The mitotic spindle of Chinese hamster ovary cells isolated in taxol-containing medium

• Published in: Journal of cell science Vol. 66; no. 1; pp. 265 - 275
• Main Authors: KURIYAMA, R, KERYER, G, BORISY, G. G
• Format: Journal Article
• Language: English
• Published: Cambridge Company of Biologists 01.03.1984
• Subjects: Alkaloids; Animals; Biological and medical sciences; Cell Fractionation - methods; Cell Line; Cell structures and functions; Cricetinae; Cricetulus; Cytoskeleton, cytoplasm. Intracellular movements; Female; Fundamental and applied biological sciences. Psychology; HeLa Cells - ultrastructure; Humans; Microtubules; Molecular and cellular biology; Ovary; Paclitaxel; Spindle Apparatus - ultrastructure; Cell line CHO; Microtubule; Electron microscopy; Mitotic spindle
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.66.1.265

Mitotic spindles from Chinese hamster ovary (CHO) cells were isolated and purified by a one-step procedure in an isolation medium containing the microtubule-stabilizing drug, taxol. Released mitotic spindles were examined by phase-contrast, polarizing and differential-interference microscopy. They were also stained with monoclonal antibody raised against yeast tubulin and examined by epifluorescence microscopy. The spindles were free from visible cytoplasmic contaminants and the chromosomes were generally lost from the preparations. Electron microscopy showed that microtubules were the dominant structural component and sodium dodecyl sulphate/gel electrophoresis showed that tubulin was the major molecular species present, although a number of minor components, possibly representing microtubule-associated proteins (MAPs), were present. The taxol procedure was also useful in obtaining other microtubule-containing structures such as the midbody or the cytoplasmic microtubule complex in interphase cells. The taxol procedure was also used to isolate mitotic spindles from HeLa cells. The HeLa spindles stained positively with an antibody specific for the 210 X 10(3) Mr microtubule-associated protein, indicating that the MAP was retained by the taxol procedure. The taxol procedure appears to be of great advantage in large-scale preparations of spindles for biochemical analysis.