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Published online 4 September 2000. doi:10.1083/jcb.150.5.975
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© The Rockefeller University Press, 0021-9525/2000//975 $5.00
The Journal of Cell Biology, Volume 150, Number 5, , 2000 975-988

Original Article

Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the Mitotic Kinesin, Eg5



Tarun M. Kapoora, Thomas U. Mayera, Margaret L. Coughlina, and Timothy J. Mitchisona,b

a Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
b Institute of Chemistry and Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115.(617) 432-3702(617) 432-3728

tarun_kapoor{at}hms.harvard.edu

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest–deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.

Key Words: monastrol • Eg5 • kinesin • MAD2 • kinetochore

© 2000 The Rockefeller University Press

The online version of this article contains supplemental material.

Abbreviations used in this paper: CSF, cytostatic factor; DIC, differential interference contrast; MAD, mitotic arrest–deficient; NuMA, nuclear/mitotic apparatus protein.


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