Microtubule-associated proteins connect microtubules and neurofilaments in vitro

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Abstract
Neuronal intermediate filaments (neurofilaments) prepared from [bovine] brain form a viscous, sedimentable complex with microtubules under suitable conditions. Under the same conditions, neurofilaments prepared from spinal cord did not form such a complex. Brain neurofilaments were shown to differ from spinal cord neurofilaments in part by having proteins that resemble microtubule-associated proteins (MAPs) attached to them. MAPs became bound to spinal cord neurofilaments when the 2 structures were incubated together. The resulting MAP-decorated neurofilaments formed a viscous complex with microtubules, showing that some component of the MAPs mediated the association between the 2 filamentous organelles. By means of gel filtration, the MAPs were separated into 2 major fractions. The large Stokes radius fraction was active in producing neurofilament-microtubule mixtures of high viscosity, while the small Stokes radius fraction was not. The dependence of the viscosity of neurofilament-microtubule mixtures upon the concentration of MAPs was found to possess a maximum. This result suggests the MAPs serve as cross-bridges between the 2 structures. Neurofilaments, with and without bound MAPs, were allowed to adhere to EM grids. The grids were then exposed to microtubules, fixed, and stained. The grids prepared with MAP-decorated neurofilaments bound numerous microtubules, each in apparent contact with 1 or more neurofilaments. The grids prepared with untreated neurofilaments lacked microtubules. These results show that 1 or more of the MAPs mediates association between microtubules and neurofilaments.