Department of Environmental Medical Biology and Institute of Tropical Medicine, Yonsei University College of Medicine, Seoul, 03722, South Korea.
Parasit Vectors. 2021 Mar 31;14(1):182. doi: 10.1186/s13071-021-04687-5.
Polo-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.
To 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.
These data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.
Polo 样激酶（PLKs）是保守的丝氨酸/苏氨酸激酶，调节细胞周期。迄今为止，利什曼原虫 PLK（GlPLK）在细胞中的作用尚未被研究。在这里，我们报告了对 GlPLK 功能的研究，以期深入了解该 PKL 在贾第虫细胞分裂中的作用，特别是在胞质分裂和鞭毛形成过程中。
为了评估 GIPLK 的功能，用 PLK 特异性抑制剂 GW843286X（GW）处理滋养体。利用在贾第虫基因组数据库中鉴定的 PLK 假定开放阅读框，我们生成了表达血凝素（HA）标记的 GlPLK 的转基因贾第虫，并使用该转基因进行免疫荧光分析（IFA）。使用抗 glplk 吗啉代寡核苷酸敲低 GlPLK 表达，观察其对细胞核数量和鞭毛长度的影响。构建了异位表达截短的 GlPLKs、激酶结构域+接头（GlPLK-KDL）或 polo 盒结构域（GlPLK-PBD）的贾第虫细胞。通过定点突变生成 GlPLK 的 Lys51、Thr179 和 Thr183 突变体，并用于激酶测定。为了阐明磷酸化 GlPLK 的作用，突变了磷酸化残基并在贾第虫滋养体中表达。
用 5μM GW 孵育滋养体后，多核细胞和长尾及前鞭毛的细胞百分比增加。IFA 表明 GlPLK 定位于基体和鞭毛，并存在于有丝分裂纺锤体中。GlPLK 敲低的吗啉代寡核苷酸导致与 GW 处理的细胞观察到的表型相同。与表达 GlPLK-PBD 的贾第虫相比，表达 GlPLK-KDL 的贾第虫在 GlPLK 定位到有丝分裂纺锤体方面存在缺陷，并且在有丝分裂细胞中基体的定位发生改变。使用突变重组 GlPLKs 的激酶测定表明，Lys51 或 Thr179 和 Thr183 突变导致激酶活性丧失。表达这些突变 GlPLK 的贾第虫也表现出细胞生长、胞质分裂和鞭毛形成缺陷。
这些数据表明 GlPLK 在贾第虫细胞分裂中发挥作用，特别是在胞质分裂过程中，并且还参与鞭毛形成。
