Life and times:synthesis, trafficking, and evolution of VSG

Evasion of the acquired immune response in African trypanosomes is principally mediated by antigenic variation, the sequential expression of distinct variant surface glycoproteins (VSGs) at extremely high density on the cell surface. Sequence diversity between VSGs facilitates escape of a subpopulation of trypanosomes from antibody-mediated killing. Significant advances have increased understanding of the mechanisms underpinning synthesis and maintenance of the VSG coat. In this review, we discuss the biosynthesis, trafficking, and turnover of VSG, emphasising those unusual mechanisms that act to maintain coat integrity and to protect against immunological attack. We also highlight new findings that suggest the presence of unique or highly divergent proteins that may offer therapeutic opportunities, as well as considering aspects of VSG biology that remain to be fully explored

Conservation and divergence within the clathrin interactome of <i>Trypanosoma cruzi</i>

Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent

Resolving the homology-function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage

Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease

Intracellular trafficking in the trypanosomatids.

Trypanosomes are members of the kinetoplastida, a group of divergent protozoan parasites responsible for considerable morbidity and mortality worldwide. These organisms have highly complex life cycles requiring modification of their cell surface together with engagement of immune evasion systems to effect survival; both processes intimately involve the membrane trafficking system. The completion of three trypanosomatid and several additional protist genomes in the last few years is providing an exciting opportunity to evaluate, at the molecular level, the evolution and diversity of membrane trafficking across deep evolutionary time as well as to analyse in unprecedented detail the membrane trafficking systems of trypanosomes

TbVps34, the trypanosome orthologue of Vps34, is required for Golgi complex segregation.

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division

Evolution of Tre-2/Bub2/Cdc16 (TBC) Rab GTPase-activating proteins

Rab GTPases serve as major control elements in the coordination and definition of specific trafficking steps and intracellular compartments. Rab activity is modulated in part by GTPase-activating proteins (GAPs), and many RabGAPs share a Tre-2/Bub2/Cdc16 (TBC)–domain architecture, although the majority of TBC proteins are poorly characterized. We reconstruct the evolutionary history of the TBC family using ScrollSaw, a method for the phylogenetic analysis of pan-eukaryotic data sets, and find a sophisticated, ancient TBC complement of at least 10 members. Significantly, the TBC complement is nearly always smaller than the Rab cohort in any individual genome but also suggests Rab/TBC coevolution. Further, TBC-domain architecture has been well conserved in modern eukaryotes. The reconstruction also shows conservation of ancestral TBC subfamilies, continuing evolution of new TBCs, and frequent secondary losses. These patterns give additional insights into the sculpting of the endomembrane system

TbVps34, the trypanosome orthologue of Vps34, is required for Golgi complex segregation.

Phosphoinositides are important regulators of numerous cellular functions. The yeast class III phosphatidylinositol 3-kinase Vps34p, and its human orthologue hVPS34, are implicated in control of several key pathways, including endosome to lysosome transport, retrograde endosome to Golgi traffic, multivesicular body formation, and autophagy. We have identified the Vps34p orthologue in the African trypanosome, TbVps34. Knockdown of TbVps34 expression by RNA interference induces a severe growth defect, with a post-mitotic block to cytokinesis accompanied by a variety of morphological abnormalities. GFP2xFYVE, a chimeric protein that specifically binds phosphatidylinositol 3-phosphate, localizes to the trypanosome endosomal system and is delocalized under TbVps34 RNA interference (RNAi), confirming that TbVps34 is an authentic phosphatidylinositol 3-kinase. Expression of GFP2xFYVE enhances the TbVps34 RNAi-associated growth defect, suggesting a synthetic interaction via competition for phosphatidylinositol 3-phosphate-binding sites with endogenous FYVE domain proteins. Endocytosis of a fluid phase marker is unaffected by TbVps34 RNAi, but receptor-mediated endocytosis of transferrin and transport of concanavalin A to the lysosome are both impaired, confirming a role in membranous endocytic trafficking for TbVps34. TbVps34 knockdown inhibits export of variant surface glycoprotein, indicating a function in exocytic transport. Ultrastructural analysis revealed a highly extended Golgi apparatus following TbVps34 RNAi, whereas expression of the Golgi marker red fluorescent protein-GRASP (Grp1 (general receptor for phosphoinositides-1)-associated scaffold protein) demonstrated that trypanosomes are able to duplicate the Golgi complex but failed to complete segregation during mitosis, despite faithful replication and segregation of basal bodies and the kinetoplast. These observations implicate TbVps34 as having a role in coordinating segregation of the Golgi complex at cell division