In the preceding paper we described pathways of mitotic spindle assembly in cell-free extracts prepared from eggs of Xenopus laevis. Here we demonstrate the poleward flux of microtubules in spindles assembled in vitro, using a photoactivatable fluorescein covalently coupled to tubulin and multi-channel fluorescence videomicroscopy. After local photoactivation of fluorescence by UV microbeam, we observed poleward movement of fluorescein-marked microtubules at a rate of 3 microns/min, similar to rates of chromosome movement and spindle elongation during prometaphase and anaphase. This movement could be blocked by the addition of millimolar AMP-PNP but was not affected by concentrations of vanadate up to 150 microM, suggesting that poleward flux may be driven by a microtubule motor similar to kinesin. In contrast to previous results obtained in vivo (Mitchison, T. J. 1989. J. Cell Biol. 109:637-652), poleward flux in vitro appears to occur independently of kinetochores or kinetochore microtubules, and therefore may be a general property of relatively stable microtubules within the spindle. We find that microtubules moving towards poles are dynamic structures, and we have estimated the average half-life of fluxing microtubules in vitro to be between approximately 75 and 100 s. We discuss these results with regard to the function of poleward flux in spindle movements in anaphase and prometaphase.
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1 March 1991
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March 01 1991
Poleward microtubule flux mitotic spindles assembled in vitro.
K E Sawin,
K E Sawin
Department of Biochemistry, University of California, San Francisco 94143.
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T J Mitchison
T J Mitchison
Department of Biochemistry, University of California, San Francisco 94143.
Search for other works by this author on:
K E Sawin
Department of Biochemistry, University of California, San Francisco 94143.
T J Mitchison
Department of Biochemistry, University of California, San Francisco 94143.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1991) 112 (5): 941–954.
Citation
K E Sawin, T J Mitchison; Poleward microtubule flux mitotic spindles assembled in vitro.. J Cell Biol 1 March 1991; 112 (5): 941–954. doi: https://doi.org/10.1083/jcb.112.5.941
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