Identification of a protein phosphatase 2A family member that regulates cell cycle progression in Trypanosoma brucei

• An essential protein phosphatase 2A family member (TbPP2A-1) is identified.
• RNAi of TbPP2A-1 prevents kinetoplast segregation and flagellar assembly from keeping pace with mitosis.
• The RNAi cells phenocopy treatment with okadaic acid.
• A cell line is constructed to facilitate live cell detection of cell cycle defects.

The cell cycle consists of an orderly sequence of events, whose purpose is to faithfully replicate and segregate cellular components. Many events in the cell cycle are triggered by protein kinases and counteracting phosphoprotein phosphatases (PPP). In Trypanosoma brucei, RNAi has been used to characterize numerous regulatory kinases, while the role of protein phosphatases has primarily been deduced with inhibitors such as okadaic acid and calyculin. In the present study, we identify for the first time a protein phosphatase 2A family member (TbPP2A-1) whose knockdown with RNAi phenocopies the effects of okadaic acid (OKA). In bloodstream forms (BF) and insect stage procyclic forms (PF) RNAi of TbPP2A-1 generates a cell population characterized by: an inhibition of cell growth, a block in cytokinesis; continued synthesis of nuclear DNA leading to aneuploidy; continued mitosis leading to cells with N > 2, and an unusual phenotype where number of kinetoplasts (and flagella) is less than the number of nuclei. An engineered cell line was constructed to further study TbPP2A-1 and to facilitate the discovery of other cell cycle regulatory genes.

An essential PP2A family member is described along with a “marker” cell line whose purpose is to facilitate live cell detection of cell cycle defects.Figure optionsDownload high-quality image (210 K)Download as PowerPoint slide