Bement et al. injected sea urchin and Xenopus embryos with a GFP reporter for RhoA activity. Using 4D imaging, they saw active RhoA accumulate in a tight band at the site of the future furrow. Nocodozole, which disrupts microtubules, prevented RhoA localization, but drugs that depolymerize actin did not. The RhoA zone preceded furrow formation in both symmetric and asymmetric divisions.
When the team repeated classical experiments that altered the location or structure of the spindle, the RhoA zone relocated in a manner similar to the spindle movement, but took several minutes to do so. The delay in RhoA movement suggests that it is not directly tethered to the microtubules but is concentrated by them.
Microtubules thus appear to direct specification of the cytokinetic apparatus by establishing a local zone of RhoA activity, which then promotes the concentration of actin filaments and myosin-2. The team hypothesizes that RhoA regulatory proteins move along the microtubules toward the site of the future zone, as suggested by previous experiments. They think the presence of both RhoA activators and inhibitors increases the rate at which RhoA moves through the GTPase cycle, driving up the rate of activity in a narrow region without causing zone spreading. Although this hypothesis remains to be tested, the team's approach does bridge the gap between classical manipulation experiments and modern molecular genetic methods.