Characterisation of TbSmee1 indicates that endocytosis is required for access of surface-bound cargo to the trypanosome flagellar pocket

Abstract

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cell's single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, may affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement, and impaired access to the pocket membrane by surface-bound cargo. Inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket.

AAK1‐like: A putative pseudokinase with potential roles in cargo uptake in bloodstream form Trypanosoma brucei parasites

“…Deletion of TbAAK1L1 correlates with a gross enlargement of the FP (Figure 5) and increased TL and ConA signal (Figure 6), coinciding with the accumulation of both TL and ConA within the FP. Endocytosis defects similar to those reported here, including blocked or slowed ConA uptake, have been previously reported following the depletion of clathrin, TbMORN1, TbSmee1 and other FPC/HC members in T. brucei (Allen et al, 2003;Broster Reix et al, 2021;Florimond et al, 2015;Halliday et al, 2021;Hung et al, 2004;Morriswood & Schmidt, 2015;Perdomo et al, 2022;Schichler et al, 2022). RNAi and null mutant cell lines exhibit similar morphological defects (Figure 4C,D), suggesting that the aberrant cells observed in the null mutant cell lines are unlikely a consequence of long-term culture adaptation.…”

“…TbAAK1L1 interacts with clathrin (Manna et al, 2017) and localizes in proximity to the FP. We confirm TbAAK1L1 localizes between the kDNA and the nDNA, where other components of the polarized endosome system are also found in T. brucei (Broster Reix et al, 2021;Field & Carrington, 2009;Florimond et al, 2015;Halliday et al, 2021;Manna et al, 2017;Morriswood & Schmidt, 2015;Perdomo et al, 2022;Schichler et al, 2022). Moreover, we demonstrate that TbAAK1L1 can be found in contact with the FP membrane and endocytic vesicles, suggestive of a role for TbAAK1L1 during cargo internalization.…”

“…We performed a second cargo internalization at 37°C by flow cytometry using concanavalin A (ConA). ConA is another lectin with specificity to mannose and capable of binding glycoproteins on the surface (i.e., representative of surface‐bound cargo; Allen et al, 2003; Schichler et al, 2022). This assay was performed on live cells to exclude cell death‐related artifacts (Figure 6C,D and Figure S7E–G).…”