Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin.
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.
Hammarton
Tansy C
TC
Wellcome Centre for Molecular Parasitology, Anderson College, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU, United Kingdom.
Clark
Jade
J
Douglas
Fiona
F
Boshart
Michael
M
Mottram
Jeremy C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D016384:Consensus Sequence; D016213:Cyclins / Q000235:genetics / Q000502:physiology*; D017931:DNA Primers; D005434:Flow Cytometry; D007621:Karyotyping; D007700:Kinetics; D008938:Mitosis / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D015800:Protozoan Proteins / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics*; D015183:Restriction Mapping; D014346:Trypanosoma brucei brucei / Q000166:cytology* / Q000235:genetics
C476455:Cyc6 protein, Trypanosoma brucei; D016213:Cyclins; D017931:DNA Primers; D015800:Protozoan Proteins; D034741:RNA, Small Interfering