1 Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, UK
DiscussionThe cell division cycle of Trypanosoma brucei is highly organised and tightly controlled, reflecting the need to co-ordinate not only nuclear division, but also the division and segregation of the mitochondrial kinetoplast DNA and its single copy organelles such as the ER, Golgi and flagellum. The temporal control of protein involved in the regulation and progression of cell cycle is essential to ensure correct growth and division, and cell cycle arrest is required for pre-adaption for transmission between hosts. Regulation of the eukaryotic cell cycle is achieved through control at multiple levels including transcriptional regulation, and despite a paucity of transcription factor mediated gene expression, T. brucei regulates it transcript abundance over the cell cycle .
We are taking an unbiased approach to investigate the role of dynamic phosphorylation in the T. brucei cell cycle by conducting global quantitative proteomic analysis of synchronised cell populations. We have optimised a centrifugal counter-flow elutriation protocol for both Pcf and Bsf cells that yields viable and proliferative cells that maintain synchronicity into subsequent cell cycles . SILAC isotopic labelling of these synchronised cells has allowed us to generate quantitative temporal profiles of protein and phosphorylation site abundance that provide an insight into the role of dynamic phosphorylation in the post-transcriptional regulation of cell cycle control.
1. Archer, S.K., et al., The cell cycle regulated transcriptome of Trypanosoma brucei. PLoS One, 2011. 6(3): p. e18425.
2. Benz, C., et al., Cell cycle synchronisation of Trypanosoma brucei by centrifugal counter-flow elutriation reveals the timing of nuclear and kinetoplast DNA replication. Sci Rep, 2017. 7(1): p. 17599.