Rac GTPases organize F-actin and myosin into an actomyosin ring between incipient reticulocyte and pyrenocyte during enucleation. An actomyosin ring made up of F-actin (A) and pMRLC (B) is shown in enucleating WT and Rac1^Δ/Δ;Rac2^−/− erythroblasts (indicated by white arrowheads). In contrast, an actomyosin ring is not formed in WT erythroblasts incubated with NSC23766 (100μM) that inhibits all Rac GTPases. In the erythroblasts observed to attempt enucleation under total Rac inhibition, actin and pMRLC continue to surround the nucleus. Three representative images are shown in each panel from at least 30 cells with similar morphology in each category studied. Images were obtained with a 40× objective lens by Imagestream^x. (C) Confocal microscopy image of WT erythroblasts incubated without and with NSC23766 confirms lagged distribution of F-actin around the nucleus when Rac-GTPases are inhibited. In the WT image (on the left), the contractile ring is not visible, probably because the cell is at the final stage of enucleation with the nucleus almost separating. F-actin was labeled with rhodamine-phalloidin (red) and nucleus with 4,6-diamidino-2-phenylindole (DAPI; blue). Images were obtained with a 100× oil-immersed objective lens, numerical aperture 1.45, and processed using Volocity Version 4.1 software to produce a 3-dimensional reconstruction of the cells visualized; 1 unit represents 4.1 μm. (D) F-actin and myosin (labeled on pMRLC) colocalize at the cleavage furrow to form an actomyosin ring. Images were obtained with a 60× objective lens by Imagestream^x. (E) Effect of pharmacologic inhibitors of actin filament assembly (cytochalasin-D), MRLC phosphorylation (ML7), and Rac GTPase activity (NSC23766) on the enucleating efficiency of WT erythroblasts in fast enucleation assay as percentile of the control sample. The absolute enucleation efficiency of the control sample in this experiment was 40.4% ± 5.3%. Data are mean ± SEM; n = 3. *P < .02 (control vs each of the inhibitor-treated samples). ‡P < .005 (control vs each of the inhibitor-treated samples). †P < .0005 (control vs each of the inhibitor-treated samples). Sample treated with both cytochalasin-D (10μM) and ML7 (25μM) demonstrated inhibition of enucleation compared with control (P < .005) but not a statistically significant difference from cytochalasin-D or ML7-alone treated samples.