MASS MAPPING OF MACROMOLECULAR ASSEMBLIES

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Neurofilaments (NFs), a neuron-specific subtype of intermediate filament (IF), are an essential structural component of the axon. Although the sequence and secondary structure of NF subunits are established, the higher-level organization of subunits into ~10-nm- diameter filaments remains unknown, in part due to compositional heterogeneity and difficulties in obtaining native preparations. Scanning transmission electron microscopy (STEM) and parallel electron energy-loss spectroscopy (PEELS) have been used to measure the mass and phosphorus distributions of native NFs isolated from the squid giant axon. It was found that squid NFs contain only eight coiled-coil heterodimers per cross section (in contrast to the 16 dimers typical of reconstituted intermediate filaments), and that each heavy chain carries at least 40 phosphate groups. The results show that electron scattering measurements can be used to characterize subunit organization and phosphorylation levels in the same protein assembly.