Kim Juri, Park Eun-Ah, Shin Mee Young, Park Soon-Jung
Department of Tropical Medicine, Yonsei University College of Medicine, Seoul, South Korea.
Microbiol Spectr. 2023 Mar 6;11(2):e0491922. doi: 10.1128/spectrum.04919-22.
Cyclin-dependent kinases (CDKs) are serine/threonine kinases that control the eukaryotic cell cycle. Limited information is available on Giardia lamblia CDKs (GlCDKs), GlCDK1 and GlCDK2. After treatment with the CDK inhibitor flavopiridol-HCl (FH), division of Giardia trophozoites was transiently arrested at the G1/S phase and finally at the G2/M phase. The percentage of cells arrested during prophase or cytokinesis increased, whereas DNA synthesis was not affected by FH treatment. Morpholino-mediated depletion of GlCDK1 caused arrest at the G2/M phase, while GlCDK2 depletion resulted in an increase in the number of cells arrested at the G1/S phase and cells defective in mitosis and cytokinesis. Coimmunoprecipitation experiments with GlCDKs and the nine putative G. lamblia cyclins (Glcyclins) identified Glcyclins 3977/14488/17505 and 22394/6584 as cognate partners of GlCDK1 and GlCDK2, respectively. Morpholino-based knockdown of Glcyclin 3977 or 22394/6584 arrested cells in the G2/M phase or G1/S phase, respectively. Interestingly, GlCDK1- and Glcyclin 3977-depleted Giardia showed significant flagellar extension. Altogether, our results suggest that GlCDK1/Glcyclin 3977 plays an important role in the later stages of cell cycle control and in flagellar biogenesis. In contrast, GlCDK2 along with Glcyclin 22394 and 6584 functions from the early stages of the Giardia cell cycle. Giardia lamblia CDKs (GlCDKs) and their cognate cyclins have not yet been studied. In this study, the functional roles of GlCDK1 and GlCDK2 were distinguished using morpholino-mediated knockdown and coimmunoprecipitation. GlCDK1 with Glcyclin 3977 plays a role in flagellum formation as well as cell cycle control of G. lamblia, whereas GlCDK2 with Glcyclin 22394/6584 is involved in cell cycle control.
细胞周期蛋白依赖性激酶（CDK）是控制真核细胞周期的丝氨酸/苏氨酸激酶。关于贾第虫属蓝氏贾第鞭毛虫的CDK（GlCDK）、GlCDK1和GlCDK2的信息有限。在用CDK抑制剂盐酸黄酮哌酯（FH）处理后，蓝氏贾第鞭毛虫滋养体的分裂在G1/S期短暂停滞，最终在G2/M期停滞。在前期或胞质分裂期间停滞的细胞百分比增加，而DNA合成不受FH处理的影响。吗啉代介导的GlCDK1缺失导致在G2/M期停滞，而GlCDK2缺失导致在G1/S期停滞的细胞数量增加以及有丝分裂和胞质分裂有缺陷的细胞数量增加。用GlCDK与9种假定的蓝氏贾第鞭毛虫细胞周期蛋白（Glcyclin）进行的免疫共沉淀实验分别确定Glcyclin 3977/14488/17505和22394/6584为GlCDK1和GlCDK2的同源伴侣。基于吗啉代的Glcyclin 3977或22394/6584敲低分别使细胞在G2/M期或G1/S期停滞。有趣的是，GlCDK1和Glcyclin 3977缺失的蓝氏贾第鞭毛虫显示出明显的鞭毛延长。总之，我们的结果表明GlCDK1/Glcyclin 3977在细胞周期控制的后期阶段和鞭毛生物发生中起重要作用。相比之下，GlCDK2与Glcyclin 22394和6584在蓝氏贾第鞭毛虫细胞周期的早期阶段起作用。蓝氏贾第鞭毛虫的CDK（GlCDK）及其同源细胞周期蛋白尚未得到研究。在本研究中，使用吗啉代介导的敲低和免疫共沉淀区分了GlCDK1和GlCDK2的功能作用。GlCDK1与Glcyclin 3977在蓝氏贾第鞭毛虫的鞭毛形成以及细胞周期控制中起作用，而GlCDK2与Glcyclin 22394/6584参与细胞周期控制。
