Polo-Like Kinase Guides Cytokinesis in Trypanosoma brucei through an Indirect Means

Abstract

Polo-like kinase in Trypanosoma brucei (TbPLK) is confined to the flagellum attachment zone (FAZ) and regulates only cytokinetic initiation. However, it apparently diffuses into the cytoplasm before the translocalization of chromosomal passenger complex (CPC) from the midzone of central spindle to FAZ, which is known to be required for initiating cytokinesis. Synchronized T. brucei procyclic cells treated with a TbPLK inhibitor, GW843682X (GW), in late S phase were found to go through a full cell cycle at a normal pace before being arrested at cytokinetic initiation in the second cycle. However, synchronized cells treated with GW in G 1 phase were arrested at cytokinetic initiation within the first cell cycle, suggesting that inhibition of TbPLK at its emergence blocks cytokinesis within the same cell cycle. To rule out potential off-target effects from GW, TbPLK RNA interference (RNAi) was induced to deplete TbPLK, and the progression of synchronized cells from late S phase was also found to be arrested at cytokinetic initiation within the first cell cycle. Apparently, TbPLK has accomplished its role in guiding cytokinesis before the late S phase, presumably by phosphorylating a certain substrate(s) during S phase, which may play a critical role in initiating the subsequent cytokinesis.

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

“…Selection of transfectants, cloning, and RNAi induction were carried out as described previously (14). TbPLK RNAi cell line and TbAUK1 RNAi cell line have been reported previously (12,17).…”

“…The small-molecule PLK inhibitor GW843682X (16) was previously demonstrated to inhibit TbPLK activity in vitro, with an in vitro IC 50 of ∼1.3 μM, and in vivo, inhibiting the insect-form trypanosomes with an IC 50 of ∼2 μM (17). The small-molecule Aurora B kinase inhibitor Hesperadin (20) was previously demonstrated to inhibit TbAUK1 activity in vitro, with an in vitro IC 50 of 40 nM, and in vivo, inhibiting the insect-form trypanosomes with an IC 50 of 0.55 μM (19).…”

“…CIF1 is a highly phosphorylated protein, containing 29 in vivo phospho-serine and phospho-threonine sites (15). To test whether CIF1 is an in vivo substrate of TbPLK, we treated the cells with GW843682X, a small-molecule Polo-like kinase inhibitor (16,17). CIF1 was detected as a doublet on the Western blot, and treatment with GW843682X reduced the level of the upper band of CIF1, which was confirmed to be the phosphorylated form of CIF1, suggesting de-phosphorylation of CIF1 by TbPLK inhibition ( Fig.…”

A Novel Basal Body Protein That Is a Polo-like Kinase Substrate Is Required for Basal Body Segregation and Flagellum Adhesion in Trypanosoma brucei

“…The full-length coding sequence of TbPLK was cloned into pET41 (18), and recombinant GST-TbPLK was purified from the soluble fraction. Purified recombinant proteins (GST-Tb-PLK and GST-SPBB1-C) were dialyzed against 50 mM Tris-Cl, pH 7.6, and 50 mM NaCl.…”

“…Inhibition of TbPLK Activity by a Small Molecule InhibitorTo inhibit TbPLK activity in trypanosomes, 5 M GW843286X (Tocris Bioscience), an inhibitor of human Plk1 (21) and trypanosome TbPLK (18), was added to trypanosome cell culture and incubated for 24 h. The phenotype caused by GW843286X treatment is similar to that caused by TbPLK RNAi (18).…”