The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei.
Mitosis and cytokinesis are events that are highly coordinated in most eukaryotic cell cycles. African trypanosomes possess a single mitochondrion and must additionally coordinate the organellar division cycle. Here we report that okadaic acid, a potent and specific inhibitor of protein phosphatases PP1and PP2A, uncouples these cycles in living trypanosomes. Cell cycle analysis of treated cells revealed elevated DNA content. Microscopic examination indicated that okadaic acid treatment yielded multinucleate cells with a single mitochondrial network indicating these cells have undergone mitosis but failed to complete cytokinesis. Immunofluorescence analysis of 5-bromo-2-deoxyuridine incorporation demonstrated that the mitochondrial DNA was replicated but did not segregate. The dose response curve for inhibition of the normal cell cycle paralleled that for the in vitro inhibition of protein phosphatase activities with IC50s of approximately 20 nM okadaic acid. These results suggest the involvement of a PP1/PP2A-like activity in coordinating mitosis, mitochondrial DNA division and cytokinesis in trypanosomes.
Das
A
A
Seattle Biomedical Research Institute, WA 98109.
Gale
M
M
Carter
V
V
Parsons
M
M
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000096:biosynthesis; D016054:DNA, Protozoan / Q000096:biosynthesis; D004988:Ethers, Cyclic / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects* / Q000235:genetics; D008938:Mitosis / Q000187:drug effects; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011494:Protein Kinases / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000235:genetics
D004272:DNA, Mitochondrial; D016054:DNA, Protozoan; D004988:Ethers, Cyclic; D019319:Okadaic Acid; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases