Developmental and morphological regulation of clathrin-mediated endocytosis inTrypanosoma brucei

Abstract

Essentially all macromolecular communication between Trypanosoma brucei and its host is confined to vesicular trafficking events occurring at or around the flagellar pocket. The vertebrate stage bloodstream form trypomastigote exhibits an extremely high rate of endocytosis required for nutrient uptake and probably also evasion of the host immune system. However, the rate of endocytosis is very low in the procyclic vector parasite, indicating that endocytosis is subject to a marked level of developmental regulation. Previous ultrastructural studies and crude biochemical fractionations have indicated the presence of coated pits and vesicles that are analogous to clathrin coats in the bloodstream form, but not in the procyclic. However, a definitive description of the components of this coat and its molecular function in T. brucei has remained elusive. We describe the molecular cloning and initial characterisation of components of the T. brucei endocytic coats: clathrin heavy chain (TbCLH) and a β-adaptin (TbAPβ1). TbCLH is markedly upregulated in the bloodstream form compared with the procyclic, whereas TbAPβ1 is subject to more limited developmental regulation. We generated antisera against both proteins and show that the clathrin coat is tightly associated with the flagellar pocket in both major life stages. However, in bloodstream parasites TbCLH is also extensively distributed throughout the posterior end of the cell on numerous large vesicular and tubular structures. By cryoimmuno EM, clathrin is localised to collecting tubules at the flagellar pocket and is also associated with the trans-Golgi network. These EM data confirm that the electron dense coats reported on trypanosome vesicles and tubules contain clathrin. The TbAPβ1 exhibits an atypical distribution relative to previously characterised adaptins, associating not only with the trans-Golgi but also with other tubular-vesicular elements. Localisation of TbAPβ1 is also subject to developmental regulation. These data describe major endocytic coat proteins in T. brucei for the first time, and indicate stage-specific expression of the clathrin heavy chain. Modulation of clathrin expression is likely to be an important factor in the developmental regulation of endocytosis and recycling in the African trypanosome.

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Fornicata

“…Given that the role for clathrin assemblies in Giardia has not been elucidated and that the nature of Gl CLC ( Gl 4259) as a clathrin light chain orthologue is dubious, we sought to shed light on the significance of this correlation by investigating the distribution of both CHC and CLC orthologues in selected eukaryotic lineages. To do this, we employed protein homology searches based on Hidden-Markov Models (HMM) (Eddy 2011) using as query an alignment of canonical and documented CHC or CLC sequences from several protozoa and metazoan species (Supplementary Tables 2 and 5) (Morgan et al 2001; Kaksonen et al 2005; Adung’a et al 2013; Kirchhausen et al 2014; Karnkowska et al 2016; Karnkowska et al 2019, Füssy et al, 2021). In our search we considered assembled read data from RNA-seq experiments (transcriptomics) as reliable as genomic sequence data (Cheon et al 2020).…”

“…This observation points to as yet uncharacterised uncoating mechanisms are present in other lineages. For example, clathrin mediated endocytosis is essential in the parasitic protist Trypanosoma brucei and CCVs have been documented in this organism (Morgan et al 2001; Allen et al 2003; Adung’a et al 2013; Link et al 2021). Clathrin and other coat proteins associated with CCVs need to be recycled.…”

Combined nanometric and phylogenetic analysis of unique endocytic compartments in Giardia lamblia sheds light on the evolution of endocytosis in Metamonada

“…This observation points to as yet uncharacterized uncoating mechanisms are present in other lineages. For example, clathrin-mediated endocytosis is essential in the parasitic protist Trypanosoma brucei and CCVs have been documented in this organism [ 70 , 71 , 114 , 115 ]. Clathrin and other coat proteins associated with CCVs need to be recycled.…”

“…orthologous) is unclear, as is the prevalence of this protein in other members of the lineage Fornicata. To investigate the occurrence of both CHC and CLC orthologues in selected eukaryotic lineages, we employed protein homology searches based on hidden Markov models (HMM) [68] using as query an alignment of canonical and documented CHC or CLC sequences from several protozoa and metazoan species (Additional file 13: Tables S2 and S5) [69][70][71][72][73][74][75]. In this search, we considered assembled read data from RNA-seq experiments (transcriptomics) as reliable as genomic sequence data [76].…”

To the Surface and Back: Exo- and Endocytic Pathways in Trypanosoma brucei

“…brucei restricts both endo-and exocytosis to the FP (Figure 2; Vickerman, 1969;Overath et al, 1997;Gull, 2003;Engstler et al, 2004;Field and Carrington, 2009). Uptake from the FP involves the formation of CCPs (Morgan et al, 2001;Allen et al, 2003;Grünfelder et al, 2003;Engstler et al, 2004), which rapidly pinch off as CCVs with a diameter of 135 nm, known as class I CCV (CCV I) (Grünfelder et al, 2003;Overath and Engstler, 2004).…”