At anaphase, the mitotic spindle provides two consecutive signals that position the cytokinesis furrow: microtubule asters provide a first signal and the spindle midzone provides a second signal. The two signals from the mitotic spindle cause a reorganization of the cortex that leads to the formation of a contractile ring and furrow ingression.
In order to understand the molecular nature of the furrow-positioning signals, we have performed an RNAi screen to identify genes that are required specifically for aster-positioned cytokinesis.
We found one gene,
let-99, that caused cytokinesis failure in the absence of a spindle midzone (
spd-1 mutant or mechanical disruption) but not in the presence of a spindle midzone (wildtype).
LET-99 has been shown to localize as a cortical band. We wondered whether the localization of LET-99 reflects its function in cytokinesis. We found that the LET-99 band precisely localizes to the site of cytokinesis furrow formation. Using mechanical displacement of the mitotic spindle, we have shown that during anaphase, the mitotic spindle positions the LET-99 band; during metaphase, however, the LET-99 band is positioned by a spindle-independent mechanism.
We then investigated the role of LET-99 in cytokinesis in more detail. Despite the lack of a detectable astral signal in
let-99 mutant embryos, they can cleave. Frequently, however, multiple furrows ingress, leading to the formation of cytoplasts. Time-lapse imaging of myosin-GFP suggests that
let-99 mutant embryos are defective in cortical flow into the furrow region during cytokinesis. Analysis of additional cortical markers suggests that
let-99 mutant embryos are defective in cortical reorganization, in general.
Taken together we conclude that the mitotic spindle positions cortical LET-99 in the presumptive furrow region, and LET-99, in turn, contributes to reorganization of the acto-myosin cortex to promote efficient furrowing.