Over the last couple of years, we have demonstrated that ion abrasion scanning electron microscopy (IA-SEM) can provide powerful new insights into subcelluar architecture. We have imaged MNT-1 melanoma cells to obtain new insights into statistical analysis of the size, shape and compositional analysis of organelles could provide valuable diagnostic markers for discriminating normal cells from abnormal cells. We reported the first application of IA-SEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Our study provided new insights into the architecture and assembly principles of both the hard (siliceous) and soft (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we showed that both micro- and nanoscale siliceous structures could be visualized at specific stages in their formation. More recently we have investigated structural origins of methylmalonic academia, a lethal inborn error of metabolism that causes mitochondrial impairment, multi-organ dysfunction and a shortened lifespan. Previous transmission electron microscope studies of thin sections from normal (Mut +/+) and diseased (Mut -/-) tissue found that the mitochondria appear to occupy a progressively larger volume of mutant cells with age, becoming megamitochondria. To assess changes in shape and volume of mitochondria resulting from the mutation, we carried out ion-abrasionscanning electron microscopy (IASEM), a method for 3D imaging that involves the iterative use of a focused gallium ion beam to abrade the surface of the specimen, and a scanning electron beam to image the newly exposed surface. Using IASEM, we showed that mitochondria are more convoluted and have a broader distribution of sizes in the mutant tissue. Compared to normal cells, mitochondria from mutant cells have a larger surface-area-to-volume ratio, which can be attributed to their convoluted shape and not to their elongation or reduced volume. The 3D imaging approach and image analysis we have developed could therefore be useful as a diagnostic tool for the evaluation of disease progression in aberrant cells at resolutions higher than that currently achieved using confocal light microscopy. Investigation of the 3D structure of HIV-infected macrophages and dendritic cells have led to surprising discoveries of the localization of HIV in these antigen-presenting cells demonstrating the importance of continued technological developents for 3D cellular imaging. Our findings show that invaginations in antigen presenting cells retain the virus in spaces that communicate with the external medium, ready for transfer to T-cells at functional virological synapses. Our electron microscopic experiments were designed to obtain structural snapshots at the earliest stages of cell-cell contact, formed under in vitro conditions similar to where most of the earlier mechanistic studies on dendritic cell-T-cell and T-cell-T-cell virological synapses have been carried out. Although the physiological relevance of HIV transmission by cell-to-cell spread in vivo is not fully established, this mode of transmission is plausible given the strong evidence from in vitro studies. Even if virus transfer to the T-cell can occur to some extent outside the context of these contacts, the finding that 50% of cell contacts at the synapse appear to involve filopodial insertions into the dendritic cell membrane suggest that viral transfer in these secluded regions will be an important factor. A possible consequence of the wrapping of the T-cell by the sheet-like envelopes is the reduction in the effective concentration of exogenously added reagents at the site of virus transfer, offering an explanation for the relative inability of neutralizing antibodies to block viral infection at dendritic cell-T-cell synapses in some instances. Inhibition of viral entry into CD4+ T-cells by therapeutic intervention may thus require higher drug concentrations and local targeting, emphasizing the importance of pursuing complementary strategies aimed at neutralizing viruses at earlier stages in the process of infection such as virus uptake and delivery to T-cells.
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| Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui et al. (2017) Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex. Cell 171:414-426.e12 |
| Subramaniam, Sriram; Earl, Lesley A; Falconieri, Veronica et al. (2016) Resolution advances in cryo-EM enable application to drug discovery. Curr Opin Struct Biol 41:194-202 |
| Matthies, Doreen; Dalmas, Olivier; Borgnia, Mario J et al. (2016) Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating. Cell 164:747-56 |
| Merk, Alan; Bartesaghi, Alberto; Banerjee, Soojay et al. (2016) Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery. Cell 165:1698-1707 |
| Glancy, Brian; Hartnell, Lisa M; Malide, Daniela et al. (2015) Mitochondrial reticulum for cellular energy distribution in muscle. Nature 523:617-20 |
| Narayan, Kedar; Subramaniam, Sriram (2015) Focused ion beams in biology. Nat Methods 12:1021-31 |
| Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken et al. (2014) Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. J Struct Biol 185:278-84 |
| Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar et al. (2014) Structural mechanism of glutamate receptor activation and desensitization. Nature 514:328-34 |
| 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 |
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