A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

Abstract

Background Polo-like kinases (PLKs) are conserved serine/threonine kinase, regulating cell cycle. Giardia lamblia PLK (GlPLK) role in its cell has not been yet studied. Here, the function of GlPLK was investigated to provide the insight of roles in Giardia cell division, especially during cytokinesis and in flagella formation. Methods To access the function of GlPLK, Giardia trophozoites were treated with PLK-specific inhibitor, GW843286X (GW) or anti-glplk morpholino, then growth of the cells was monitored and phenotypic characteristics of GlPLK-inhibited cells were observed by using mitotic index and flow cytometry assay. Transgenic G. lamblia expressing GlPLK as a hemagglutinin (HA)-tagging was constructed and used for immunofluorescence assay to detect the localization of GlPLK, followed by the subcellular fractionation. Furthermore, kinase assay was performed to assess the phosphorylation activities of GlPLK by purified proteins or in vitro synthesized proteins. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoites. Results After incubating trophozoites with 5 µM GW, the percentages of cells with four nuclei and/or longer flagella were increased. Immunofluorescence assays indicated that GlPLK was mainly localized at basal bodies and transiently localized at mitotic spindles in the dividing cells. Fractionation experiments demonstrated that GlPLK is present in the nuclear fraction, as did the centromeric histone H3. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells, i.e., increased mitotic index and flagella length. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis, and flagella. Conclusions These data indicated that GlPLK plays roles in Giardia cell division, especially during cytokinesis, and in flagella formation.