We have implemented the technique of automated electron tomography in a 300 kV field-emission TEM equipped with a post-column energy-filter to determine the three-dimensional arrangement of supramolecular assemblies and subcellular cellular organelles. Series of unfiltered or energy-filtered digital images each containing 2048 x 2048 pixels were acquired using a cooled CCD camera over a specimen tilt range from -70 degrees to +70 degrees and for dual tilt axes rotated by 90 degrees. These image series were analyzed using the IMOD program (University of Colorado) to obtain three-dimensional reconstructions. ? ? We have applied electron tomography to determine the three-dimensional arrangement of proteins in post-synaptic densities (PSDs) of cultured neurons from rat brain prepared by high-pressure freezing, freeze-substitution and heavy metal staining. The structures of interest associated with the PSDs include CAM kinase II, scaffolding proteins as well as cytoskeletal fibers. ? ? To complement studies being conducted by NIDDK investigators to understand defects in the endocytic trafficking pathway that occur in Niemann-Pick Type C Disease, we are determining the cellular ultrastructure of endosomal vesicles and endosomal tubules in cultured fibroblasts and CHO cellls containing wild-type or mutant NPC protein. The reconstructions show multi-lamellar tubules that appear to correlate with late endosomal tubules previously observed in fluorescent light microscopy of labeled cells.
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| Daniell, Sarah J; Kocsis, Eva; Morris, Edward et al. (2003) 3D structure of EspA filaments from enteropathogenic Escherichia coli. Mol Microbiol 49:301-8 |
