AN ASSAY FOR THE ACTIVITY OF MICROTUBULE-BASED MOTORS ON THE KINETOCHORES OF ISOLATED CHINESE-HAMSTER OVARY CHROMOSOMES

This chapter describes an assay for examining the behavior of microtubule based motors on the kinetochores of isolated Chinese hamster ovary (CHO) chromosomes. The assay of kinetochore activity relies on specific capture of microtubules by kinetochores, and trapping by chromosome arms obscures the assay. This trapping appears to be caused by adhesion of cytoskeletal fragments to the chromosome arms, not to chromatin. In general, the cleaner the chromosome preparation, the fewer the number of microtubules attached to the chromosome arms. The clean chromosomes are obtained only if the cells lyse within the first few strokes of the pestle. The assay is very sensitive to variables, such as time of incubation and washing. The low-ionic strength buffer minimizes extraction of loosely bound kinetochore components. The isolated chromosomes are adsorbed to the glass slide of a perfusion chamber. To assay the microtubule-based motor activity of the kinetochores of these isolated chromosomes, polarity-marked microtubules are attached to the kinetochores. After addition of ATP, the movement of these microtubules over the kinetochores of the isolated chromosomes is followed by video microscopy. © 1993, Elsevier Science Publishers, B.V.