In vitro polymerization of microtubules from HeLa cells.

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Abstract
Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro is routine, the application of this method to non-neural, cultured cells is less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. A method was developed for preparing microtubules from [human cervical cancer] HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through 2 complete cycles of polymerization at 37.degree. C and depolymerization at 4.degree. C in vitro. The microtubules obtained are morphologically similar to brain microtubules in EM, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the 2nd polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after 2 cycles of polymerization and depolymerization has an apparent MW of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.