Positioning the Plane of Cell Division: Spatiotemporal Regulation of RhoA at the Cortex

During cytokinesis, a cell furrows at a unique position between segregating chromosomes to successfully divide into two daughter cells. The mechanism by which a single, well-focused cleavage furrow is generated is inadequately understood. The small GTPase RhoA promotes the cortical recruitment and organization of actomyosin for furrow ingression. RhoA is activated by signals from the mitotic spindle. In particular, the centralspindlin complex, a heterotetramer of a kinesin-like protein ZEN-4 and a RhoGAP protein CYK-4, associates with the RhoGEF ECT-2 at the central spindle, an interaction that is required to activate RhoA. However, because the spindle is not adjacent to the cortex, it is unclear how RhoA activation is achieved and fine-tuned at the cell cortex.
To gain insight into this process, we examined early C. elegans embryos deficient in PAR-5/14-3-3, which were previously shown to have dramatically increased contractility through an unknown mechanism (Morton et al., 2002). We find that the hypercontractile phenotype is CYK-4 dependent and is associated with increased accumulation of CYK-4 and ZEN-4 on the cortex. This demonstrates that PAR-5 is an inhibitor of centralspindlin-dependent contractility. CYK-4 and ZEN-4 are interdependent for their cortical localization, though surprisingly they are not microtubule dependent. It has previously been shown in HeLa cells, that 14-3-3 binding to ZEN-4 prevents centralspindlin clustering, thereby inhibiting its stable localization to the central spindle (Douglas et.al., 2010). This inhibition is released upon phosphorylation of ZEN-4 by Aurora B kinase. Intriguingly, we have found that while Aurora B is required for centralspindlin-mediated furrowing in wild-type embryos, it is not required in PAR-5-depleted embryos. These results are congruent with the pathway shown to regulate centralspindlin oligomerization in HeLa cells. Thus, both central spindle and cortical localization of the centralspindlin complex may be modulated through its clustering activity. Centralspindlin oligomerization may promote cortical activation of RhoA, and spatial regulation of this activity could help focus the position of cleavage furrow formation to a single site.