Synthesis of a Colchicine-Binding Protein During the Hela Cell Life Cycle

Cells or tissues exposed to colchicine- 1 H bind the isotope in direct relation to the morphologically visible number of microtubules that they manifest in the electron microscope (1). Those species, such as Physarum polycephalum, which have no discernible microtubules do not bind colchicine. Homogenization of tissues followed by sucrose density gradient analysis have demonstrated that the colchicine-binding activity runs as a 6S protein (1). When the central pair of microtubules in sea urchin sperm tails is selectively solubilized with EDTA, the extracted protein is rich in colchicine-binding activity, migrates as a single peak on acrylamide gels and, like the colchicinebinding activity from tissue homogenates, has a sedimentation coefficient of 6S (2). The apparently selective binding of colchicine to microtubule protein provides a convenient means of identifying it in heterogeneous preparations. We have thus used a combination of colchicine- 1H binding and leucine- 14C incorporation to trace the synthesis of the colchicine-binding protein during the cell life cycle in synchronized HeLa cells. Since the mitotic spindle is composed of microtubules, we make the tentative assumption that the pattern of synthesis demonstrated for colchicine-binding protein is applicable to the mitotic spindle. MATERIALS AND METHODS HeLa cells of the Sa strain were routinely maintained in spinner culture as previously described. For synchronization, they were placed in monolayer culture containing only the Ca++ present in the serum supplement (3). Mitotic cells were selectively detached by gentle rocking as previously described (3) and placed in spinner culture. Aliquots of 107 cells were removed from spinner at 0, 3, 7, 9, and 15 hr after collection, washed in leucine-free medium and pulsed for 20 min with 15 °c of leucine C (New England Nuclear Corp., Boston) in 20 ml of leucinefree medium. These time points were chosen so that the populations were in the mitotic, G 1, early S, mid-S, and late S-G2 phases of their life cycle, respectively (3). Following exposure to the label, the cells were pelleted, washed, swollen in 2 ml of 0.01