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


CONCLUSIONS
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.
Wong
Raymond
R
Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, TMDT Building, East Tower, Toronto, Ontario, Canada. re.wong@utoronto.ca <re.wong@utoronto.ca>
Fabian
Lacramioara
L
Forer
Arthur
A
Brill
Julie A
JA
D000199:Actins / Q000378:metabolism*; D000478:Alkylation / Q000187:drug effects; D000818:Animals; D001381:Azepines / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D048749:Cytokinesis / Q000187:drug effects; D004331:Drosophila melanogaster / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology; D005033:Ethylmaleimide / Q000737:chemistry / Q000494:pharmacology; D008297:Male; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009281:Naphthalenes / Q000737:chemistry / Q000494:pharmacology; D010742:Phospholipid Ethers / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011760:Pyrrolidinones / Q000494:pharmacology; D013090:Spermatocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000199:Actins; D001381:Azepines; D004791:Enzyme Inhibitors; D004963:Estrenes; D018994:Myosin Light Chains; D009281:Naphthalenes; D010742:Phospholipid Ethers; D011760:Pyrrolidinones; C070571:ML 7; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C026659:edelfosine; D009219:Myosin-Light-Chain Kinase; D010738:Type C Phospholipases; D005033:Ethylmaleimide