Investigating the involvement of proteins ATG24 (Tb927.9.13380) and Raptor N-terminal CASPase (Tb927.1.2190) in Trypanosoma brucei autophagy pathway

Abstract

This study investigates the roles of proteins ATG24 and Raptor in autophagy, particularly in the context of nutritional shortage, in Trypanosoma brucei, a crucial process for breaking down and recycling cell components.We employed CRISPR-Cas9 technology to endogenously tag ATG24 and Raptor in T. brucei, enabling visualization of their localization to autophagosomes during nutrient deprivation. Growth curves assessed the impact of ATG24 on cell viability, while fluorescence microscopy and Western blotting confirmed successful tagging and protein expression. Experimental conditions included nutrient starvation with and without L-lysine to investigate autophagy induction. Results indicated that the development of autophagosomes under nutrient-limiting conditions is dependent on ATG24, as demonstrated by the co-localization seen in fluorescence microscopy. The fraction of cells with autophagosomes increased significantly in response to starvation, especially after 60 minutes of lysine deficiency. On the other hand, Raptor's tagging attempt was a failure, which restricted the examination of its function in autophagy. The significance of ATG24 in nutrient-restricted autophagy in T. brucei is highlighted by this study, contributing to our comprehension of autophagic mechanisms in kinetoplastids. Even though Raptor was hard to tag, the results point to ATG24's potential use as a marker in autophagy research. Future research should focus on developing trustworthy markers and examining the regulation mechanisms of autophagy in various environmental settings.