| In this thesis, we examine interactions between neurofilaments (NFs), intermediate filaments which represent the most abundant cytoskeletal element of large, myelinated axons. One model for the organization of axonal NFs proposes that the C-terminal sidearm domains of NFs are unstructured and mediate interfilament repulsion through mutual steric exclusion (so-called polymer brush repulsion). Here, we assess that model through analysis and simulation of axonal NF distributions and experiments with isolated mammalian NFs. The first part of this thesis describes a novel analytical framework in which the cross-sectional organization of NFs in axons is characterized by radial distribution functions and occupancy probability distributions interpreted using information theory. This analysis reveals that NF organization in the axon can be represented in terms of NF-NF pair correlations. We then develop NF-NF pair potentials from polymer brush theory and conduct two-dimensional Monte Carlo simulations to explore the relationship between the biochemical properties of the sidearms and NF organization. These simulations permit the recapitulation of key features of NF organization starting from an NF-NF pair potential. We find that NF-NF repulsion determined by the thickness of NF sidearm layers, mediated in turn by the degree of sidearm phosphorylation, is capable of serving as a strong determinant of NF organization.; In the second part of the thesis, we apply our method to explore the biophysical origin of aberrant NF organization in mice genetically deficient in myelin-associated glycoprotein (MAG −/−). Surprisingly, we find that NFs in MAG −/− axons are more weakly organized in spite of the fact that they are more densely packed. This result is best explained by a model in which the NFs interact through repulsive brush-like forces and in which MAG −/− NFs are less heavily phosphorylated than wild-type NFs, repel one another more weakly, and are therefore less strongly organized.; Finally, we present experimental studies in which NF sidearm structure is manipulated through changes in buffer conditions and enzymatic dephosphorylation. We find that NF excluded volume falls in response to enzymatic dephosphorylation, increasing ionic strength and increasing divalent cation concentration. Atomic force microscopy imaging reveals that native NFs produce strong steric exclusion even at high ionic strength whereas dephosphorylated NFs do not. Together, the experimental data are consistent with a model in which sidearm phosphorylation serves as a graded switch which controls sidearm thickness via modulation of local steric and electrostatic interactions. |
