Microtubule-associated proteins connect microtubules and neurofilaments in vitro

Neuronal intermediate filaments (neurofilaments) prepared from brain form a viscous sedimentable complex with microtubules under suitable conditions [Runge, M.S., Laue, T.M., Yphantis, D.A., Lifsics, M.R., Saito, A., Altin, M., Reinke, K., & Williams, R.C., Jr. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 1431-1435]. 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 two 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 two filamentous organelles. By means of gel filtration, the MAPs were separated into two 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 that the MAPs serve as cross-bridges between the two structures. Neurofilaments, with and without bound MAPs, were allowed to adhere to electron microscope 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 one or more neurofilaments. The grids prepared with untreated neurofilaments lacked microtubules. These results show that one or more of the MAPs mediates association between microtubules and neurofilaments.