Focused ion beam scanning electron microscopy (FIB-SEM), also referred to as ion abrasion scanning electron microscopy (IA-SEM), is a technology that we have been developing in the lab to image cells and tissues in 3D at high resolution. Imaging cells and tissues by FIB-SEM at high resolution offers many exciting possibilities for biological research; however, at high resolution, this technology produces enormous amounts of data, and is extremely slow. Moreover, one of the most promising aspects of this technology is the ability to quantitatively analyze ultrastructural morphology. Thus in addition to using FIB-SEM to study 3D architecture in cells and tissues, we have also been developing imaging methods and techniques that align the technology with the goal of automated, quantitative analysis of 3D structure at electron microscopy resolutions. While developing these techniques, we have also applied the FIB-SEM technology to new biological problems. One exciting area of FIB-SEM analysis has been a collaboration with the Balaban laboratory in NHLBI to visualize the 3D mitochondrial network in muscle cells. In a 3D study of the mitochondria in skeletal muscle we published in 2015, we showed that the mitochondrial reticulum network extends directly and contiguously from the area surrounding the blood vessel deep into the muscle fiber, enabling direct energy transfer via electrical conduction within the mitochondria. This past year, we have followed up our skeletal muscle study with a similar investigation of mitochondrial networks in cardiac muscle. Skeletal and cardiac muscle have mitochondrial networks with similar architectures; interestingly, our 3D EM work showed a much higher concentration of mitochondria in the perivascular region in cardiac muscle than in skeletal muscle, which aligns with the high and constant energy requirements for this tissue type. Unlike skeletal muscle, where all the mitochondria compose a single network, the cardiac muscle is divided into smaller subnetworks running along the long axis of the cell, which are only occasionally joined by an intermitochondrial junction. This structure, critically, appears to protect the cardiac muscle tissue from cascading damage. For example, while dysfunction in a single area of the mitochdronial network in skeletal muscle could perpetuate throughout the entire cell via the interconnected network, the subnetworks we observed in cardiac muscle do not allow for electrical conduction between subregions of the network, thus limiting the scope of any mitochondrial dysfunction in this critical tissue. Building upon these advances, we are now engaged in an active collaboration with Dr. Luigi Ferrucci at the National Institute of Aging, aimed at understanding the differences in skeletal muscle morphology in humans during aging. Although this investigation is still in its early stages, the use of FIB-SEM technology to explore differences in mitochondrial density, network formation, and connectivity at different stages during the aging process is already beginning to provide new and unexpected insights into the biological processes that may be relevant in aging.
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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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