Isolation of mitotic spindles from Chinese hamster ovary cells.

Abstract

ABSTRACT. Mitotic spindles were isolated from Chinese hamster ovary cells with an isolation medium that contained 5% glycerol, 1% dimethyl sulfoxide, 5% polyethylene glycol, 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM ethylene glycol bis(f3-aminoethylether)tetraacetic acid, 0.5 mM MgSO4, 0.5% Triton X-100, 0.5 mM GTP and 0.5 mM dithiothreitol at pH 6.8. The chromosomes generally were lost from the preparation; thus, electron microscopy of whole-mount preparations showed isolated spindles with a microtubule array in the spindle that attached to the centrosomes at each pole.An increase in the concentration of MgSO4 to 5 mM stabilized the chromosomal structure, and mitotic spindles with chromosomes attached in almost the same configuration as in vivo could be isolated.A general discussion of the procedures for isolating mitotic spindles from tissue cultured cells is included.The isolation of mitotic spindles from dividing cells essentially involves four separate processes : preparation of pure mitotic cell populations, stabilization of the spindle structure, lysis of cells, and separation of the whole structure of the stabilized spindles from the other cellular components. Most studies on spindle isolataion have been done with dividing sea urchin eggs. These cells are particularly suitable for the isolation of mitotic cell populations because of their natural synchrony and because they are easily lysed by gentle agitation or by exposure to a slightly hypotonic solution.Mitotic spindles are, in general, very labile and should be stabilized before their release from the cells. In the first successful preparation of the mitotic apparatus from sea urchin eggs, Mazia and Dan (16) used cold ethanol to fix the structure. Subsequent attempts to improve the isolating procedure have been focussed on developing better stabilizing agents. Of those tried, hexylene glycol-containing solution has been one of the most widely used in studies that have provided a wealth of information on this organelle. This method, originally devised by Kane in 1965 (11) for sea urchin eggs, also has been adapted to other cleaving eggs and to embryos of invertebrates (1,(18)(19)(20).Abbreviations used: DMSO, dimethyl sulfoxide; PEG, polyethylene glycol; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); EGTA, ethylene glycol bis(/3-aminoethylether) tetraacetic acid; DTT, dithiothreitol; CHO, Chinese hamster ovary Present Address: