Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization.

Abstract

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 .mu.m) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tublin. Only the supernates from differentiated N18 cells were polymerization competent. EM observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at .apprx. 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 MW proteins. The inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.