Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle.

Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 degrees C to 26 degrees C in the presence of citrate and cis-aconitate.