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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010827-05
Application #
8349190
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2011
Total Cost
$298,293
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Borgnia, Mario J; Banerjee, Soojay; Merk, Alan et al. (2016) Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase. Mol Pharmacol 89:645-51
Schauder, David M; Kuybeda, Oleg; Zhang, Jinjin et al. (2013) Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci U S A 110:5921-6
Milne, Jacqueline L S; Borgnia, Mario J; Bartesaghi, Alberto et al. (2013) Cryo-electron microscopy--a primer for the non-microscopist. FEBS J 280:28-45
Tran, Erin E H; Borgnia, Mario J; Kuybeda, Oleg et al. (2012) Structural mechanism of trimeric HIV-1 envelope glycoprotein activation. PLoS Pathog 8:e1002797
Khursigara, Cezar M; Lan, Ganhui; Neumann, Silke et al. (2011) Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response. EMBO J 30:1719-29
Butan, Carmen; Hartnell, Lisa M; Fenton, Andrew K et al. (2011) Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus. J Bacteriol 193:1341-50
Heymann, Jurgen A W; Shi, Dan; Kim, Sang et al. (2009) 3D imaging of mammalian cells with ion-abrasion scanning electron microscopy. J Struct Biol 166:1-7
Hildebrand, Mark; Kim, Sang; Shi, Dan et al. (2009) 3D imaging of diatoms with ion-abrasion scanning electron microscopy. J Struct Biol 166:316-28
Milne, Jacqueline L S; Subramaniam, Sriram (2009) Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol 7:666-75
Borgnia, Mario J; Subramaniam, Sriram; Milne, Jacqueline L S (2008) Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography. J Bacteriol 190:2588-96

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