Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Abstract

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole-associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.