Fig. 2. Effect of C3 exoenzyme treatment on Citron-K localization during cell division. Recombinant C3 exoenzyme was introduced into HeLa cells enriched in the S-phase by electroporation, and its effects on cell division and localization of endogenous Citron-K were examined. (A) Failure of cytokinesis in HeLa cells treated with C3 exoenzyme. Almost all the treated cells became binucleate 16 hours after electroporation as shown by DAPI staining (blue). Microtubules are stained in green. (B) Effect of C3 exoenzyme treatment on Citron-K localization during cell division. HeLa cells without (left-hand pairs of panels) or with (middle and right-hand pairs of panels) C3 exoenzyme treatment were fixed in various phases of cell division and stained with anti-Citron antibody (green). Microtubules and DNA were stained with anti-ß-tubulin antibody (red) and TOPRO3 (blue), respectively. The left panels of each pair represent merged images. Note that Rho inactivation by C3 exoenzyme treatment did not affect Citron-K localization in prometa- and metaphase, but prevented the transfer of Citron-K to the cortex in telophase, which instead was associated with the spindle midzone (middle bottom pairs of panels). The Citron-K signal in the spindle midzone was abolished in the presence of the antigenic peptide (the right bottom pair of panels). Bars, 10 µm.