Inhibition of cytokinesis and altered contractile ring morphology induced by cytochalasins in synchronized PtK2 cells

PtK2 cells and antigen affinity-purified antibodies to actin and tubulin were used to study the effects on mitosis of cytochalasin B (CB) and dihydrocytochalasin B (H₂CB). PtK2 cells were synchronized in S phase by a double thymidine block and CB or H₂CB was added at various concentrations at the time of release from the block. CB- and H₂CB-treated populations, and control populations not treated with either drug, progressed synchronously through G2 and into mitosis with similar time courses. By both phase contrast and immunofluorescence microscopy, CB- and H₂CB-treated cells appeared normal in terms of chromosome condensation, spindle formation and spindle dynamics throughout prophase, metaphase and early anaphase. At late anaphase, contractile ring staining with actin antibody was not normal. High actin antigenicity remained localized in the region of the contractile ring; however, it appeared atypically as a punctate line of fluorescence across the midzone. Although some degree of furrowing was often seen to occur, at suitable concentrations of CB or H₂CB only binucleate G1 cells formed. Scanning electron microscopy (SEM) of normal and CB- and H₂CB-treated cells verified that cleavage furrowing did not proceed normally in treated cells. Large numbers of microvilli and surface blebs occurred in the normally smooth furrow region in these treated populations. These results suggest that intact microfilament function is not necessary for progression from S phase into mitosis, for spindle formation or for chromosome movement. They indicate that CB and H₂CB lead to formation of binucleated cells by causing aberrant cleavage furrowing and inhibition of contractile ring microfilaments.