Stage-specific Differences in Cell Cycle Control in Trypanosoma brucei Revealed by RNA Interference of a Mitotic Cyclin

• Published in: The Journal of biological chemistry 2003-06, Vol.278 (25), p.22877-22886
• Main Authors: Hammarton, Tansy C, Clark, Jade, Douglas, Fiona, Boshart, Michael, Mottram, Jeremy C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cell Cycle - genetics; Cell Cycle - physiology; Cloning, Molecular; Consensus Sequence; Cyclins - genetics; Cyclins - physiology; DNA Primers; Flow Cytometry; Karyotyping; Kinetics; Mitosis - genetics; Mitosis - physiology; Molecular Sequence Data; Open Reading Frames; Protozoan Proteins - genetics; Protozoan Proteins - physiology; Restriction Mapping; RNA, Small Interfering - genetics; Trypanosoma brucei brucei - cytology; Trypanosoma brucei brucei - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M300813200

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.