Inhibition of PLK activity affects the cell cycle and flagella biogenesis in G. lamblia
In order to define the role of PLK, we treated G. lamblia trophozoites with various concentrations of GW843682X (GW), an ATP-competitive inhibitor of PLK1 and PLK3 (Additional file 1: Fig. S1) and observed that the inhibition of growth of G. lamblia was inversely related to the GW concentration, with an IC50 of 7 μM.
To determine the effect of PLK inhibition on Giardia cell division, the cells were treated with 5 µM GW for various lengths of time (range 3–24 h), then stained with Giemsa. The stained cells (> 300 cells for each of the three independent experiments) were scored for the number and position of the nuclei, as follows: cells with one nucleus, with two nuclei in the normal position, with two nuclei in an abnormal position, with three nuclei, with four nuclei and with > 4 nuclei (Additional file 2: Table S1). Based on these data, treatment with 5 µM GW for 18 h was selected to determine the number of nuclei in GW-treated cells and control cells (treated with DMSO for 18 h) (Fig. 1a). A representative cell for each category is shown in Fig. 1a. A majority of the cells were found to have two nuclei in the normal position (97%), and the percentage of these cells decreased to 87%. The number of cells with one nucleus, two abnormally positioned nuclei or three nuclei increased, but this change was not statistically significant. The most notable increase was the percentage of cells with ≥ 4 nuclei, in particular to 5.8 and 2.2% (from 1 and 0.7% of the control cells, respectively), indicating that GW induced cell cycle arrest during cytokinesis.
Among the GW-treated Giardia trophozoites with two nuclei in the normal position, we found cells with condensed nuclei that which appeared larger and more deeply stained. Thus, the percentages of cells with condensed nuclei were also monitored in the cells treated with 5 µM GW for various lengths of time (3–24 h) (Fig. 1b). The percentage of cells with condensed nuclei significantly increased in cells treated with 5 μM GW for 6 h compared to DMSO-treated cells.
In order to determine the effect of GlPLK inhibition on DNA content of Giardia, we treated trophozoites with 0.1% DMSO or 5 μM GW for various lengths of time (6, 12, 18 and 24 h) and then analyzed the tropozoites by flow cytometry (Fig. 1c). The presented data are representatives of three independent samples per each condition. Control cells (untreated cells and DMSO-treated cells) were found to be a mixture of G1/S-, and G2/M-phase cells, with the cells at the G2/M phase the most dominant (72–73%), as reported previously . For cells treated with GW for 6 and 12 h, the percentage of cells at G2/M phase increased to 86%, which was not a statistically significant difference. Interestingly, a greater number of cells treated with 5 μM GW for longer times, 18 and 24 h, were present in the G1/S phase (63–66%), as compared with the control cells (26%). These results indicated that the inhibition of PLK in Giardia causes cell cycle arrest at the G2/M phase or the G1/S phase, depending upon the treatment time with GW.
In addition, we examined whether the GW treatment affected the flagella formation of Giardia trophozoites (35 cells per each condition, and 3 independent experiments) by quantitatively measuring the length of the membrane-bound and cytoplasmic portion of all four pairs of flagella (Fig. 2a). The cytoplasmic portion of the ventral flagella could not be measured in this assay. The length of the cytoplasmic posterolateral flagella was 5.2–7.2 μm. The anterior and caudal flagella had longer cytoplasmic portions (7.9–9.2 and 7.9–10.8 μm, respectively). None of the three flagella showed any significant change in the length of their cytoplasmic part in response to GW.
With respect to the membrane-bound region, the lengths of the four flagella were more variable. Under normal conditions, that is, DMSO treatment for various time periods, the membrane-bound ventral flagella were the longest (13.8 μm). The caudal flagella demonstrated the shortest membrane-bound region (7.2 μm). The lengths of the posterolateral and anterior membrane flagella were 7.9 and 9.0 μm, respectively. The effect of GW on the length of the membrane-bound flagella was determined using data derived from the cells treated with GW for 18 h and their counterpart control cells (Fig. 2b). GW treatment did not induce a significant increase in the length of the membrane-bound portion of the posterolateral and ventral flagella while, in contrast, GW-treated cells clearly showed an extension of the anterior and caudal flagella in their membrane-bound parts of up to 11.3 and 11.1 µm, respectively. These data clearly showed that the GW treatment affected the formation of these two flagella among the four types of flagella present in G. lamblia.
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 the putative G. lamblia PLK, GlPLK. Amino acid sequences deduced from the ORF were aligned with those of human and Trypanosoma brucei PLKs (GenBank accession numbers NP_005021.2 and Tb927.7.6310, respectively), showing 31–34% identity (Additional file 3: Fig. 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 contains a serine/threonine kinase domain (KD) at the amino-terminal portion (from amino acid residue no. 20 to 309). In addition, blocks of amino acids near the carboxyl terminus were proposed as the PBDs (amino acid residues no. 432–517 and 563–640), 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.neo, was constructed (Fig. 3a) 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 80 kDa (Fig. 3b). In contrast, the extracts of G. lamblia carrying the vector control, pKS-3HA.neo, 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. 3c). GlPLK was found in the basal bodies, flagella, axonemes, an adhesive disc and median bodies of Giardia trophozoites at interphase. Localization at the basal bodies was maintained in the dividing cells (cells at metaphase, anaphase and telophase), as well as at cytokinesis. In cells at anaphase, GlPLK was also present in the mitotic spindles and axonemes of the dividing 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‐acetylated-α‐tubulin antibodies, respectively (Fig. 3d). In Giardia cells at interphase and anaphase, GlPLK was found together with MTs in the basal bodies, axonemes, median bodies and flagella. Giardia cells at anaphase also demonstrated the 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 , and the MTOC can be observed by staining for its marker, centrin. Additional IFAs for Giardia expressing HA-tagged GlPLK were performed using antibodies against HA and G. lamblia centrin (GlCentrin) (Fig. 3e). These double‐stained Giardia cells clearly showed the co‐localization of GlPLK and GlCentrin during cell division as well as at interphase.
As mentioned above, GlPLK comprises two regions: the KD and two PBDs (Fig. 4a). The region between the KD and PBDs was named the linker. To examine whether the KD and/or PBDs play a role in GlPLK localization, we constructed two plasmids, pGlPLKKDL.neo and pGlPLKPBD.neo, that expressed the KD linker and the PBDs of GlPLK, respectively. Western blotting using anti-HA antibodies revealed the expression of the truncated GlPLK proteins, KD linker and PBDs, in Giardia trophozoites in the form of immunoreactive bands with a molecular weight of 60 and 40 kDa, respectively (Fig. 4b). On the other hand, Giardia carrying the vector plasmid pKS-3HA.neo did not show any immunoreactive bands.
Giardia lamblia cells carrying pGlPLKKDL.neo were double-stained with anti-HA and anti-acetylated-α-tubulin antibodies or with anti-HA and anti-GlCentrin antibodies (Fig. 4c) in order to observe whether this truncated GlPLK-KDL was correctly localized in mitotic spindles and basal bodies, respectively. In interphase Giardia cells, double staining with antibodies against acetylated-α-tubulin and HA resulted in the labeling of flagella, axonemes, a median body and basal bodies (n = 5). In contrast, GlPLK-KDL was not found in the mitotic spindles of the dividing Giardia at anaphase (n = 5). Double staining of G. lamblia cells carrying pGlPLKKDL.neo with anti-HA and anti-GlCentrin antibodies revealed the co-localization of these two proteins in basal bodies [cells at metaphase (n = 2) and cells at anaphase (n = 6)]. However, the relative position of these structures with DAPI-stained nuclei indicated an incorrect localization of the basal bodies, as presented in an extended view.
In contrast, Giardia cells expressing truncated GlPLK-PBD demonstrated the same pattern of co-localization with the full-length GlPLK with respect to α-tubulin (Fig. 4d). In addition to being found in the flagella, axonemes and median bodies of the interphase cells (n = 3), GlPLK-PBD was found at the mitotic spindles in dividing Giardia cells (n = 7). Double staining of Giardia cells expressing GlPLK-PBD revealed the co-localization of this protein with GlCentrin in basal bodies (n = 7). Extended views of these dividing cells indicated that double-stained basal bodies positioned themselves in the correct positions for mitosis. These results suggest that the PBD of GlPLK is required for GlPLK localization in mitotic spindles and the correct positioning of basal bodies 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 oligomer) was also synthesized and transfected by electroporation into G. lamblia trophozoites carrying pGlPLK.neo (Table 2). When the cells were harvested at various time-points, ranging from 12 to 48 h, and analyzed for GlPLK inhibition, the cells at 24 h post-transfection demonstrated a maximal inhibition of GlPLK expression (data not shown). However, we chose the cells harvested at 18 h after transfection for further studies (Fig. 5a). In cells treated with an anti-glplk morpholino, the amount of HA-tagged GlPLK at 18 h post-transfection had decreased to 59.5% of that in cells treated with the control morpholino (P = 0.0003). In addition, these extracts were examined to determine their intracellular GlPLK-HA and GlPLK levels by western blotting using anti-GlPLK antibodies. In cells treated with an anti-glplk morpholino, the amounts of GlPLK-HA and GlPLK at 18 h post-transfection had decreased to 56 and 55% of those in cells treated with the control morpholino, respectively.
The effect of GlPLK knockdown on cell division was determined based on the nuclear phenotypes, which included the number of nuclei and the condensation of the DNA in the cells (Fig. 5b). The percentage of cells with two normally positioned nuclei decreased from 98.7 to 90.7%. Among these cells, the number of cells showing nuclear condensation increased from 0.4 to 4.6%. The percentages of cells with one nucleus, two nuclei in abnormal position, three nuclei and four nuclei were slightly increased in cells treated with an anti-glplk morpholino, without any statistical significance. Only the percentage of cells with more than four nuclei showed a statistically significant increase in anti-glplk morpholino-treated cells (P = 0.02).
The effect of GlPLK depletion on the DNA ploidy of Giardia cells was also determined by flow cytometric analysis (Fig. 5c). Cells treated with control morpholino for 6 or 18 h showed a similar proportion of cells in the G1/S and G2/M phases (19 and 79%, respectively). In cells treated with anti-glplk morpholino, the percentage of G2/M-phase cells increased to 84% after 6 h post-transfection. In contrast, a greater number of cells treated with anti-glplk morpholino for 18 h were present in the G1/S phase (25%), as compared with the control cells (17%). The percentage of G2/M-phase cells decreased to 69% after 18 h post-transfection of anti-glplk morpholino, compared 79% in control cells.
GlPLK depletion also resulted in the formation of Giardia trophozoites with longer flagella (Fig. 5d). The length of the membrane-bound portion of the caudal flagella in cells treated with an anti-glplk morpholino increased to 12.1 μm compared to 6.5 μm in the control morpholino cells (P = 0.003). GlPLK-depleted cells also showed extension of the anterior and ventral flagella in their membrane-bound parts of up to 11.0 and 14.8 µm compared to 8.7 and 13.0 µm, respectively, in control cells.
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 GlPLK expression varies in a phase-dependent manner. Giardia cells were treated with nocodazole to prepare G2/M-phase cells (91%) or sequentially with nocodazole and aphidicolin to acquire G1/S-arrested cells (86%). The stage of the resulting Giardia cells carrying pGlPLK.neo was confirmed by flow cytometry (Additional file 4: Fig. S3A). Control Giardia trophozoites treated with 0.01% DMSO were found to be a mixture of G1/S- and G2/M-phase cells, G2/M-phase cells being the dominant cell type (78%).
Western blotting of these cell extracts using anti-HA antibodies demonstrated an increased amount (1.5-fold) of GlPLK in G2/M-phase and interphase cells in comparison with G1/S-phase cells (Additional file 4: Fig. S3B). The immunoreactive band was absent from the extracts prepared from Giardia cells carrying pKS-3HA.neo. Western blotting of the same blot using anti-GlPDI1 antibodies served as a loading control.
Increased expression of the glplk transcript was also examined using an alternative method, quantitative reverse transcription (RT)-PCR (Additional file 4: Fig. S3C). The relative level of glplk transcripts to glactin transcripts remained increased (2.4-fold) in G2/M-phase cells compared to G1/S-phase cells (p = 0.01). To monitor the phases of our samples, the assays included two G1/S phase marker genes encoding histone H3 and histone H4, which showed a decreased expression in the G2/M-phase cells . In addition, the transcript level of Glγ-giardin was measured in these cells and found to show increased expression in G2/M phase, as expected .
Subcellular localization of GlPLK in G. lamblia
In order to function properly during mitosis, PLK1 should be localized to specific sites through differential interaction with various scaffold proteins . The nucleus is one of the subcellular locations where PLK1 localizes in the G2 phase .
Giardia extracts were prepared from Giardia cells expressing HA-tagged GlPLK at interphase, the G1/S phase and the G2/M phase, and then further divided into cytoplasmic and membrane fractions, which may include nuclear fractions. These extracts were analyzed by western blotting using anti-HA antibodies (Additional file 5: Fig. S4). In addition, extracts were evaluated for G. lamblia glyceraldehyde 3-phosphate dehydrogenase (Gl50803_6687; GlGAP1), G. lamblia centrin (Gl50803_104685; GlCentrin) and G. lamblia centromeric histone H3 (GL50803_20037; GlCenH3) as markers for cytoplasmic, membrane and nuclear proteins, respectively. Because the amino acid sequence alignment of the three histone H3 proteins of G. lamblia demonstrated 33–46% identity among them, it is unlikely that anti-GlCenH3 reacts with the other two histone H3 proteins. Even though there is some cross-reactivity of these antibodies against the other histone H3 proteins, it did not interfere with this experiment in that all three hisone H3 proteins are located in the nuclei of G. lamblia .
GlPLK was found in both the cytoplasmic and membrane fractions in all examined phases. As expected, GlGAP1 was mainly present in the cytoplasmic fraction, and GlCentrin and GlCenH3 were found only in the membrane fraction.
Both G1/S- and G2/M-phase cells demonstrated GlPLK localization in the membrane fraction, and more GlPLK was found in the G2/M-phase cells than in the G1/S-phase cells. A constant amount of GlGAP1 was present in the cytoplasmic fraction of all examined phases, whereas more GlCentrin and GlCenH3 were found in the membrane fraction of the G2/M-phase cells than in the G1/S-phase cells.
Expression and localization of phosphorylated GlPLK in G. lamblia
In addition to the expression of GlPLK, GlPLK activity is important for its role in Giardia cell division; this role may be regulated by its activation status, possibly by phosphorylation. We examined whether GlPLK phosphorylation was modulated in a cell phase-dependent manner (Fig. 6a). Giardia cells carrying pGlPLK.neo were used to prepare the cell extracts at interphase, G1/S phase and G2/M phase, and then analyzed by western blotting using anti-phospho-PLK, anti-HA, anti-GlPLK and anti-GlPDI1 antibodies. In the western blot analysis with anti-phospho-PLK, both HA-tagged GlPLK and endogenous GlPLK were detected, and the amount of both proteins increased fivefold and 2.5-fold during the G2/M phase, respectively. In the same manner, western blotting using anti-GlPLK and anti-HA antibodies demonstrated more than a twofold increase in the expression of both HA-tagged GlPLK and endogenous GlPLK during the G2/M phase.
Giardia trophozoites were double-stained with anti-HA and anti-phospho-PLK antibodies (Fig. 6b). Both anti-HA and anti-phospho-PLK antibodies stained basal bodies in the interphase and dividing cells. Localization of phospho-GlPLK in the cytoplasmic portion of anterior flagella, median bodies, and flagella tips was distinct. In dividing cells, phospho-GlPLK was also found at mitotic spindles, as with the HA-tagged GlPLK.
In vitro autophosphorylation of GlPLK and identification of critical amino acid residues for its autophosphorylation
The putative amino acid sequence of GlPLK indicates a serine/threonine KD at the amino terminus and two PBDs at the carboxyl terminus (Fig. 7a). 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.
Kinase assays were also performed using recombinant GlPLK (rGlPLK), which was synthesized using in vitro transcription and translation systems, and expression was confirmed by western blotting with anti-c-Myc antibodies (Fig. 7b). Upon incubation with [γ-32P]ATP, rGlPLK was radiolabeled due to autophosphorylation.
To define the amino acid residues that are critical for GlPLK autophosphorylation, several recombinant GlPLK proteins were synthesized using in vitro transcription/translation systems and used for kinase assays (Fig. 7c). 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 autophosphorylated, although the efficiency of autophosphorylation 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 autophosphorylation ability. Conversion of Lys51 to Arg abolished the autophosphorylation 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
Subsequent experiments were performed to determine the physiological roles of GlPLK. Transgenic G. lamblia carrying pGlPLKK51R.neo was constructed. In addition, Giardia cells ectopically expressing mutant GlPLK (T179AT183A) were prepared. Western blotting demonstrated that the transgenic cells expressed HA-tagged GlPLK proteins (Fig. 8a).
The growth of various Giardia cells (ectopically expressing GlPLK, mutant GLPLKK51R, mutant GlPLKT178AT183A or carrying empty vector) was determined (Fig. 8b). The growth of Giardia cells overexpressing wild-type GlPLK was slightly affected when compared with that of the control cells. Interestingly, Giardia cells expressing mutant GlPLKs showed dramatic growth inhibition compared to those carrying the vector plasmid.
These cells were then evaluated for their number of nuclei as described earlier (Fig. 8c). The majority of the control cells carrying vector plasmid and cells overexpressing wild-type GlPLK (98%) had two nuclei in the correct position. The percentage of cells with two nuclei in the correct position were decreased to 88–89% when mutant GlPLKs were ectopically expressed. Among these cells, the number of cells showing nuclear condensation increased to 4.7 and 2.6% from 0.2 to 0.3% (control and GlPLK-overexpressing cells, respectively) in the case of Giardia expressing GLPLKK51R and GlPLKT179AT183A. In contrast to a slight increase in the number of cells with one nucleus, two abnormally located nuclei and three nuclei, the cells expressing mutant GlPLKs showed a dramatic increase in percentage of cells carrying four nuclei and more than four nuclei. 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. GlPLK overexpression did not affect the length of flagella. In contrast, the ectopic expression of mutant GlPLK resulted in the extension of the lengths of three flagella, except for the posterolateral flagella (Fig. 8d). These data indicate that GlPLK plays a role in regulating flagella morphogenesis and cell cycle in Giardia and that GlPLK phosphorylation is critical for its in vivo function.