitchable fluorophores separated in time. We further extended the then two-dimensional capability to three-dimensional (3D) STORM by determining both axial and lateral positions of individual fluorophores with nanometer accuracy using optical astigmatism. Iterative, stochastic activation of photo-switchable probes enables high-precision 3D localization of each probe and thus the construction of a 3D image without scanning the sample. We achieved an image resolution of 20 - 30 nm in the lateral dimensions and 50 - 60 nm in the axial dimension. This development allowed us to resolve the 3D morphology of nanoscopic cellular structures. Enabled by the super-resolution imaging capability, we used 3D STORM in conjunction with biochemical assays to study structures and dynamics in live bacteria. Bacterial chromosomes are confined in submicron-sized nucleoids. Chromosome organization is facilitated by nucleoid-associated proteins (NAPs), but the structure of the chromosome and the molecular mechanisms underlying its organization are poorly understood, in part due to the lack of appropriate tools for visualizing the chromosome in vivo. Using STORM, we found that four NAPs, HU, Fis, IHF, and StpA, were largely scattered throughout the E. coli nucleoid. In contrast, H-NS, a global transcriptional silencer, formed two compact clusters per chromosome driven by oligomerization of DNA-bound H-NS, through their N-terminal domain interactions. H-NS sequestered the regulated operons into these clusters and juxtaposed numerous DNA segments broadly distributed throughout the chromosome. Deleting H-NS led to substantial chromosome reorganization. These observations demonstrate that H-NS plays a key role in global chromosome organization in E. coli. Finally, we describe the use of the same sub-diffraction localization for single-particle tracking to study MreB paralogs (actin-like proteins in bacteria) in B. subtilis. We found that MreB and the elongation machinery moved circumferentially around the cell, perpendicular to its length, with nearby synthesis complexes and MreB filaments moving independently in both directions. Inhibition of cell wall synthesis by various methods blocked the movement of MreB. Thus, bacteria elongate by the uncoordinated, circumferential movements of synthetic complexes that insert radial hoops of new peptidoglycan during their transit, possibly driving the motion of the underlying MreB filaments.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:9453705
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