Mitotic spindles from Drosophila melanogaster embryos

Milsted, Amy; Cohen, William

doi:10.1016/0014-4827(73)90064-5

Cited by 10 publications

(3 citation statements)

References 14 publications

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“…For one, CANDE et al (2) and MCINTOSH et al (19) have studied partially lysed cells in which chromosome movement was maintained; however, this system has not yet been developed so that one can study the chemistry of the movement process. In another approach, mitotic apparatus (MA) have been isolated using brain tubulin as stabilising agent (15,20,21), but chromosome movement has not been reported in such isolated MA. In yet another approach, MA have been isolated using glycerol-dimethylsulphoxide (7,8,9,10) or using glycerol alone (22,23,24,25) as lysing-stabilising medium.…”

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Stability of Isolated Mitotic Apparatus During Storage*

Forer¹,

Zimmerman²

1978

Dev Growth Differ

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Mitotic apparatus (MA) were isolated from sea urchin zygotes using various isolation procedures, and various properties of the isolated MA were studied and compared. MA isolated using hexylene glycol had birefringences which depended on the pH of the isolation medium, the lower the pH the higher the MA birefringence. The stability of the MA birefringence also depended on the pH of the hexylene glycol isolation medium (the lower the pH the slower the rate of decay of birefringence), as did the final birefringence reached after prolonged storage. MA isolated using glycerol-dimethylsulphoxide (MTME) had much more stable birefringence than MA isolated using hexylene glycol; their birefringence decay rates were about 1000 times slower than those of MA isolated using hexylene glycol.Birefringence which remained after extraction of MA with H20, or 0.5 M KCl was also studied; the results depended on the MA isolation medium, on the medium the MA were stored in, and on the amount of time the MA were stored after isolation, as described in detail in the text.These results are discussed, and it is suggested that several components (including, perhaps, oriented ribosomes) contribute to birefringence of isolated MA.

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confidence: 99%

Stability of Isolated Mitotic Apparatus During Storage*

Forer¹,

Zimmerman²

1978

Dev Growth Differ

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“…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) Further progress on microtubule polymerization in vitro has made it possible to isolate the mitotic apparatus under conditions that are close to those of the native state. Isolation media containing glycerol (22), glycerol and dimethyl sulfoxide (DMSO) (4), or microtubule polymerization buffer only (10,21), have been shown to be effective for the preparation of a mitotic apparatus that resembles that of the living cell.…”

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confidence: 99%

“…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: …”

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Isolation of mitotic spindles from Chinese hamster ovary cells.

Kuriyama

1982

Cell Struct. Funct.

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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:

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Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo

Warn

1986

Experimental Cell Research

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Mitotic spindles from Drosophila melanogaster embryos

Cited by 10 publications

References 14 publications

Stability of Isolated Mitotic Apparatus During Storage*

Stability of Isolated Mitotic Apparatus During Storage*

Isolation of mitotic spindles from Chinese hamster ovary cells.

Microtubule arrays present during the syncytial and cellular blastoderm stages of the early Drosophila embryo

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