Inhibition of PLK activity affects the cell cycle and flagella biogenesis in G. lamblia
In order to define the role of PLK, G. lamblia trophozoites were treated with various concentration of GW843682X (GW), an ATP-competitive inhibitor of PLK1 and PLK3 (Additional file 1: Figure S1a). The growth of G. lamblia was inhibited proportionally to the GW concentration, and the 50% inhibitory concentration for cell death (IC50) was 5 µM. Control cells, i.e. Giardia trophozoites treated with 0.05% dimethyl sulfoxide (DMSO), were found to be a mixture of G1/S-, and G2/M-phase cells, and the cells at G2/M-phase were dominant (72%), as reported previously (Additional file 1: Figure S1b) (Poxleitner et al., 2008). Flow cytometry of the DNA content of the 5 µM GW-treated cells also demonstrated that more cells were present at the G1/S-phase (up to 70%) than that in untreated cells (16%), whereas the percentages of G1/S-phase cells decreased proportionally to GW concentration (Additional file 1: Figure S1b). These results indicated that the inhibition of PLK in Giardia causes cell cycle arrest, eventually leading to growth inhibition of Giardia trophozoites.
To determine the effect of PLK inhibition on Giardia division, 5 µM GW-treated cells were observed by DAPI staining (Fig. 1a). As a result, the nuclei of most cells appeared larger than those of DMSO-treated cells. The percentage of cells with four nuclei was significantly increased to 6.3% (from 1.6% of the control cells; P = 0.0018), indicating that GW induced cell cycle arrest at cytokinesis. These cytokinesis-defective Giardia cells were further classified into the following four sequential phenotypes: disorganized cells impertinent for cytokinesis, cells defective in furrow formation, arrested cells at cytokinesis, and cells failed at abscission step (Fig. 1b). The percentages of cells showing disorganization were increased to 18% from 9% (control) (P = 0.0018). On the contrary, the percentages of Giardia cells defective in the subsequent three steps were not significantly affected by GW-treatment.
To observe the morphology of PLK-inhibited Giardia trophozoites, 5 µM GW-treated cells were stained with Giemsa. Interestingly, GW treatment was found to trigger the formation of Giardia trophozoites with longer flagella (Fig. 1c). The extension of flagella in the GW-treated cells was documented by quantitatively measuring caudal flagella length. These data clearly showed that GW-treated cells had longer caudal flagella (8.6 µm) than the untreated cells (5.4 µm) (P = 0.0023).
Localization of GlPLK and definition of domains required for its localization in Giardia trophozoites
A homology search in the Giardia database indicated an ORF (GL50803_104150), as a putative G. lamblia PLK, GlPLK. Amino acid sequences deduced from the ORF were aligned with those of human and Trypanosoma brucei PLKs, and PLKs were deduced from the nucleotide sequences (GenBank accession number NP_005021.2 and Trypanosoma database ORF number Tb927.7.6310, respectively), showing 31–34% identity (Additional file 2: Figure S2). The ORF was postulated to encode a protein of pI = 8.8, and a search of domains within this ORF using the Entrez program (http://www.expasy.org/) indicated that it does contain a serine-threonine kinase domain (KD) at the amino-terminal portion (from amino acid residue No. 20 to 309). In addition, a block of amino acids near the carboxyl terminus was proposed as PBDs (from amino acid residue No. 432 to 517 and 563 to 640), which had been conserved in diverse PLKs . Based on the alignment of GlPLK with other PLKs, Lys51 was suggested as a residue that initially receives phosphate from ATP, and Thr179 and Thr183 residues were proposed as target sites that are subsequently phosphorylated.
A plasmid, pGlPLK.pac, was prepared (Fig. 2a) and used to construct transgenic Giardia trophozoites expressing HA-tagged GlPLK. Western blotting of the resulting G. lamblia extracts confirmed the expression of HA-tagged GlPLK as an immunoreactive band with a molecular weight of 75 kDa (Fig. 2b). In contrast, the extracts of G. lamblia carrying the vector control, pΔ.pac, did not produce any immunoreactive bands in the same analysis. Western blotting of the same membrane with anti-GlPDI1 antibodies  served as a loading control for the total amount of protein in the extracts used for this assay.
The localization of GlPLK was determined using Giardia expressing HA-tagged GlPLK (Fig. 2c). In Giardia, GlPLK was found in basal bodies and axonemes at the interphase. Localization at basal bodies was maintained in the dividing cells, i.e., cells at metaphase, anaphase, and telophase as well as cytokinesis. In cells at anaphase and telophase, GlPLK was also present in mitotic spindles and possibly in the midbody between two daughter cells.
To confirm the localization of GlPLK, Giardia cells expressing HA-tagged GlPLK were double-stained for GlPLK and microtubules (MTs) using anti‐HA and anti‐α‐tubulin antibodies, respectively (Additional file 3: Figure S3a). In Giardia cells at interphase as well as during division, GlPLK was found together with MTs in the basal bodies and axonemes. In addition, Giardia cells during cell division demonstrated co-localization of GlPLK with MTs in the mitotic spindles present between two separated groups of basal bodies.
Basal bodies serve as the MT-organizing center (MTOC) in G. lamblia , which can be observed by staining for its marker, centrin. Additional immunofluorescence assays (IFAs) for Giardia expressing HA-tagged GlPLK were performed using antibodies against HA and G. lamblia centrin (GlCentrin) (Additional file 3: Figure S3b). These double-stained Giardia cells clearly showed co‐localization of GlPLK and GlCentrin during cell division as well as at interphase.
As mentioned above, GlPLK comprises two regions, containing a KD and two PBDs (Additional file 4: Figure S4a). The region between KD and PBDs was named linker. To examine whether KD and/or PBD play a role in GlPLK localization, two plasmids were constructed, i.e., pGlPLKKDL.neo and pGlPLKPBD.neo (expression of the KD-linker and PBDs of GlPLK, respectively). The truncated GlPLK proteins, KD-linker and PBDs, were observed in the form of immunoreactive bands with a molecular weight of 60 and 40 kDa, respectively, by western blotting using anti-HA antibodies (Additional file 4: Figure S4b).
G. lamblia cells carrying pGlPLKKDL.neo or pGlPLKPBD.neo were double-stained with anti-HA and anti-α-tubulin antibodies (Additional file 4: Figure S4c, d) or anti-HA and anti-GlCentrin antibodies (Additional file 4: Figure S4e, f) in order to observe whether these truncated GlPLKs were correctly localized in mitotic spindles and basal bodies. Both the truncated GlPLK proteins were present in the basal bodies, as was the full-length GlPLK. However, both proteins showed defective localization in the mitotic spindles during cell division. These results suggested that both KD and PBD are required for GlPLK localization in mitotic spindles during Giardia cell division.
Effect of GlPLK knockdown on cell division and flagella biogenesis in G. lamblia
To define the role of this putative GlPLK in G. lamblia, we designed an anti-glplk morpholino to block the translation of glplk mRNAs (Table 1). A control morpholino (non-specific oligomers) was also synthesized and transfected into G. lamblia trophozoites by electroporation (Table 1). These extracts were examined to determine their intracellular GlPLK levels at 24 h post-transfection by western blotting using anti-GlPLK antibodies (Fig. 3a). In cells treated with anti-glplk morpholino, the amount of GlPLK at 24 h post-transfection had decreased to 37% of that in cells treated with control morpholino (P = 0.011).
The effect of GlPLK knockdown on cell division was determined based on the mitotic index, which showed that the proportion of cells with four nuclei increased from 2% in control-morpholino-treated cells to 8% in cells treated with anti-glplk morpholinos (Fig. 3b; P = 0.0024). The second assay was used to distinguish between Giardia at the different stages of cytokinesis (i.e., disorganized, no furrow, cytokinesis, and abscission) (Fig. 3c). The percentage of disorganized cells was significantly increased in GlPLK-knockdown cells (14% compared to 9% of control cells; P = 0.019). However, cell numbers at the subsequent steps were not affected by anti-glplk morpholino. GlPLK depletion also resulted in the formation of Giardia trophozoites with longer flagella (Fig. 3d). The length of caudal flagella in cells treated with anti-glplk morpholino was increased to 8.1 µm compared to 5.2 µm in the untreated cells (P = 0.0006). As the phenotypes of cells with morpholino-mediated depletion of the putative glplk gene and of GW-treated cells coincided, these results clearly demonstrated that this putative ORF encodes PLK in G. lamblia.
Expression pattern of GlPLK at G1/S- and G2/M-phase of the Giardia cell cycle
As human PLK1 is highly expressed during mitosis , we examined whether the expression of GlPLK varies in a cell phase-dependent manner. Giardia cells were treated with nocodazole to prepare G2/M-phase cells or sequentially with nocodazole and aphidicolin to acquire G1/S-arrested cells. The stage of the resulting Giardia cells carrying pGlPLK.pac was confirmed by flow cytometry (Additional file 5: Figure S5a). Control cells, i.e. Giardia trophozoites treated with 0.05% DMSO, were found to be a mixture of G1/S-, and G2/M-phase cells, and the cells at G2/M-phase were dominant (76%).
Western blotting of these extracts using anti-HA antibodies demonstrated a constant amount of GlPLK in G2/M- and G1/S-phase cells as well as interphase cells (Additional file 5: Figure S5b). The immunoreactive band was absent from the extracts prepared from Giardia carrying pΔ.pac. Western blotting of the same blot using anti-GlPDI1 antibodies served as a loading control.
Constitutive expression of GlPLK was also examined using an alternative method, quantitative RT-PCR (Additional file 5: Figure S5c). The relative level of glplk transcripts to glactin transcripts remained constant (60–64%) in G1/S-, G2/M-phase, and interphase cells.
Localization of GlPLK into nucleus of G. lamblia
In order to function properly during mitosis, PLK1 should be localized into specific sites through differential interaction with various scaffold proteins . The nucleus is the one of the subcellular locations where PLK1 localizes at G2 phase . Therefore, we investigated whether GlPLK exists in the nuclei of Giardia trophozoites. Giardia extracts were prepared from Giardia cells expressing HA-tagged GlPLK, and then further divided into cytoplasmic and nuclear fractions. These extracts were analyzed by western blotting using anti-HA antibodies (Fig. 4a). In addition, extracts were evaluated for G. lamblia glyceraldehyde 3-phosphate dehydrogenase (Gl50803_6687; GlGAP1) and G. lamblia centromeric histone H3 (GL50803_20037; GlCENH3) as a marker for cytoplasmic and nuclear proteins, respectively. GlPLK was found in both cytoplasmic and nuclear fractions. As expected, GlGAP1 was mainly present in the cytoplasmic fraction, and GlCENH3 was found only in the nuclear fraction.
Giardia cells at G1/S-phase and G2/M-phase were prepared and analyzed for nuclear localization of GlPLK (Fig. 4b). Both G1/S- and G2/M-phase cells demonstrated GlPLK localization in the nuclei and more GlPLK was found in the nuclear fraction of G2/M-phase cells than in that of G1/S-phase cells. GlGAP1 was present in the cytoplasmic fraction of all examined phases, whereas GlCENH3 was found in the nuclear fraction of the G1/S-phase and G2/M-phase cells.
Localization of phosphorylated GlPLK in G. lamblia
Constitutive expression of GlPLK (Additional file 5: Figure S5b, c) suggests that the activity of GlPLK may be regulated by its activation status, possibly by phosphorylation. Giardia trophozoites were double-stained with anti-HA and anti-phospho-PLK antibodies against phosphorylated T210 of human PLK1 (Additional file 6: Figure S6). Both anti-HA and anti-phospho-PLK antibodies stained the basal bodies in both interphase and dividing cells. In dividing cells, phospho-GlPLK was found at mitotic spindles, where localization was limited to the middle region, whereas HA-tagged GlPLK was more widely present in the mitotic spindles of the dividing cells.
In vitro auto-phosphorylation of GlPLK and identification of critical amino acid residues for its auto-phosphorylation
The putative amino acid sequence of GlPLK indicates a serine-threonine KD at the amino terminus and two PBDs at the carboxyl terminus (Fig. 5a). Based on comparison with other PLKs, it was predicted that Lys51 is the primary binding site for ATP, and that the phosphate of Lys51 is eventually transferred to Thr179 and Thr183 in the activation loop.
Immunoprecipitated (IP) extracts were prepared from Giardia expressing HA-tagging GlPLK. These GlPLK IP extracts were reacted with [γ-32P]ATP to radiolabel the protein (75 kDa) (Fig. 5b). Control IP extracts were prepared in the same manner by incubating Giardia extracts with mouse preimmune serum instead of anti-HA antibodies. Incubation of the control IP extracts with [γ-32P]ATP did not result in the labeling of GlPLK.
Kinase assays were also performed using recombinant GlPLK (rGlPLK), which was synthesized using in vitro transcription and translation systems (Fig. 5c). Upon incubation with [γ-32P]ATP, rGlPLK was radiolabeled due to auto-phosphorylation.
To define the amino acid residues that are critical for the auto-phosphorylation of GlPLK, several recombinant GlPLK proteins were also synthesized using in vitro transcription/translation systems and used for kinase assays (Fig. 5d). Specifically, the two putative phosphorylation sites were mutated to Ala, and the resulting mutant GlPLK proteins (GlPLKT179A and GlPLKT183A) were used for kinase assays. In an additional mutant GlPLK, the putative ATP binding site of Lys51 was mutated to Arg (GlPLKK51R). Both GlPLKT179A and GlPLKT183A proteins were auto-phosphorylated, although the efficiency of auto-phosphorylation was lower than that of wild-type GlPLK. When both Thr179 and Thr183 were mutated to Ala in GlPLK, the resulting protein exhibited a dramatic decrease in its ability for auto-phosphorylation. Conversion of Lys51 to Arg abolished the auto-phosphorylation of rGlPLK. This result demonstrated that both Thr179 and Thr183 in the activation loop of GlPLK were phosphorylated. As expected, Lys51 of GlPLK was confirmed to serve as an ATP binding site.
Role of GlPLK phosphorylation in cytokinesis and flagella biogenesis in G. lamblia
The subsequent experiments were performed to define the physiological roles of GlPLK. Transgenic G. lamblia carrying pGlPLKK51R.neo was constructed. In addition, Giardia cells ectopically expressing mutant PLK (T179AT183A) were prepared. Western blotting demonstrated that the transgenic cells expressed HA-tagged GlPLK proteins (data not shown).
The growth of various Giardia cells (ectopically expressing GlPLK, mutant GLPLKK51R, mutant GlPLKT178AT183A, or carrying vector control) was determined (Fig. 6a). The growth of Giardia cells overexpressing wild-type GlPLK was similar to that of the control cells. However, Giardia cells expressing mutant GlPLKs showed inhibited growth as compared with those expressing wild-type GlPLK.
These cells were then used to evaluate mitotic indices by determining the percentages of cells with four nuclei (Fig. 6b). The percentage of cells with four nuclei was increased to 6.7% in transgenic G. lamblia expressing mutant GlPLKK51R compared with that in cells transfected with the vector control (1.7%) or GlPLK-expressing plasmid (1.4%) (P = 0.003). G. lamblia ectopically expressing mutant GlPLKT179AT183A also showed arrest at cytokinesis (7.3%) (P = 0.010). These results indicate that Lys51 as well as two Thr residues (Thr179 and Thr183) in GlPLK may play a role in cell division in Giardia. In addition, ectopic expression of these mutant GlPLK resulted in the extension of the length of caudal flagella from 5% (vector and wild-type GlPLK) to 8-8.2% (mutant GlPLK) (Fig. 6c). These data indicated that GlPLK plays a role in regulating the cell cycle in Giardia, and that the phosphorylation of GlPLK is critical for its in vivo function.