Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5

Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin , Eg5, arrests cells in mitosis with monoastral spindles. Here, we use mona strol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplica tion. The mitotic arrest due to monastrol is also rapidly reversible. Chrom osomes 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 sub set of kinetochores, suggesting the activation of the spindle assembly chec kpoint in these cells. Mad2 localizes to some kinetochores that have attach ed microtubules in monastrol-treated cells, indicating that kinetochore mic rotubule 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 spindle s that form. Imaging bipolar spindles disassembling in the presence of mona strol allowed direct observations of outward directed forces in the spindle , orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to s tudy mitotic processes, detection and correction of chromosome malorientati on, and contributions of Eg5 to spindle assembly and maintenance.