Keith Gull

Antigenic variation in Trypanosoma brucei ( T. brucei ) requires monoallelic expression of one variant surface glycoprotein (VSG) from one of the subtelomeric bloodstream form (BSF) expression sites (BESs). This transcription is unusually mediated by RNA polymerase I (RNA Pol I) and occurs in a specialized nuclear body, the expression site body (ESB). While factors promoting active BES transcription and silencing inactive BESs are known, how these opposing activities are integrated remains unknown. Here, we identify ESBX (Tb927.3.1660) as a BSF-specific ESB protein necessary for this coordination. We show that ESBX RNAi knockdown prevents RNA Pol I localizing to the ESB and reduces active BES transcription, while also derepressing inactive BESs with low processivity transcription. Conversely, ESBX overexpression weakly activates inactive BESs in a distinct manner from ESBX knockdown, leading to processive transcription, without disrupting the active BES or forming supernumerary ESBs. ESBX knockdown causes a similar transcriptomic defect to ESB1 and VEX2 knockdown combined, establishing ESBX as a key factor linking transcriptional activation of the active BES with inactive BES silencing through the VSG exclusion (VEX) phenomenon. This allows us to suggest models for understanding the establishment and maintenance of monoallelic expression critical for parasite immune evasion.

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African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible polymerase chain reaction-based protein tagging in trypanosomes, which enabled the subcellular mapping of 89% of the trypanosome proteome. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an extended set of >100 plasmids that incorporate universal primer annealing sequences, enabling protein tagging with a range of fluorescent, biochemical and epitope tags, using five different selection markers. We evaluated the suitability of various fluorescent proteins for live and fixed cell imaging, fluorescent movies, and we demonstrate the use of tagging plasmids encoding tandem epitope tags to support expansion microscopy approaches. We show that this series of plasmids is functional in other trypanosomatid parasites, significantly increasing its value. Finally, we developed a new plasmid for tagging glycosylphosphatidylinositol-anchored proteins. We anticipate that this will be an important toolset for investigating trypanosomatid protein localization and function.

Keith Vickerman was a parasitologist and protozoologist who made major contributions to our understanding of the biology of African trypanosomes, the causative agents of human sleeping sickness and nagana in cattle. His first academic post was at University College London, where he quickly mastered the techniques of electron microscopy (EM) and produced some of the best electron micrographs of parasitic protozoa at that time. He was a great believer in observation and deduction, and what began as an exercise in EM led him to investigate two of the then outstanding problems of trypanosome biology: how the parasites manage the transition from the tsetse fly vector to its mammalian host, and how they evade the host's immune response. Morphological changes, he found, were correlated with changes in the single mitochondrion and ensuing biochemical changes during the transition from a glucose-rich environment in mammalian blood to the glucose-poor tsetse gut. It was while comparing bloodstream and tsetse forms that he observed that the trypanosomes possessed a thick surface coat in the blood, which he subsequently identified as the variable antigen that was repeatedly formed and reformed and that this was the basis of antigenic variation—findings that stimulated a vast amount of interest among immunologists, biochemists and geneticists. In his later career a new problem emerged, and he found that a disease devastating stocks of the commercially important Norway lobster, Nephrops norvegicus, thought to be caused by a virus was actually caused by a protozoan, Hematodinium. Keith will always be remembered as one of the founders of modern parasitology.

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1–G2 tether and S–M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells. Significance Centriole/basal body (BB) connections involve two concepts: a tether and a linker. Studies have revealed essential components of the tether, while linker components remain largely unknown. Here, we describe BBLP, a trypanosome protein that is a cytoskeletal component of the linker, localizing between the BB and pro-BB throughout the cell cycle. Importantly, RNA interference (RNAi) depletion of BBLP results in a splitting of the BB–pro-BB pair subtending only the new flagellum, and perturbs mitochondrial DNA inheritance. Our data identify BBLP as the first specific protein component of the BB/centriole linker in eukaryotic cells. We provide a unifying model revealing how aspects of centriole/BB ontogeny and lineage can operate at different points of the cell cycle in evolutionarily diverse cells.

Trypanosomes have complex life cycles within which there are both proliferative and differentiation cell divisions. The coordination of the cell cycle to achieve these different divisions is critical for the parasite to infect both host and vector. From studying the regulation of the proliferative cell cycle of the Trypanosoma brucei procyclic life cycle stage, three subcycles emerge that control the duplication and segregation of ( a) the nucleus, ( b) the kinetoplast, and ( c) a set of cytoskeletal structures. We discuss how the clear dependency relationships within these subcycles, and the potential for cross talk between them, are likely required for overall cell cycle coordination. Finally, we look at the implications this interdependence has for proliferative and differentiation divisions through the T. brucei life cycle and in related parasitic trypanosomatid species.

Summary: Differentiation of Trypanosoma brucei, a flagellated protozoan parasite, between life cycle stages typically occurs through an asymmetric cell division process, producing two morphologically distinct daughter cells. Conversely, proliferative cell divisions produce two daughter cells, which look similar but are not identical. To examine in detail differences between the daughter cells of a proliferative division of procyclic T. brucei we used the recently identified constituents of the flagella connector. These segregate asymmetrically during cytokinesis allowing the new‐flagellum and the old‐flagellum daughters to be distinguished. We discovered that there are distinct morphological differences between the two daughters, with the new‐flagellum daughter in particular re‐modelling rapidly and extensively in early G1. This re‐modelling process involves an increase in cell body, flagellum and flagellum attachment zone length and is accompanied by architectural changes to the anterior cell end. The old‐flagellum daughter undergoes a different G1 re‐modelling, however, despite this there was no difference in G1 duration of their respective cell cycles. This work demonstrates that the two daughters of a proliferative division of T. brucei are non‐equivalent and enables more refined morphological analysis of mutant phenotypes. We suggest all proliferative divisions in T. brucei and related organisms will involve non‐equivalence.

Significance: Leishmaniaalternates between an insect vector and human host; in these different environments, the parasite adopts different forms. There are important commonalities between these different forms, particularly the flagellar pocket (FP) and associated flagellum attachment zone (FAZ). We show that the FAZ is important in different forms ofLeishmaniafor FP shape and function, which are altered in mutants lacking a FAZ protein, FAZ5. FAZ5 deletion did not affect parasite proliferation and differentiation in culture; however, it dramatically reduced parasite proliferation in the sand fly and mouse. These results demonstrate the importance of the FAZ for FP function and architecture, and show that deletion of one FAZ protein can have a dramatic effect onLeishmaniadevelopment and pathogenicity.

Most motile flagella have an axoneme that contains nine outer microtubule doublets and a central pair (CP) of microtubules. The CP coordinates the flagellar beat and defects in CP projections are associated with motility defects and human disease. The CP nucleate near a ‘basal plate’ at the distal end of the transition zone (TZ). Here, we show that the trypanosome TZ protein ‘basalin’ is essential for building the basal plate, and its loss is associated with CP nucleation defects, inefficient recruitment of CP assembly factors to the TZ, and flagellum paralysis. Guided by synteny, we identified a highly divergent basalin ortholog in the related Leishmania species. Basalins are predicted to be highly unstructured, suggesting they may act as ‘hubs’ facilitating many protein-protein interactions. This raises the general concept that proteins involved in cytoskeletal functions and appearing organism-specific, may have highly divergent and cryptic orthologs in other species.

Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement.Leishmaniaexhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation inLeishmaniaby deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis.Δift140cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures ofΔift140cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus,Leishmaniapromastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure, echoing the process that occurs during the promastigote to amastigote differentiation.