Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins

Abstract

In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations.