Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin
Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin
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DOI：
10.1074/jbc.m300813200
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发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Mottram, JC
中科院分区：
文献类型：
--
作者：
Hammarton, TC;Clark, J;Mottram, JC
非洲锥虫有一个紧密协调的细胞周期，以有效地分离它们的单个细胞器，即核、鞭毛和动泡体。为了研究锥虫细胞周期的调控，在布氏锥虫中发现了一个有丝分裂周期蛋白基因(Cycl6)。我们发现，在体内，CyC6与锥虫CDK1同源物CRK3形成活性激酶复合体。利用RNA干扰，我们证明了在昆虫原周期和哺乳动物的血流形式中，缺乏CyC6 mRNA会导致有丝分裂障碍和生长停滞。在原周期形式中，CyC6 RNA干扰产生只有一个动泡体的无核细胞，而在血流形式的锥体中，观察到具有一个核和多个动泡体的细胞。荧光激活的细胞分选分析表明，血流而不是前循环锥体能够在没有有丝分裂的情况下重新启动核S期。综上所述，这些数据表明，原环锥体可以在没有完成有丝分裂的情况下进行胞质分裂，而血流形式的锥体中的有丝分裂阻断会抑制胞质分裂，但不会抑制动质复制和分离，也不会产生额外的一轮核DNA合成。这表明布鲁氏毛滴虫在生活周期形式之间在细胞周期控制方面存在根本差异，高等真核生物中存在的关键细胞周期检查点在锥虫中缺失。
African trypanosomes have a tightly coordinated cell cycle to effect efficient segregation of their single organelles, the nucleus, flagellum, and kinetoplast. To investigate cell cycle control in trypanosomes, a mitotic cyclin gene (CYC6) has been identified in Trypanosoma brucei. We show that CYC6 forms an active kinase complex with CRK3, the trypanosome CDK1 homologue, in vivo. Using RNA interference, we demonstrate that absence of CYC6 mRNA results in a mitotic block and growth arrest in both the insect procyclic and mammalian bloodstream forms. In the procyclic form, CYC6 RNA interference generates anucleate cells with a single kinetoplast, whereas in bloodstream form trypanosomes, cells with one nucleus and multiple kinetoplasts are observed. Fluorescence-activated cell sorting analysis shows that bloodstream but not procyclic trypanosomes are able to reinitiate nuclear S phase in the absence of mitosis. Taken together, these data show that procyclic trypanosomes can undergo cytokinesis without completion of mitosis, whereas a mitotic block in bloodstream form trypanosomes inhibits cytokinesis but not kinetoplast replication and segregation nor an additional round of nuclear DNA synthesis. This indicates that there are fundamental differences in cell cycle controls between life cycle forms of T. brucei and that key cell cycle checkpoints present in higher eukaryotes are absent from trypanosomes.