MAP2 competes with MAP1 for binding to microtubules.

A question whether MAP1 and MAP2 (the major microtubule associated proteins from mammalian brain) bind to common or distinct sites on the microtubule surface was studied. Microtubules were assembled from tubulin and MAP1 and then centrifuged through a layer of MAP2 solution under conditions where no repolymerization of tubulin with MAP2 could occur. During centrifugation, MAP2 displaced most of MAP1 on the microtubules. This implies that MAP1 is reversibly bound to microtubules and that MAP2 binding interferes with MAP1 binding. The latter means that binding sites for MAP1 and MAP2 are identical or overlap.

High molecular weight protein MAP 2 promoting microtubule assembly in vitro is associated with microtubules in cells.

Brain high molecular weight (HMW) protein promoting microtubule assembly in vitro and identical to MAP 2 (one of the proteins which copurify with tubulin through microtubule assembly-disassembly cycles), is shown to be associated with microtubules in interphase and mitotic cells. This HMW protein was purified earlier (Kuznetsov et al., 1978), directly from bovine brain without previous obtaining total microtubule protein. Now we have obtained a monospecific antibody against it. Identity of the HMW protein with MAP 2 is inferred from SDS-electrophoresis and immunological tests; its intracellular localization is determined by indirect immunofluorescent staining of cultured bovine tracheal epithelium. the anti-MAP 2 antibody stains the same structures in the cells as the tubulin antibody: it stains the fibrillar network in interphase cells, mitotic spindle, and the stem body. No fibrillar structures in the cells treated with colchicine or vinblastine were stained with the antibody against MAP 2. Anti-MAP 2 also stains tubulin-containing paracrystals which have been formed in the vinblastine-treated cells. Therefore HMW protein MAP 2 which promotes tubulin polymerization in vitro is associated with microtubules in vivo.

Microtubular system in cultured mouse epithelial cells.

Previous observations had shown that colcemid does not affect locomotion of epithelial cells. Nevertheless, cultured mouse kidney cells forming epithelial sheets were found to contain a well-developed microtubular system sensitive to colcemid. The orientation of microtubules in the epithelial cells was not correlated with the stable or active state of the cell edges. It is suggested that microtubular system of the epithelial cells forming coherent sheets, in contrast to that of individually moving fibroblasts, is not essential for stabilization of the lateral cell edges.

The effect of non-ionic detergent tween 80 on colcemid-resistant transformed mouse cells in vitro.

The effect of the non-ionic detergent tween 80 on one colcemid-resistant and one sensitive subline of mouse L cells has been studied. The colcemid-resistant subline was also resistant to colchicine and Vinca alkaloids. Tween 80 at concentrations of 0.01% (v/v) or higher increased the sensitivity of drug-resistant cells to the antimitotic effect of colcemid, colchicine and vinblastine. Tween 80 also potentiated the initial rate and the maximal level of the (3-H)-cholchicine uptake by both sensitive and resistant cells. However, the detergent at concentrations of up to 1% had no effect on (3-H)-colchicine binding by cell homogenates. Thus it appears that the effects of tween 80 were due to an increase in cell membrane permeability to the drugs. The effect was completely reversible. The cells did not become adapted to the sensitizing action of tween 80 even after prolonged incubation in medium containing the detergent. A considerable increase in the permeability to the drug was obtained with doses of tween 80 which were non-toxic and which had no effect on cell proliferation, morphology and locomotion. The increase in membrane permeability caused by tween 80 at these concentrations was selective, the membrane permeability to ions and to (3-H)-2-deowy-D-glucose being, in fact, unchanged.