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Roles of end-binding 1 protein and gamma-tubulin small complex in cytokinesis and flagella formation of Giardia lamblia.

ABSTRACT: Giardia lamblia is a unicellular organism with two nuclei, a median body, eight flagella, and an adhesive disk. ?-Tubulin is a microtubule (MT)-nucleating protein that functions in the ?-tubulin small complex (?-TuSC) in budding yeast. In this study, G. lamblia ?-tubulin (Gl?-tubulin) was found to bind to another MT-binding protein, namely G. lamblia end-binding protein 1 (GlEB1), via both in vivo and in vitro assays. Hemagglutinin (HA)-tagged Gl?-tubulin localized to the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. The knockdown of Gl?-tubulin expression using an anti-Gl?-tubulin morpholino resulted in a decreased growth rate and an increased failed cytokinesis cells of Giardia. The formation of median bodies was affected, and the central pair of MTs in flagella was frequently missing in the Giardia treated with an anti-Gl?-tubulin morpholino. G. lamblia ?-tubulin complex protein 2 (GlGCP2) and GlGCP3, which are putative components of ?-TuSC, were co-immunoprecipitated with HA-tagged Gl?-tubulin in Giardia extracts. The knockdown of GlGCP2 and GlGCP3 expression also resulted in decreased formation of both the median body and flagella MTs. Knockdown of Gl?-tubulin, GlGCP2, and GlGCP3 expression affected localization of GlEB1 in G. lamblia. In addition, decreased level of GlEB1 caused reduced formation of median body and the central pair of flagella MTs. These results indicated that Gl?-tubulin plays a role in MT nucleation for median body formation and flagella biogenesis as a component of Gl?-TuSC in Giardia and GlEB1 may be involved in this process.

SUBMITTER: Kim J

PROVIDER: S-EPMC6562232 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Roles of end-binding 1 protein and gamma-tubulin small complex in cytokinesis and flagella formation of Giardia lamblia.

Kim Juri J Park Soon-Jung SJ

MicrobiologyOpen 20181014 6

Giardia lamblia is a unicellular organism with two nuclei, a median body, eight flagella, and an adhesive disk. γ-Tubulin is a microtubule (MT)-nucleating protein that functions in the γ-tubulin small complex (γ-TuSC) in budding yeast. In this study, G. lamblia γ-tubulin (Glγ-tubulin) was found to bind to another MT-binding protein, namely G. lamblia end-binding protein 1 (GlEB1), via both in vivo and in vitro assays. Hemagglutinin (HA)-tagged Glγ-tubulin localized to the basal bodies, axonemes, ...[more]

PMID: 30318753

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Project description:BackgroundPolo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation.MethodsTo assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. 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 percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. 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 formation.ConclusionsThese data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.

Dataset Information

Roles of end-binding 1 protein and gamma-tubulin small complex in cytokinesis and flagella formation of Giardia lamblia.

Publications

Roles of end-binding 1 protein and gamma-tubulin small complex in cytokinesis and flagella formation of Giardia lamblia.

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