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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

Keywords:
TEM, tomography, synapses, vesicles, proteins

Abstract:
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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