Fig. 2. RhoA activity and microtubule organization are required for RhoA localization at the equatorial cortex. (A-C) Quantification of RhoA density at the equatorial cortex during metaphase or anaphase in HeLa cells. (A) Typical RhoA immunofluorescence images at metaphase (meta.) and anaphase (ana.). (B) Schematic drawing of cells used to measure RhoA fluorescence intensity. Stars represent points for measuring RhoA density at the cell pole and the equator. Dark ovals represent nuclei. (C) Fluorescence intensities of RhoA at the pole and the equator. RhoA density at the equator significantly increases after metaphase (n=10). (D) RhoA activity is required for localization at the equatorial cortex. HeLa cells were injected with Botulinum C3 exoenzyme (C3), RhoGDI or fluorescein dextran (control) before metaphase and incubated until control cells entered anaphase. No RhoA accumulation is evident in C3- or RhoGDI-injected cells. (E) Microtubule organization is required for RhoA localization at the equatorial cortex during cytokinesis. HeLa cells were treated with 200 µM latrunculin A or with 100 µM blebbistatin before metaphase onset and incubated until control cells entered anaphase. RhoA normally accumulated at the equatorial cortex (+LAT-A, + blebb., arrowheads). A6 cells expressing ß-tubulin-GFP were incubated with 16 µM nocodazole just after anaphase onset for 30 minutes. RhoA did not localize at the equatorial cortex but scattered on the cortex (+ Noc., arrowheads). Bars, 10 µm.