Knockdown of APC/C-associated genes and its effect on viability and cell cycle of protozoan parasite of Trypanosoma brucei

Abstract

In the eukaryotic pathogen of Trypanosoma brucei, the anaphase promoting complex or cyclosome (APC/C) is composed of ten subunit proteins which are conserved in kinetoplastid protozoan parasites. During the course of APC/C characterization by PTP tagging and mass spectrometry, some other proteins were found to be associated in substoichiometric ratio to APC/C. These proteins could not be assigned as APC/C core components as they are below the threshold imposed by mass spectrometry identification and therefore they are termed non-core APC/C-associated proteins. Here in this study, functional roles of these proteins were investigated through reverse genetics approach. mRNAs of protein-encoding genes were individually knocked down by RNA interference and the resulting phenotypes were assayed through functional assays such as growth curve, cell cycle progression by flow cytometry, and DNA profiles by DAPI staining and microscopy examination. Based on the presented data, these proteins are playing essential functions in the cell biology of T. brucei; and more specifically, in regulating its cell cycle progression. Thus, the non-core APC/C-associated proteins appear to play important roles in complementing APC/C specialized function in the cell cycle of T. brucei.