Drug-synchronized monopolar cytokinesis in HeLa cells

Cytokinesis, the fi nal step of cell division, requires regionally specialized activities of the microtubule and actin cytoskeletons that are precisely regulated in space and time. We now have a nearly complete “ parts list ” of cytokinesis proteins ( Eggert et al., 2006 ); however, our understanding of the wiring diagram that interconnects cytokinesis proteins is still rudimentary. For example, we do not know why regulators such as kinesin-6 are selectively delivered on some microtubules and not others. This selectivity presumably requires some unknown biochemical differentiation of microtubules oriented toward the equator. Moreover, most reviews discuss a linear pathway from microtubules to actomyosin ( D ’ Avino et al., 2005 ; Glotzer, 2005 ; Yuce et al., 2005 ). Our data calls this linear view into question and implies the existence of positive feedbacks with a central role in dynamic organization. Bipolar microtubule organization plays a central role in furrow positioning. The midpoint between two asters tends to organize a furrow ( Rappaport, 1996 ). It was thus a surprise when Canman et al. (2003) showed that a single aster could initiate a furrow on one side of PtK cells, prompting the question of how the symmetric aster could position an asymmetrical furrow. Monopolar mitotic PtK cells tend to cluster chromosomes on one side of the aster and the furrow always forms on the same side as the chromosomes. They proposed that asymmetrically located chromosomes stabilized microtubules in their vicinity and that these stable microtubules triggered furrow assembly. However, this model of signaling from chromosomes was not proven and signaling molecules were not identifi ed. The system developed by Canman et al. (2003) was infl exible in that it required microinjection. We developed a convenient drugsynchronization protocol in HeLa cells. HeLa cells arrest in monopolar mitosis with chromosomes symmetrically distributed around the aster, so there is no preexisting directional bias as in PtK cells. Triggering monopolar cytokinesis caused HeLa cells to break symmetry and polarize essentially every aspect of their internal organization. We investigated polarization mechanisms and discuss their implications for normal cytokinesis.