Phosphorylation and subunit organization of axonal neurofilaments determined by scanning transmission electron microscopy

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Abstract
Phosphorylation plays a critical role in controlling the function of cytoskeletal assemblies but no direct method yet exists to measure the phosphorylation state of proteins at the level of individual molecules and assemblies. Herein, we apply scanning transmission electron microscopy in combination with electron energy loss spectroscopy to measure the distributions of mass and phosphorus in neurofilaments (NFs) isolated from the squid giant axon. We find that native squid NFs, in contrast to typical reconstituted intermediate filaments, are a relatively homogeneous population containing only eight coiled-coil dimers per cross section. The measured stoichiometry of ∼1:1 for light/heavy peptides strongly suggests that squid NFs are composed of heterodimers. Furthermore, each heavy chain of the dimers carries at least 100 phosphate groups and is, therefore, near-maximally phosphorylated. These results also demonstrate that scanning transmission electron microscopy combined with electron energy loss spectroscopy at the nanometer scale is capable of characterizing the level and distribution of phosphorylation in individual mass-mapped protein assemblies.