Over the last year we have continued to make progress both using ion abrasion scanning electron microscopy and cryo-electron tomography. Collaborative projects on 3D imaging of melanoma cells, diatoms, macrophages and dendritic cells have successfully demonstrated that this is a very powerful approach for quantitative description of subcellular architecture. These studies have shown that the resolution of imaging is high enough to locate individual HIV particles within cells of the immune system. Mapping of patterns of their localization in whole cells has provided surprising insights into HIV distribution and delivery to T-cells. Ongoing studies to extend these studies to map mitochondrial architecture and shape changes in normal and diseased cells are underway. Cryo-electron tomographic studies to map the structure of the interior of Bdellovibrio cells have also been very productive. Visualizing the 3D structure of the bacterial nucleoid under near native conditions is essential to map dynamic changes in nucleoid structure that occur during cell growth and division. Bacterial chromosomes have been proposed to form ring-like structures or coiled spiral arrangements. Our new results provide the first in-depth visualization of bacterial nucleoids in wild-type cells and in a mutant strain in which the normal MreB2 gene has been replaced with a GFP-tagged version. The mutant cells have a striking helical arrangement of the nucleoid, which is more open than that typically seen in wild-type cells. The more open structure of the MreB2-mTFP nucleoids enabled the in situ visualization of 10 nm-wide domains within and at the edge of the nucleoid that were decorated with ribosomes. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Unexpectedly, our immunolocalization experiments provide evidence that a bacterial MreB is associated with the edges of the nucleoid. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles, and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest other roles for MreB, and raise the intriguing possibility that bacterial MreB proteins may act not merely as cytoskeletal elements, but rather as regulators of nucleoid architecture, with a range of potential functions, as is the case with eukaryotic nuclear actins.
Showing the most recent 10 out of 11 publications
