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Vol. 18, Issue 3, 850-863, March 2007

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Mad2-independent Spindle Assembly Checkpoint Activation and Controlled Metaphase–Anaphase Transition in Drosophila S2 Cells

Bernardo Orr^*, Hassan Bousbaa, and Claudio E. Sunkel^*,

*Instituto de Biologia Molecular e Celular, 4150-180 Porto, Portugal; Instituto Superior de Ciências da Saúde-Norte, Grupo de Biologia Molecular e Celular, 4580 Gandra PRD, Portugal; and Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, 4000 Porto, Portugal

Submitted July 10, 2006; Revised November 27, 2006; Accepted December 3, 2006
Monitoring Editor: Orna Cohen-Fix

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase–anaphase transition and normal patterns of chromosome segregation.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Claudio E. Sunkel (cesunkel{at}ibmc.up.pt)

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