Myosin II ATPase activity is enhanced by the phosphorylation of MRLC (myosin II regulatory light chain) in non-muscle cells. It is well known that pMRLC (phosphorylated MRLC) co-localizes with F-actin (filamentous actin) in the CR (contractile ring) of dividing cells. Recently, we reported that HeLa cells expressing non-phosphorylatable MRLC show a delay in the speed of furrow ingression, suggesting that pMRLC plays an important role in the control of furrow ingression. However, it is still unclear how pMRLC regulates myosin II and F-actin at the CR to control furrow ingression during cytokinesis. In the present study, to clarify the roles of pMRLC, we measured the turnover of myosin II and actin at the CR in dividing HeLa cells expressing fluorescent-tagged MRLCs and actin by FRAP (fluorescence recovery after photobleaching). A myosin II inhibitor, blebbistatin, caused an enhancement of the turnover of MRLC and actin at the CR, which induced a delay in furrow ingression. Furthermore, only non-phosphorylatable MRLC and a Rho-kinase inhibitor, Y-27632, accelerated the turnover of MRLC and actin at the CR. Interestingly, the effect of Y-27632 was cancelled in the cell expressing phosphomimic MRLCs. Taken together, these results reveal that pMRLC reduces the turnover of myosin II and also actin at the CR. In conclusion, we show that the enhancement of myosin II and actin turnover at the CR induced slower furrowing in dividing HeLa cells.

Excessive centrosomes often lead to multipolar spindles, and thus probably to multipolar mitosis and aneuploidy. In Caenorhabditis elegans, ∼70% of the paternal emb-27^APC6mutant embryonic cells contained more than two centrosomes and formed multipolar spindles. However, only ~30% of the cells with tripolar spindles formed two cytokinetic furrows. The rest formed one furrow, similar to normal cells. To investigate the mechanism via which cells avoid forming two cytokinetic furrows even with a tripolar spindle, we conducted live-cell imaging in emb-27^APC6mutant cells. We observed that the chromatids were aligned on only two of the three sides of the tripolar spindle, and the angle of the tripolar spindle relative to the long axis of the cell correlated with the number of cytokinetic furrows. Our numerical modeling showed that the combination of cell shape, cortical pulling forces, and heterogeneity of centrosome size determines whether cells with a tripolar spindle form one or two cytokinetic furrows.

Cytokinesis D is known as the midwife mechanism in which neighboring cells facilitate cell division by crossing the cleavage furrow of dividing cells. Cytokinesis D is thought to be mediated by chemotaxis, where midwife cells migrate toward dividing cells by sensing an unknown chemoattractant secreted from the cleavage furrow. In this study, to validate this chemotaxis model, we aspirated the fluid from the vicinity of the cleavage furrow of a dividing Dictyostelium cell and discharged it onto a neighboring cell using a microcapillary. However, the neighboring cells did not show any chemotaxis toward the fluid. In addition, the cells did not manifest an increase in the levels of intracellular Ca²⁺, cAMP, or cGMP, which are expected to rise in chemotaxing cells. From several lines of our experiments, including these findings, we concluded that chemotaxis does not contribute to cytokinesis D. As an alternative, we propose a cortical-flow model, where a migrating cell attaches to a dividing cell by chance and is guided toward the furrow by the cortical flow on the dividing cell, and then physically assists the separation of the daughter cells.