This is a proposal to renew a P41 Research Resource grant that supports the Boulder Laboratory for 3-D Electron Microscopy of Cells and Macromolecular Structures. Our goals for research in technology (R&D) include the development of methods for reliable specimen preparation, image acquisition, and image processing. In addition, we provide service, training and dissemination of software and technology to the scientific community. Our methods comprise 3-D electron microscopy aimed to resolve the structure and function of cellular organelles and macromolecular assemblies. Our facility is unique in a sense that we are working on the interface between most advanced cryo-technologies applied to frozen-hydrated specimens revealing best possible molecular resolution in 3-D, and large-scale 3-D imaging typically provided by freeze-substitution and plastic embedding of cells and tissue. This way we provide a direct link between cellular structures and macromolecular detail and atomic-scale interpretation. Freeze-substitution and plastic embedding are excellent method for high-throughput 3-D imaging of large complex systems and entire cells to a resolution of approx. 4-5 nm. This is sufficient to unambiguously detect cellular structures such as microtubules, actin filaments, mitochondria, ER systems, and even macromolecular structures such as ribosomes or other large enzyme complexes. Tomographic 3-D imaging on frozen-hydrated specimens aims for most accurate molecular detail down to 2nm resolution or even beyond. That, however, requires focusing on much smaller volumes. In this renewal we propose a series of new methods that should improve our interpretations of frozen-hydrated specimens by employing novel high-electron dense labeling techniques, specifically designed for vitrified macromolecular and cellular samples, and correlative approaches combining fluorescence light microscopy with 3-D electron microscopy. We propose new computational procedures in our software packages such as volume-averaging of 3-D data picked from tomograms and a variety of processes that will improve the resolution of tomographic 3-D reconstructions, both, during data acquisition on the microscope, and for the alignment and image correction procedures.

Public Health Relevance

Curing a pathologic condition requires detailed molecular knowledge of the healthy state, which is a major goal of pre-clinical biomedical research like the work of our lab. Nevertheless, several of our collaborations are directly relevant to an understanding of pathologic processes in humans, such as our large-scale structural analyses of cardiomyocytes and of virus entry and replication. The data we will obtain will constitute an essential scientific basis that will in turn lead to new cures and treatments of many diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-CB-J (40))
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Swain, Amy L
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University of Colorado at Boulder
Schools of Arts and Sciences
United States
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Winey, Mark; O'Toole, Eileen (2014) Centriole structure. Philos Trans R Soc Lond B Biol Sci 369:
Winey, Mark; Meehl, Janet B; O'Toole, Eileen T et al. (2014) Conventional transmission electron microscopy. Mol Biol Cell 25:319-23
O'Toole, Eileen T; Dutcher, Susan K (2014) Site-specific basal body duplication in Chlamydomonas. Cytoskeleton (Hoboken) 71:108-18
Hepperla, Austin J; Willey, Patrick T; Coombes, Courtney E et al. (2014) Minus-end-directed Kinesin-14 motors align antiparallel microtubules to control metaphase spindle length. Dev Cell 31:61-72
Basta, Tamara; Wu, Hsin-Jui; Morphew, Mary K et al. (2014) Self-assembled lipid and membrane protein polyhedral nanoparticles. Proc Natl Acad Sci U S A 111:670-4
Maurer, Sebastian P; Fourniol, Franck J; Hoenger, Andreas et al. (2014) Seeded microtubule growth for cryoelectron microscopy of end-binding proteins. Methods Mol Biol 1136:247-60
Wu, Yumei; O'Toole, Eileen T; Girard, Martine et al. (2014) A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis. Elife 3:e01621
Briegel, Ariane; Pilhofer, Martin; Mastronarde, David N et al. (2013) The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards. J Struct Biol 183:95-8
Nazarova, Elena; O'Toole, Eileen; Kaitna, Susi et al. (2013) Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly. Mol Biol Cell 24:3238-50
Kamasaki, Tomoko; O'Toole, Eileen; Kita, Shigeo et al. (2013) Augmin-dependent microtubule nucleation at microtubule walls in the spindle. J Cell Biol 202:25-33

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