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Conical Tomograph: A Method for the Study of Macromolecular Assemblies

L. Zampighi, N. Fain, S. Lanvazecchia, G.A. Zampighi
University of California, Los Angeles School of Medicine, US

TEM, tomography, synapses, vesicles, proteins

We have developed conical tomography to study the structure of macromolecular assemblies and organelles in their native cellular environment. Unlike the other related types of tomography, conical tomography has a higher throughput and more importantly, produces reconstructions that are isotropic in the x-y plane with a resolution of 2-3 nm. We have been able to collect complete conical series from a variety of sources, including thin sections and freeze-fractured replicas of chemical synapses from the rat somato-sensory cortex as well as isolated synaptic vesicle anti-SV2 complexes. We used volumetric representations and density segmentation methods to analyze sections, revealing a number of highly detailed structures: tubes and cisterns were found in the presynaptic terminal, as well as protein cages formed of SV-2 rings, circumscribing the synaptic vesicles, and a number of docked vesicles and omega figures. While conical tomography is a general technique, theoretically applicable to any types of structure on the molecular scale, we are currently focused on the synaptic terminal and the distribution of the ~60 proteins associated with it. Subsequent analysis promises to provide information about it's structure/function, including clues as to it's role in Alzheimer's, Parkinson's, drug addiction, and other diseases.

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