Abstract
BackgroundThe Trypanosoma brucei cell cycle is regulated by combinations of cyclin/CRKs (cdc2 related kinases). Recently, two additional cyclins (CYC10, CYC11) and six new CRK (CRK7-12) homologues were identified in the T. brucei genome database [1,2].ResultsIndividual RNAi knockdowns of these new proteins in the procyclic form of T. brucei showed no apparent phenotype except for the CRK9 depletion, which enriched the cells in G2/M phase. But a similar CRK9 knockdown in the bloodstream form caused no apparent phenotype. CRK9 lacks the typical PSTAIRE motif for cyclin binding and the phenylalanine "gatekeeper" but binds to cyclin B2 in vitro and localizes to the nucleus in both forms of T. brucei. CRK9-depleted procyclic-form generated no detectable anucleate cells, suggesting an inhibition of cytokinesis by CRK9 depletion as well. The knockdown enriched cells with one nucleus, one kinetoplast and two closely associated basal bodies with an average distance of 1.08 mm in between, which was shorter than the control value of 1.36 μm, and the cells became morphologically deformed and rounded with time.ConclusionCRK9 may play a role in mediating the segregation between the two kinetoplast/basal body pairs prior to cytokinetic initiation. Since such a segregation over a relatively significant distance is essential for cytokinetic initiation only in the procyclic but may not be in the bloodstream form, CRK9 could be specifically involved in regulating cytokinetic initiation in the procyclic form of T. brucei.
Highlights
The Trypanosoma brucei cell cycle is regulated by combinations of cyclin/cdc2-related kinases (CRKs)
RNA interference (RNAi) mediated ablation of cyclin and CRK homologues in procyclic form T. brucei The expression of newly identified homologues of cyclins (CYC9-10 accession Tb08.11J15.340, and Tb08.11J15.300) and CRKs (CRK7-12, accession Tb07.43M14.340, Tb11.02.5010, Tb927.2.4510, Tb03.48K5.160, Tb06.5F5.880, and Tb11.01.4130, respectively) [1,2] were each silenced in RNAi experiments for potential effect on cell cycle progression in procyclic-form T. brucei
The level of CRK9 mRNA before and after 2 days of RNAi were further examined by real time quantitative RT-PCR
Summary
The Trypanosoma brucei cell cycle is regulated by combinations of cyclin/CRKs (cdc related kinases). The most extensively studied class of protein kinases is the cyclin-dependent kinases (Cdks) such as the prototypic Cdc regulating the cell cycle in budding yeast [3,4,5,6,7,8,9,10]. Trypanosoma brucei is a parasitic protozoan and the causative agent of sleeping sickness, a devastating disease responsible for considerable morbidity and mortality in sub-Saharan Africa. It is considered a deeply branched eukaryote with many unique biological features [13,14].
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