Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis

摘要

Background Phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for successful completion of cytokinesis. In addition, both PIP₂ and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP₂ to yield diacylglycerol (DAG) and inositol trisphosphate (IP₃), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP₂ must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP₂ regulates cytokinesis remains to be elucidated. Results Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH₃ (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH₃ produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH₃. Conclusion We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP₂ hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.