Distinct cytoskeletal modulation and regulation of G1-S transition in the two life stages of Trypanosoma brucei.

Procyclic-form Trypanosoma brucei is arrested in G1 phase with extended and/or branched posterior morphology when expression of its cdc2-related kinases 1 and 2 (CRK1 and CRK2) is knocked down by RNA interference. Transmission electron microscopy indicated that the mitochondrion in the cell is also extended and branched and associated with cortical microtubules in each elongated/branched posterior end. This posterior extension is apparently driven by the growing microtubule corset, as it can be blocked by rhizoxin, an inhibitor of microtubule assembly. In the bloodstream form of T. brucei, however, a knockdown of CRK1 and CRK2 resulted only in an enrichment of cells in G1 phase without cessation of DNA synthesis or elongated/branched posterior ends. A triple knockdown of CRK1, CRK2 and CycE1/CYC2 in the bloodstream form resulted in 15% of the cells arrested in G1 phase, but no cells had an abnormal posterior morphology. The double and triple knockdown bloodstream-form cells were differentiated in vitro into the procyclic form, and the latter thus generated bore the typical morphology of a procyclic form without an extended/branched posterior end, albeit arrested in the G1 phase as the bloodstream-form precursor. There is thus a major distinction in the mechanisms regulating G1-S transition and posterior morphogenesis between the two life stages of T. brucei.