Biology subjects

Gull, K.

Publications and source records attributed to Gull, K..

4 recordsLinked to original sources

bioRxiv2023

Unlocking Trypanosome Biology: A Comprehensive Protein-Tagging Toolkit for Localization and Functional Analysis

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 protein tagging in trypanosomes, which enabled the localisation of all proteins in the trypanosome cell. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an expanded set of >100 vectors that utilizes universal primer annealing sequences, enabling protein tagging with a range of fluorescent and biochemical tags using five different selection markers. We evaluated the suitability of various fluorescent proteins for live cell imaging and determined their brightness and stability under different fixation conditions. Finally, we determined the optimal fluorescent protein for a set of specific experimental conditions demonstrating the utility of this toolkit.

cell biology↗

bioRxiv2022

Genome-wide subcellular protein localisation in the flagellate parasite Trypanosoma brucei

Trypanosoma brucei is a prototypical trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is hindered since, as with most eukaryotic microbes, [~]50% of the proteins encoded in the genome have completely unknown function. Using fluorescence microscopy and cell lines expressing endogenously tagged proteins we mapped the subcellular localisation of 89% of the proteome, giving clues to function, defining the lineage-specific organelle adaptations for obligate parasitism and mapping the ultra-conserved cellular architecture of eukaryotes. This includes the single flagellum, vital for morphogenesis and pathology: the first comprehensive cartographic analysis of the flagellum in any organism. To demonstrate the power of this resource, we identify novel specialisation of organelle molecular composition through the cell cycle and in specialised subdomains. This is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

microbiology↗

bioRxiv2021

Monoallelic antigen expression in trypanosomes requires a stage-specific transcription activator

Monoallelic expression of a single gene family member underpins a molecular "arms race" between many pathogens and their host, through host monoallelic immunoglobulin and pathogen monoallelic antigen expression. In Trypanosoma brucei, a single, abundant, variant surface glycoprotein (VSG) covers the entire surface of the bloodstream parasite1 and monoallelic VSG transcription underpins their archetypal example of antigenic variation. It is vital for pathogenicity, only occurring in mammalian infectious forms1. Transcription of one VSG gene is achieved by RNA polymerase I (Pol I)2 in a singular nuclear structure: the expression site body (ESB)3. How monoallelic expression of the single VSG is achieved is incompletely understood and no specific ESB components are known. Here, using a protein localisation screen in bloodstream parasites, we discovered the first ESB-specific protein: ESB1. It is specific to VSG-expressing life cycle stages where it is necessary for VSG expression, and its overexpression activates inactive VSG promoters. This showed monoallelic VSG transcription requires a stage-specific activator. Furthermore, ESB1 is necessary for Pol I recruitment to the ESB, however transcript processing and inactive VSG gene exclusion ESB sub-domains do not require ESB1. This shows that the cellular solution for monoallelic transcription is a complex factory of functionally distinct and separably assembled sub-domains.

microbiology↗

bioRxiv2018

Basalin: an evolutionary unconstrained protein revealed via a conserved role in basal plate function

Most motile flagella have an axoneme that contains nine outer microtubule doublets and a central pair (CP) of microtubules. The CP is thought to coordinate the flagellar beat and defects in CP projections are associated with loss of motility and human disease. In most cilia, 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 inefficient recruitment of CP assembly factors to the TZ, loss of the CP and flagellum paralysis. Guided by synteny, we identified highly divergent basalin orthologs in the genomes of related Leishmania species. Basalins are predicted to be highly unstructured, suggesting that they may act as hubs facilitating many protein-protein interactions. This raises the general concept that proteins involved in cytoskeletal functions and apparently appearing organism-specific, may have highly divergent and cryptic orthologs in other species.

cell biology↗