Figure 3.
Rac1 inactivation by CYK4 is required for cytokinesis. (A) HeLa cells stably expressing inducible Myc-tagged copies of CYK4 or CYK4R385A were treated for 36 h with an siRNA directed to the 3′-UTR of CYK4 to deplete the endogenous protein or a control siRNA. At the same time, the cells were treated with doxycycline to induce the Myc-tagged transgenes (induced) or left untreated (uninduced). The cells were fixed and then stained with antibodies to tubulin, the Myc epitope, or DAPI to detect DNA. The number of binucleate cells was counted for each condition and plotted as a bar graph. Error bars show the standard deviation from the means (n = 3). Asterisks mark binucleate cells that have failed cytokinesis. (B) HeLa cells stably expressing inducible EGFP-tagged copies of Rac1 or activated Rac1Q61L were treated for the time indicated with doxycycline to induce the EGFP-tagged transgenes. The cells were fixed and then stained with antibodies to tubulin or DAPI to detect DNA. Rac1 was directly visualized by EGFP fluorescence. The number of binucleate cells was counted for each condition and plotted as in the line graph. (C) Alternatively, the cells were fixed using TCA and stained for RhoA and with antibodies to EGFP to detect EGFP-Rac1. DAPI was used to detect DNA. siControl, nonsilencing control; WT, wild type. Bars, 10 µm.